Referenten
Prof. Dr. Alexander Rohrbach
Prof. Dr. Thorsten Hugel
Heureka!
Zusammenfassung
Aus der Sichtweise eines Ingenieurs besteht der menschliche Körper aus einem steifen, tragenden Gerüst
(Skelett), welches durch zusammenziehbare Bündel (Muskeln) in Form gebracht und bewegt werden kann.
Verbindungselemente (Sehnen, Gelenke) und verschiedene Maschinen (Organe) dienen dessen Unterstützung
und Versorgung. Obwohl die jeweiligen Strukturen und Mechanismen verschieden sind, gilt dasselbe Konzept
auch für einzelne Zellen. Mechanisch werden Zellen von einer filamentartigen Struktur, genannt Zytoskelett,
dominiert. Dies ist eine genau abgestimmte, hochdynamische Maschine, die in fast allen essentiellen
Prozessen im Leben einer einzelnen Zelle eine wichtige Rolle spielt. Beispiele hierfür sind etwa die
Fortbewegung und Zellteilung. Fehlfunktionen des Zytoskeletts gehen häufig mit schweren Erkrankungen
einher, dennoch sind viele grundlegende Prozesse erst wenig verstanden. Im Zuge unterschiedlicher
wissenschaftlicher Fragestellungen beschäftigt sich die vorliegende Arbeit mit zwei verschiedenen
zytoskelettbasierten Modellsystemen, die mit Hilfe von zeitlich gemultiplexten optischen Pinzetten untersucht
werden.
Optische Pinzetten nutzen stark fokussiertes Laserlicht um mikrometergroße Objekte zu fangen und zu
manipulieren. Neben ihrer vielseitigen und weitreichenden Anwendung in vielen biophysikalischen
Fragestellungen der letzten Jahre wurden sie hauptsächlich in Kombination mit sphärischen Sonden in
statischen Konfigurationen benutzt. Dynamische bzw. zeitlich gemultiplexte Pinzetten werden durch schnelles
Verfahren der Falle zwischen verschiedenen Fokuspositionen erzeugt. Dadurch können im zeitlichen Mittel
nahezu beliebige Fallenkonfigurationen erzeugt werden.
Im ersten Teil der vorliegenden Arbeit wird ein linear oszillierender Focus genutzt um ein helikales
Bakterium zu fangen und dessen Windungen abzubilden. Technische Aspekte werden dabei analytisch
beschrieben, experimentell überprüft und mit Simulationen verglichen. Hierauf basierend wird dann die
Dynamik der Fortbewegung einzelner Bakterien in verschiedenen experimentellen Situationen analysiert und
dies als Basis zur Entwicklung eines biophysikalischen Models verwendet, welches den zugrundeliegenden,
krafterzeugenden molekularen Motor beschreibt. Dieser Motor ist einzigartig in der Natur. Seine
Funktionsweise ist bisher nicht verstanden, könnte aber wichtige Auswirkungen auf die Konstruktion von sich
autonom fortbewegenden, künstlichen Mikromaschinen haben. Der zweite Teil der Arbeit nutzt ein Raster aus
optischen Fallen um synthetisierte Zytoskelettnetzwerke mit einer definierten Struktur in einem bottomup
Ansatz flexibel zu konstruieren, zu testen und zu analysieren. Um das Detektieren und Weiterleiten von
Kräften im Inneren von eukaryotischen Zellen besser zu verstehen, werden MikrorheologieTechniken
angewendet, um das viskoelastische, frequenzabhängige Antwortverhalten verschiedener Netzwerke auf eine
einwirkende mechanische Last zu untersuchen. Auch hier werden wieder analytische Beschreibungen des
Systems mit Simulationen kombiniert, um die dahinterliegenden, grundsätzlichen Prinzipien zu verstehen.
4
Abstract
From an engineer’s perspective, the human body is a stiff, load bearing scaffold (skeleton), kept in shape
and actuated by contractile bundles (muscles), supported by connecting elements (e.g., tendons or joints) and
supplied by different engines (organs). The same concept holds for single cells, although structure and
mechanisms are different. Mechanically, single cells are dominated by a filamentous structure called
cytoskeleton; a precisely tuned, highly dynamic machine involved in nearly all essential processes in the life of
a single cell, e.g., cell movement or proliferation. Even though a malfunctioning is often related to severe
diseases, most of its basic functions are still poorly understood. The present work investigates two different
cytoskeletal model systems by timemultiplexed optical tweezers configurations pursuing different scientific
objectives.
Optical tweezers make use of focused laser light to trap and manipulate micronsized objects. Besides their
versatile and farreaching application to numerous biophysical problems in recent years, they have mainly
been used in combination with spherical probes in a static manner. Dynamical or timemultiplexed optical
tweezers are created by rapidly scanning the optical trap between different focal positions to generate nearly
arbitrary, timeshared trapping configurations.
In the first part of the present thesis, a linescanning focus is used to trap helicallyshaped bacteria by a
light tube and to image individual positions of their windings. Technical aspects are described analytically,
verified experimentally and compared to simulations. Based on this technique, the dynamics of the swimming
pattern of single bacterial cells is then studied under different conditions and used as basis to develop a
biophysical model describing their cytoskeletonbased molecular motor generating propulsion forces. This
motor is unique in nature  its detailed functioning is yet unknown but may have an important impact on
constructing artificial, micronsized machines able to move autonomously. In the second part of this thesis, a
grid of multiplexed tweezers is used to flexibly construct, probe and analyze synthesized cytoskeletal networks
with a defined topology in a bottomup approach. In the light of understanding force sensing and transmission
inside eukaryotic cells, microrheology techniques are used to study the viscoelastic, frequencydependent
response of different networks to an applied mechanical load. Again, analytical descriptions of the system are
used in combination with simulations to understand the basic principles and mechanisms behind.
5
Special functions and abbreviations
The following convention is used throughout this thesis: a scalar parameter or variable x is printed in italic,
numbers are nonitalic, a vector x is bold nonitalic, common functions and parameters such as the sine
function (sin) and pi () are nonitalic and nonbold to distinguish between variables introduced in the special
context of this thesis. Besides, the following functions are used:
LCM(a, b) is the least common multiple of the quantities a and b.
<f(x)> is the average of the function f with respect to x.
AC(f(x)) and PSD(f(x)) are the autocorrelation function and power spectral density of the function
f(x).
²/DOF is a commonly used quantity to assess the quality of a fit. 2 ( fit ( xi ) f ( xi )) 2 / i2 is the
summed deviation of the fit function fit(xi) to the measured data points f(xi) relative to the individual
standard deviation i of each data point. DOF = Nd – Nf is the number of degrees of freedoms, i.e.,
the number of data points Nd subtracted by the number of fit parameters Nf. Thus, 2/DOF = 1 is an
indication that the fit function describes the physical process expressed by the measured data points.
a.u. = arbitrary unit
x = FT(x) denotes the Fourier transformation of x.
Re(x) and Im(x) denote the real and imaginary part of the complex quantity x = Re(x) + i·Im(x).
≡ identical
≈ approximately
~ proportional
6
Publications
The following publications contain results and concepts from this thesis:
1. Koch, M. and A. Rohrbach (2012) 'Objectadapted optical trapping and shapetracking of energy
switching helical bacteria'. Nature Photonics, 6(10): p. 680686. DOI: 10.1038/nphoton.2012.232
2. Koch, M. and A. Rohrbach (2014) 'How to calibrate an objectadapted optical trap for force sensing
and interferometric shape tracking of asymmetric structures'. Optics Express, 22(21): p. 25242
25257. DOI: 10.1364/OE.22.025242.
3. Koch, M., J. Roth, and A. Rohrbach (2014) 'Bakterien gefangen im Licht'. BIOspektrum, 20(4): p. 386
389. (Invited article)
4. Roth, J., M. Koch, and A. Rohrbach ‘A unique propulsiongererating bacterial proteinchain motor –
theoretical model and optical tweezers based characterization’.
5. Koch, M., J. Roth and A. Rohrbach ‘Instant and reversible switching of bacterial dynamics by light’
6. Koch, M. and A. Rohrbach ‘Microrheology off single microtubule filaments reveals power law
stiffening at high freqeuncies’
7
Table of contents
Zusammenfassung .................................................................................................................................. 4
Abstract ................................................................................................................................................... 5
Special functions and abbreviations ....................................................................................................... 6
Publications ............................................................................................................................................. 7
1. Introduction ..................................................................................................................................... 11
2. Optical tweezers background .......................................................................................................... 19
2.1. Brownian dynamics and the Langevin equation ...................................................................... 19
2.2. Optical forces ........................................................................................................................... 20
2.2.1. Gradient force .................................................................................................................. 22
2.2.2. Scattering force ................................................................................................................ 23
2.2.3. Multiplexed optical trapping ............................................................................................ 23
2.3. Optical tracking by back focal plane interferometry ............................................................... 25
2.4. Detector and trap calibration techniques ................................................................................ 29
2.4.1. Equipartition theorem ...................................................................................................... 29
2.4.2. Langevin calibration ......................................................................................................... 30
2.4.3. Piezo scan ......................................................................................................................... 31
2.4.4. Laser scan ......................................................................................................................... 32
2.5. Optical setup ............................................................................................................................ 33
3. Spiroplasma melliferum ................................................................................................................... 35
3.1. Optical forces and potentials of a linetrapped helix............................................................... 37
3.1.1. Analytical description ....................................................................................................... 37
3.1.2. Simulation......................................................................................................................... 39
3.1.3. Measurement ................................................................................................................... 44
3.2. 3D imaging and force sensing of helical cells by line optical tweezers .................................... 45
3.2.1. General image generation and data processing .............................................................. 45
3.2.2. System calibration ............................................................................................................ 47
3.2.3. Correction factors for measured cell diameters .............................................................. 52
3.2.4. Signal of a kinked cell ....................................................................................................... 56
3.2.5. Summary and calibration receipt ..................................................................................... 57
3.3. Motor characteristics – experiments ....................................................................................... 58
3.3.1. The freeswimming cell .................................................................................................... 58
3.3.2. The trapped cell  induced cell death ............................................................................... 58
3.3.3. The trapped cell  hindered death .................................................................................... 62
3.3.4. Modulation of laser power ............................................................................................... 64
3.3.5. Chemical disturbance ....................................................................................................... 65
3.3.6. Additional experimental observations ............................................................................. 67
3.4. The linear motor complex – theoretical model ....................................................................... 70
3.5. Summary and discussion .......................................................................................................... 78
4. Microtubules .................................................................................................................................... 85
4.1. Background and preparatory experiments .............................................................................. 87
4.1.1. Buckling beam .................................................................................................................. 87
4.1.2. Microrheology ................................................................................................................. 88
4.1.3. Optical tweezers grid ........................................................................................................ 93
4.1.4. Signal processing .............................................................................................................. 95
4.1.5. Calibration of an array of beads ....................................................................................... 97
4.1.6. Oscillatory excitation of an actor bead and hydrodynamic interaction ........................... 99
8
4.2. Single filament experiments .................................................................................................. 102
4.2.1. Buckling (pushing forces) ............................................................................................... 102
4.2.2. Stretching (pulling forces) .............................................................................................. 104
4.2.3. Oscillatory driving force – active microrheology .......................................................... 107
4.3. Microtubule networks............................................................................................................ 113
4.3.1. Linear configuration ....................................................................................................... 113
4.3.2. Triangular configuration ................................................................................................. 115
4.4. Modeling microrheology results ........................................................................................... 119
4.5. Summary and discussion ........................................................................................................ 121
5. Summary and final conclusions ..................................................................................................... 133
6. Appendix ........................................................................................................................................ 135
6.1. Analytical derivation of QPD signals ...................................................................................... 135
6.1.1. Lateral ............................................................................................................................. 135
6.1.2. Axial ................................................................................................................................ 138
6.2. Simulation of QPD signals for nonspherical scatterers ......................................................... 140
6.3. Optical setups......................................................................................................................... 145
6.4. Acoustooptic deflectors ........................................................................................................ 148
6.5. ROS scavenging systems ........................................................................................................ 151
6.6. Sample preparation................................................................................................................ 154
6.6.1. Microtubules .................................................................................................................. 154
6.6.2. Spiroplasma .................................................................................................................... 156
6.7. Supplementary figures to forces and potentials of a helix .................................................... 156
6.8. Dark field imaging in reflection mode.................................................................................... 158
6.9. Supplementary information for Spiroplasma motor model .................................................. 160
6.10. Microtubule buckling amplitude and viscous drag ................................................................ 162
6.11. Axial component of buckling force ........................................................................................ 165
6.12. 2pAMR of beads in water....................................................................................................... 166
6.13. Single filament oscillation ...................................................................................................... 167
6.13.1. QPD signals ..................................................................................................................... 168
6.13.2. Analytical description ..................................................................................................... 169
6.13.3. Simulated signals ............................................................................................................ 172
6.13.4. Rheology results for single filaments ............................................................................. 173
6.13.5. Estimation of total viscous drag force during buckling .................................................. 175
6.13.6. Persistence length according to Pampaloni ................................................................... 176
Bibliography ........................................................................................................................................ 178
Conference contributions ................................................................................................................... 190
Scientific acknowledgements ............................................................................................................. 192
Dedicated to… ..................................................................................................................................... 193
9
1. Introduction
1. Introduction
The apparent diversity of life is maybe nature’s most impressive feature. There are likely up to one
1
hundred million different species on our planet – and this is just an estimation {Alberts 2004}. Today, one and
2
a half million species are catalogued, excluding large groups such as bacteria, fungi and beetles {Wilson 2000}.
In the endeavor to get a deeper understanding of life in general, the basic functional building blocks and
1
constituents of all species are searched. Comparable to the atom in physics or chemistry, this is called a ‘cell’,
since a cell cannot be further divided without loss of function. To stick with this picture: the basic building
blocks of cells are proteins – the same way elementary particles, i.e., quarks, leptons and gauge particles, are
the constituents of atoms or matter in general. While the variety of cells is again beyond words, they all share
some commonalities. Among these are the cellular organization and primary functions such as replication,
locomotion and metabolism. These tasks, among a multitude of others, are highly complex and rely on a finite
number of concerted components and mechanisms that were brought to perfection during evolution. Their
understanding is a major goal in order to identify and treat diseases or to realize new synthetic, bioinspired
materials that are able to cope with the increasingly complex demands of future technologies.
The cytoskeleton
Despite the fact that all cells have evolved from the same ancestral cell, they are divided in two major
categories: eukaryotes, comprising human, animal, plant and fungus cells and prokaryotes, further divided into
bacteria and archaea. In eukaryotic cells, most of the primary functions are carried out by diverse organelles
2
and a complex, versatile filamentous protein meshwork called cytoskeleton . The organelles can be regarded
as the counterparts to human organs inside the cell, whereas the cytoskeleton would be bones and muscles
1
{Alberts 2004}. The latter comprises three different classes of protein filaments: microtubules, which are
closest to be the bones of the cell since they are the stiffest filaments. Their role is mainly to keep the
organelles in position, to guide organelle growth and act as cellular highways for molecular motors carrying
cargo. Microtubules are dynamic, meaning that they exist in a fragile balance between polymerization and de
8
polymerization {Desai 1997}. They continuously undergo growth and shrinkage and are predominantly aligned
1
Atom originates from the Greek word ‘atomos’ and means impartible.
2
The term ‘cytoskeleton’ was mentioned first in the 1930s {Needham4 1936; Wintrebert5 1931}, however, the scientific necessity of a
cytoskeletallike structure, that is able to position organelles dawned already hundred years earlier {Dujardin6 1835}. A good essay article
about the prehistory of the cytoskeletal concept can be found in {Zampieri7 2014}.
11
1. Introduction
radially inside the cell, growing out of the centrosome (located near the cell center and close to the nucleus) to
the edge of the cell (see Fig. 1 and Fig. 3B). Actin, on the other hand, can be regarded as the muscles. In most
cells, there is a thin sheet of actin underneath the cell membrane called actin cortex (see Fig. 1 and Fig. 3A).
Individual filaments are flexible but are mainly crosslinked by a variety of proteins such as molecular motors
that can generate forces to move the cell and tremendously increase the stiffness of the crosslinked network
compared to a single filament. Similar to microtubules, actin is highly dynamic, a feature that is used by cells to
rapidly adapt to changes in the environment. The third class consists of intermediate filaments which mainly
reinforce the cell against mechanical stress. Like ropes, they have a great tensile strength but are flexible
under compressive forces. Unlike actin and microtubules, they are not highly dynamic but are more robust
against toxins and unfavorable conditions. Beside these main functions, all three components also fulfill
different other tasks. Microtubules, for example, are used in the flagellum of sperm for propulsion or to
9
position chromosomes during cell division in animal cells {Olmsted 1973} while actin forms the socalled
10
contractile ring {Scholey 2003} (see Fig. 2). In the cytoskeleton, all three components are crosslinked and
dynamically interact with each other, the organelles and the cell membrane, rendering it a complex system
3
able to fulfill numerous tasks inside the cell {Huber 2013}.
While the motivation to research eukaryotic, e.g., mammalian, cells is obvious, bacteria are of interest due
to their short reproduction cycles, their small genome and their relative simplicity in contrast to eukaryotic
cells. Moreover, bacteria are related to a lot of severe and often deadly diseases such as tuberculosis
(‘Mycobacterium tuberculosis’), the plague (‘Yersinia pestis’) or cholera (‘Vibrio cholera’). Similar to eukaryotes,
they possess a cytoskeleton and different organellelike structures, as well. Although there is some striking
11 12
evidence that the eukaryotic cytoskeleton evolved from bacteria or archaea {Erickson 2007; Wickstead
2011}, the prokaryotic cytoskeleton is less well studied compared to the eukaryotic counterpart because it has
13
been discovered only relatively recently {Bi 1991}. Homologs to all three major eukaryotic cytoskeletal
components are identified today, and beyond that, it seems that the cytoskeletal elements are even more
14
diverse in bacteria than in eukaryotes {Shih 2006}. Similar to its analog, the bacterial cytoskeleton is also
15 16
responsible for a broad range of cellular functions such as cell shape {Gitai 2005; Margolin 2009},
17 18 19 20
locomotion {Wolgemuth 2006; Kurner 2005} or cell division {Errington 2003; Margolin 2005}.
1
Structural research on cells, no matter of which domain, is mainly based on optical imaging techniques .
Besides conventional bright field and fluorescence microscopy, there are also more advanced methods like
2 21
confocal ({Shotton 1989} and references therein), fluorescence excitation by an evanescent field of total
1
Besides this, electron microscopy techniques are also used frequently for structural analysis. However, measurements typically take
place under vacuum conditions and probes have to be treated in special ways which renders this technique unsuitable for live cell
research.
2
Here, a gain in resolution by a factor 2 is obtained by overlapping the pinhole filtered point spread functions of the detection and
illumination beam. The main advantage of this technique is a good optical sectioning due to the pinholes that block out of focus light.
12
1. Introduction
22 1 23 2
internal reflected light (TIRF) {Axelrod 1981}, two photon {Denk 1990}, dark field microscopy, selective
3 24 4 25
plane illumination (SPIM) {Huisken 2004} or differential interference contrast (DIC) microscopy ({Shribak
2006} and references therein). Since most of the cellular constituents, such as the components of the
cytoskeleton, are too small to be resolved with conventional light microscopy approaches, the invention of
5 26 27 6
techniques that outsmart Abbe’s resolution limit {Abbe 1873; Pawley 2006} are of great importance .
Among these techniques, the most prominent might be the stimulated emission depletion (STED) of
29 30 27
fluorophores {Hell 1994; Klar 2000}, the structured illumination (SIM) of a probe {Pawley 2006} and
31
photoactivatable localization microscopy (PALM) {Betzig 2006} which is similar to stochastic optical
32
reconstruction microscopy (STORM) {Rust 2006} (see Fig. 3A).
A B
However, at least since Newton formulated one of the most basic laws of physics ‘actio = reactio’, it is clear
that structure alone is at most one side of the coin. The manifold nature of forces exerted by or on cells,
35
ranging between femto and nano Newtons, led to the invention of different probing techniques {Suresh
2007}. In traction force microscopy, for example, overall deformations of thin, twodimensional substrates
36 37
{Harris 1980} or threedimensional elastic hydrogels {Legant 2010} caused by cells are measured. The
deformation of a cell membrane as a response to a defined pressure applied by micropipette aspiration
38
{Hochmuth 2000} gives rise to mechanical properties of cell membranes. However, the most widespread
39 40
methods currently used are atomic force microscopy (AFM) {Müller 2008; Binnig 1986}, which is an
41
excellent tool for surface imaging at atomic resolution and measuring the elastic properties of cells ({Rotsch
2000} and references therein) by measuring the deflection of a microneedle attached to a cantilever (see Fig.
4A). Secondly, there are magnetic tweezers (MT) which are used to actuate magnetic probes (small beads) and
42
measure their displacement by optical microscopy {Smith 1992} (see Fig. 4B). The third and maybe most
versatile technique exploits optical forces acting on particles (beads) inside a focused laser beam (see Fig. 4C)
1
Similar to confocal microscopy, the PSF of the illumination is squared because two low frequency photons have to excite the fluorophore
at the same time. There is again an improved sectioning compared to conventional fluorescence microscopy. Furthermore, the long
wavelength photons are less scattered and can penetrate deeper into tissue.
2
This is a nonfluorescence technique where samples are illuminated from the side and only light scattered at the probe is detected.
3
Here, fluorescent probes are illuminated by a sheet of light which is either generated statically by SLMs or cylindrical lenses or
dynamically by beam scanning. Detection is done under an angle of 90° which results in excellent sectioning and reduced photobleaching.
4
Here, linearly polarized light is split into two beams with perpendicular polarization by a birefringent material (Wollaston prism) on the
illumination side. On the detection side, the light is united again by a Wollaston prism and filtered by a linear polarizer. If the phase and
thus the polarization is not changed, the light is blocked. Only light that has passed an object reaches the camera which results in sufficient
contrast even for nearly transparent structures.
5
A common measure of resolution is the minimal distance between two pointlike scatterers before their images, i.e., their point spread
functions, overlap. Per definition, this is the FWHM or the position of the first root of the airy spot, i.e., x = y = 0.61 / NA, in lateral
direction. The spot size in axial direction is z ≈ 2 / n(1cos() with NA = n sin().
6
This is also emphasized by the Nobel committee which awarded the 2014 Nobel Prize to the inventors of the two most important super
resolution techniques, namely STED and PALM {Nobelprize.org28 2014}.
13
1. Introduction
43
and is called optical trap or optical tweezers (OT) {Ashkin 1986}. While magnetic tweezers represent the
most forcesensitive technique that allows applying moderate forces, they are not as versatile as AFMs or OTs
since the ability to combine other research methods is lacking or cumbersome. AFMs on the other hand allow
applying very large forces but their cantilevers are rather stiff and can easily damage soft biological surfaces by
scratching. A nice overview, comparison and typical applications of these and more approaches is given by
35 44
{Suresh 2007; Neuman 2008}.
The stiffness and maximal trapping force of optical tweezers, otherwise, can be tuned very flexibly and
instantaneously by changing the incident laser power. Forces can be applied and measured together with
displacements in three dimensions at the same time. Beyond this, it can easily be combined with other
45
techniques such as complementary imaging methods {Heller 2013}. The potential applicability of optical
46
tweezers to trap and analyze biological specimen such as single bacteria and viruses {Ashkin 1987} and whole
47
cells {Ashkin 1987} has been shown immediately after its invention in 1986. Since this, optical traps have
been used in a wide range of applications  from the assistance during imaging bacteria in different
48
orientations {Carmon 2014}, the chemical stimulation and manipulation of single cells by optically
49 50
manipulated microsources {Kress 2009}, the study of the flagella beating of bacteria {Min 2009},
51
microtubule pushing forces during polymerization {Kerssemakers 2003}, molecular motor stall forces and
52 53
velocity {Visscher 1999}, elasticity measurements of single bacteria {Wang 2010} to basepair stepping
54
during DNA transcription {Abbondanzieri 2005}.
A B C
Fig. 4 The three major techniques used in biophysical studies. Adapted from
44
{Neuman 2008}. (A) Atomic force microscopy (AFM). Deflections of a laser
beam reflected on a cantilever bent by a biological specimen are measured
with photosensitive devices (PSD). (B) Magnetic tweezers (MT) use magnetic
field gradients to trap magnetic particles by either permanent magnets (top)
or thin magnetic, laser manufactured foils (bottom). (C) Optical tweezers
(OT) formed by a focused laser beam pulling on DNA with one (top) or two
(bottom) beads as handles.
In the present study, two specialized optical tweezers configurations are used to investigate a prokaryotic
and a eukaryotic cytoskeletal model system, pursuing different scientific objectives as outlined in the following
two paragraphs.
The first system is the unique and so far little investigated contractile, propulsion generating molecular
motor of the bacterial genus Spiroplasma. These helicallyshaped cells are often plant and arthropod
55
pathogens which lack a stiff cell wall in contrast to most other bacteria {Lo 2013}. They are able to deform
14
1. Introduction
themselves intensively, a property that is used for propulsion by generating a pair of kinks propagating down
56
the length of the cell body (see Fig. 5A)  thus representing a linear motor {Trachtenberg 1998} in harsh
57
contrast to other driving motors which are mainly of rotary nature (see Fig. 5C) {Chevance 2008}. Kinks are
generated by a cytoskeletal ribbon attached to the cell membrane and spanning the entire cell (see Fig. 5B).
The ribbon’s subunits, composed of the unique protein Fibril, are able to change their elliptical diameter by
58
conformational changes, which is used to shorten or lengthen the cytoskeletal band {Trachtenberg 2006}. In
contrast to common rotary motors, which consist of approximately a dozen of different parts (see Fig. 5C), this
linear motor is much simpler which might have important implications for the construction of an autonomous,
artificial microswimmer. However, the functional principle and the mechanics of the Fibril ribbon have not yet
been understood. The relative structural simplicity of the motor also reflects the rather primitive cell itself,
which has one of the smallest genomes known. Thus, research on these cells might give insights in the basic
biophysical principles of complex life in general.
In general, Spiroplasma’s movement is not restricted to a one or twodimensional environment since they
are freeswimming cells. Depending on the properties of the surrounding medium, they are able to move at
59
speeds up to a couple of microns per second {Shaevitz 2005}, thus, they are able to rapidly swim out of focus
when investigated under a microscope. Secondly, their small body dimensions (~200nm in diameter and 3
60
5µm in length) {Trachtenberg 2014} and rapid body deformations necessitates a fast and precise microscopy
technique which, in order to characterize the motor’s mechanical properties, should also be able to extract
forces and energies exerted by the cell. In this context, optical tweezers are used for the first time to trap,
track and manipulate a biological specimen with a geometry more complex than spherical or rod shaped
48
{Carmon 2014}. Due to the overall longish and thin form of the cell, line optical tweezers are used. This
61
approach has already been applied to research particle interaction {Tränkle 2012} and fulfills all of the
abovementioned requirements. Here, the trapping and tracking system is fully described and characterized
analytically, by simulations and experimentally. This is then used as a basis for probing the bacterial motor
under different conditions shedding light on the physical details of the motor which is also modeled
theoretically and compared to other common bacterial locomotive systems.
A B C
Fig. 5. Bacterial driving motors. (A) DIC images of a kink sequence (arrows
point to kinks) of a Spiroplasma cell squeezed between two coverslips.
59
Adapted from {Shaevitz 2005}. (B) Reconstruction of cytoskeletal ribbon
18
(green, red, yellow) inside the cell body. Adapted from {Kurner 2005}. (C)
Fluorescence image of a single E.Coli cell with four flagella /bottom left).
Electron micrograph (top left) and principle structure of rotary flagellar
62 63
motor (right) of E. Coli. Adapted from {Berg 2000} and {Turner 2000}.
The second system investigated in this thesis addresses the question how single cells can sense and
respond to their mechanical environment, or, in particular, how mechanical forces are conducted inside the
15
1. Introduction
64
cell {Vogel 2006}. The mechanisms behind are commonly summarized by the term mechanotransduction, a
65 th
process every cell is capable of {Jaalouk 2009}. Julius Wolff, by formulating Wolff’s law in the end of the 19
66
century ({Frost 1990} and references therein), was the first who formulated a theory based on
mechanotransduction. He stated that bone cells will adapt to physical loads, i.e., remodel themselves in order
to increase the strength a bone can withstand. Indeed, even basic human senses like hearing, balance or touch
67 65
{Orr 2006} rely on mechanotransduction and a lot of diseases, ranging from deafness to cancer {Jaalouk
2009}, are related to a malfunctioning of the mechanotransduction process. The overall cellular properties, like
the ability to withstand external loads, are regulated and altered by gene expression in the cell nucleus, thus
external stimuli have to be transported to this organelle. A common model is the conversion of a physical
stimulus to a chemical signal by mechanosensitive complexes situated in or near the cell membrane, i.e., at the
68 65 69
cell’s outside margin {Geiger 2009}. These can trigger a signal cascade {Jaalouk 2009; Chien 2007} of
which the product is transported to the nucleus either by passive diffusion or active transport by means of
molecular motors (see Fig. 6). Both processes need seconds up to a minute before the cell is able to respond.
However, different experiments have shown nearly immediate (T < 1s) reactions at distant sites to a defined
70 71
physical stimulus ({Wang 2009; Isermann 2013} and references therein). This gives rise to different and
72 73 74
much faster pathways. {Ingber 1993 and 2003 and 2003} formulated a tensegrity model based on a stiff
cytoskeleton, which would be able to transport a physical stimulus by stress waves within microseconds (see
75 76
Fig. 6). Since the cytoskeleton is mechanically connected {Maniotis 1997} to the cell membrane {Geiger
71 77
2001} as well as to the nucleus ({Isermann 2013; Shivashankar 2011} and references therein), this model
has been attracted growing interest in recent years. Ingber suggested prestressed actin filament bundles as
70
mediator {Wang 2009}. However, microtubules, as stiffest part of the cytoskeleton, are also able to fulfill this
77 78
task {Shivashankar 2011; Na 2008}.
In this context, the second part of this thesis introduces a new bottom up approach to study the frequency
dependent mechanical properties of single microtubule filaments and small networks thereof (see Fig. 7).
Again, optical tweezers are used to create an array of optically trapped beads acting as anchor points for
biochemically attached microtubule filaments. This approach allows to construct networks with a defined
topology and to successively increase the complexity of the system. A frequency modulated force can be
applied to the network via a single anchor point by a spatial oscillation of its trap, thus serving as force actor.
The frequencydependent displacement of the remaining beads, acting as sensors, is measured
interferometrically at high spatial and temporal precision. Established microrheology methods are then used
to analyze the frequencydependent behavior of the microtubule network acting as a force transducer. This
approach allows testing a possible transport pathway of a physical stimulus by the cytoskeleton itself, which
would be much faster than a chemical transport.
16
1. Introduction
The second chapter starts with a fundamental introduction to optical trapping, tracking and calibration
techniques. A general optical tweezers setup is presented.
The third chapter addresses the biomechanics and energetics of the bacterium Spiroplasma melliferum. It
is shown how back focal plane interferometric tracking can be used to image the slope of this helical cell with
nanometer precision at rates up to several kHz. An analytical description is derived and proven by simulations
and measurements. In the same manner, the forces and potentials of the optical trap, acting on the cell, are
described analytically and simulations thereof are compared to experimental results. A theory for the
functioning of the unique molecular motor used by the cell for selfpropulsion is described and different
measurements to confirm the theory are presented.
The fourth chapter concerns the biomechanics of artificial microtubule networks. After a technical
introduction, different experiments on single microtubule filaments are presented, serving as solid basis to
understand their mechanical behavior. Microrheology techniques are introduced to analyze the frequency
dependent response of a single filament to an external force. The conclusions and principle findings made here
are compared to findings of small networks. Theoretical aspects used to explain experimental observations are
supported by simulations and physiologically relevant implications are drawn.
The fifth chapter briefly summarizes the main results of the thesis and draws some more fundamental and
wideranging conclusions.
17
2. Optical tweezers background
Fext describes any external force acting on the particle as, for example, an optical trap Fext = x (see
chapter 2.2.1). The Langevin equation can be solved in Fourierspace, where its power spectrum (PSD)
84
describes a Lorentzian {Wang 1945}.
Fth ( ) 2k BT 1
x( ) PSD ( ) x ( )
2
(2.4)
i 2 c2
The corner frequency c = / describes the characteristic frequency of the drop PSD(c) = ½PSD(0) and
relates the viscous drag of the particle to the stiffness of the optical trap (see chapter 2.4). However, for
1
Except for high frequencies where the spectral amplitude drops (coloured noise). White noise is an idealized case of a purely random
process that does not occur in nature {Coffrey80 2004}.
2
The Reynolds number Re of a body with characteristic size d moving at a velocity v in a fluid with density and viscosity is defined as
the ratio Re = vd / of inertial to viscous forces. For a macroscopic body, e.g., a human swimming in water, Re ≈ 104 and inertial forces
dominate. The picture is completely different at microscopic scales, where a micron sized bacterium (E. Coli for example) has Re ≈ 105. If
such a cell moves at v = 30µm/s and the propulsion generating mechanism (flagella) suddenly stops, the cell coasts within 0.3µs a distance
of 0.1Å – only a percentage of an atom’s diameter {Purcell86 1977}.
19
2. Optical tweezers background
times shorter than 1 / c, the trapped particle diffuses freely but is confined by the optical trap for larger
times.
The particle displacement x = xn+1  xn inside an optical trap due to the continuous bombardment by the
molecules of the immersion medium during the time step t can be described numerically {Grassia 1995;
85
87
Kress 2006} by
24 k BT
xn 1 xn 1 Fext rn t (2.5)
t
Here, the maximum amplitude Ath of the thermal force is given by
2 k BT 24 k BT
Ath (2.6)
t r n
2
t
where rn2 112 the is the variance of the pseudorandom number rn reflecting the stochastic nature of the
force.
between the incident ki and the scattered ks photon. The sum of momentum change of all photons (i.e., from
every angle of incidence) leads to a net momentum (and force) pointing towards the focus center. This is the
socalled gradient force Fgrad since it points along the gradient of the incidence intensity, i.e., towards the
brightest point of the focus. Despite the beauty of this picture, quantitative predictions are difficult and it
2
neglects backscattering and absorption of light by the particle. Backscattered light causes radiation pressure
resulting in a force pointing in propagation direction of light, thus counteracting the gradient force. Due to its
origin, this force is commonly called scattering force Fscat.
1
Snell’s law is the conservation of momentum kx,1 = kx,2 in lateral direction (x) at an interface of two different materials (1 and 2) with
refractive indices n1 and n2. In particular: k0 n1 sin(1) = k0 n2 sin(2).
2
This effect can also be observed when a comet passes our solar system. Some of them have a tail, often with a length up to 100 million
kilometers, which does not point in direction of the actual movement of the comet. This is caused by radiation pressure from sunlight
acting on the particles of the gaseous tail.
20
2. Optical tweezers background
ks,2 ks,1
k1
ks,1
ki,1
b k2
k1 = ki,1  ks,1 rL k
k2 = ki,2  ks,2 ks,2
k = k1 + k2 ki,2
ki,1 k k1
.
k2
ki,2
The ray optics picture is only valid if the particle’s diameter R » is much larger than the wavelength of
trapping light . A general description of optical forces can be derived using Mie theory. It is a complete
analytical solution of Maxwell’s equations describing the scattering of light at spherical particles of any size
97
{Hecht 2002}. Due to its complexity, detailed calculations can become complicated. Since particles used in
optical tweezers experiments are typically smaller (diameter 10nm ≤ d ≤ 1000nm) than the wavelength of
trapping lasers (800nm ≤ ≤ 1100nm), forces can be also estimated in an easier and more illustrative way.
92 98
The following description bases on {Rohrbach 2001; Friedrich 2011}. A dielectric particle with refractive
index np immersed in a medium with refractive index nm is polarized by an external electromagnetic field and
molecular dipoles are excited. Every dipole then interacts with the electric E and magnetic B field of the light
resulting in Coulomb and Lorentz forces
p
Fd (p )E B (2.7)
t
The dipole moment p of a small volume element V in the local electric field E = Ei + Es (the superposition
of the incident Ei and scattered Es field) is given by
p 0 nm2 V E (2.8)
with the vacuum permittivity 0 = 8.85pF/m and the polarizability according to the ClausiusMosotti
relation with m= np / nm
m2 1
3 (2.9)
m2 2
Together with the vector identity (E )E 12 E2 E ( E) and the Maxwell equation E t B ,
the force acting on a dipole can be written as
21
2. Optical tweezers background
A laser wavelength = 1064nm (typically used for optical tweezers) results in a frequency f = c / ≈
280THz, thus only the temporal average of Eq. (2.10) is significant and the second term vanishes. For
monochromatic light, E(r, t) = E0(r)eit can be used and Eq. (2.10) further simplifies to
0 nm2 V Re( )
Fd E02 r (2.11)
4
This dipole force can now be identified as a force density f = Fd / V and integrated over the volume V of the
scatterer, resulting in the total optical force Fopt on the whole particle. Remembering that the local field E is
the superposition of the incident and the scattered field, the total force further splits up into two parts that
88
are identified with the gradient and the scattering force Fgrad and Fscat, respectively {Rohrbach 2005}.
0 nm2 Re( ) 0 nm2 Re( )
Fopt fdV Ei dV E
Ei E*s E*i E s dV
2 2
s (2.12)
V
4 V
4 V
Fgrad Fscat
The first term in Eq. (2.12) can also be expressed by the incident intensity I 12 c 0 nm Ei . For small
2
particles (d < ), the gradient of the focal intensity varies only little within the particle’s volume at position b
relative to the focal center and the integral can be solved
nm Re( )V
Fgrad I (b) (2.13)
2c
The intensity profile at position r = (rx, ry, rz) in every direction (j = x, y, z) can be described by a Gaussian
in very good approximation up to a large spatial extent. The Gaussian widths j directly reflect the width of the
PSF x = y ≈ x = y = 0.61/ NA and z ≈ z = 2/ nm(1  cos()) of the focus where NA = nm sin() reflects
the focusing angle of the lens
r j2
I0 2 2j
I j ( rj ) e (2.14)
2 j
Thus, the intensity gradient can be calculated and a first order Taylor expansion results in a linear force
dependence for small particle displacements.
13 0 0
nm Re( )VI 0 0
x
22
2. Optical tweezers background
The gradient force showed to be independent of the scattering at the particle which does not hold for the
scattering force as can be seen from Eq. (2.12). The scattered field Es can be determined by Rayleigh or
1
RayleighGans theory depending on the size of the scatterer. A detailed description based on an extension of
88
the latter theory is given in {Rohrbach 2005}. Fscat can be expressed by the scattering and extinction cross
sections Cscat and Cext, respectively.
A B
x, y
z
Fig. 9. Simulated trapping efficiency Q ~ F(b). (A) Lateral direction and (B)
99
axial direction. Adapted from {Rohrbach WS 2008/2009}.
Besides the simple possibility of using multiple lasers simultaneously to generate multiple optical traps,
they can be generated out of a single laser source by multiplexing, as well. This can be achieved by beam
100 101
steering devices such as spatial light modulators (SLM) {Curtis 2002}, acoustooptic deflectors (AOD) {Ruh
102 103 104
2011; Visscher 1998}, electrooptic deflectors (EOD) {Valentine 2008}, scan mirrors {Fällman 1997} or
105 106
combinations thereof {Akselrod 2006; Čižmár 2011}. These devices can be also used to create
61 107 108 109
geometrically complex shaped traps such as line {Tränkle 2012; Brunner 2004; Crocker 1999; Koch
110 111 112
2012} or circular traps {Faucheux 1995} with different optical potential profiles {Faucheux 1995; Koch
113 2
2014; Nambiar 2004}. For trapping purposes, multiplexing can be performed in space or time domain .
Spacemultiplexing (SLM)
In the first case, the wave front is shaped to form multiple foci at the same time. This is usually done by a
SLM. The main advantage of this method is that dozens of traps can be generated and manipulated
simultaneously. Besides, individual traps can also be displaced axially and the beam profile can be shaped
114 115
arbitrarily {Grier 2003}, for example to transfer angular momentum to a trapped particle {Padgett 2011}.
1
Depending on the relation of the diameter d of a particle relative to the wavelength of light, there are different approximations of the
general Mie theory: Rayleigh scattering for d « and RayleighGans scattering for d ≤ .
2
In telecommunication applications, for example, frequency and polarization multiplexing is used, as well.
23
2. Optical tweezers background
The main disadvantage is the maximum frame rate a SLM can be refreshed. This limits any time dependent
change of an optical trap and therewith the bandwidth of the trapped particle to actuation frequencies below
1
100Hz . Besides this, spatial multiplexing relies on video based microscopy for detection of particle or probe
displacements. Although this technique has been substantially improved in the last years, especially by the
117 2
availability of faster cameras {Hirvonen 2014}, video microcopy can still not compete with more advanced
tracking techniques such as back focal plane interferometry (see next chapter). The main drawbacks are the
maximum frame rate and position accuracy. Fast cameras and short acquisition times need a lot of photons to
get enough contrast in every picture. This might cause damage to a biological probe within very short time.
Another drawback of video based particle tracking is the axial tracking which is only possible with advanced
118 119 120
tracking algorithms or strategies {Jesacher 2014; Cheong 2010; Speidel 2003} and is still not as precise
as back focal plane interferometry.
Nevertheless, SLMbased holographic optical tweezers are a powerful tool particularly when it comes to
the trapping of a very large set (N > 20) of objects and especially if a precise threedimensional position
tracking is not necessary.
A B
Fig. 10. Holographic optical tweezers generated by an SLM. (A) The phase
ei() of an incoming laser beam (green) is modulated to form outgoing
beams under different angles creating an array of 20 x 20 optical traps in the
sample volume. (B) ac: Twentysix particles trapped in different trap
arrangements. d, e: Two identical particles are moved past each other in two
different axial planes. fh: Seven identical particles are trapped in seven
114
different axial positions and moved up and down. Adapted from {Grier
2003}.
Timemultiplexing (AOD)
Timemultiplexing, in contrast, relies on fast beam steering devices and the consecutive scanning of
3
different focal positions. If a single beam is displaced fast enough the particle can neither follow the motion of
the focus nor escape the virtual trap during the absence of the focus. This yields a mean or timeshared, so
101 109 121
called effective optical force {Ruh 2011; Koch 2012; Speidel 2009}
1
Devices based on ferro electric liquid crystals are much faster (~1.5kHz) but suffer other shortcomings such as binaryonly phase shifts,
i.e., the phase of every pixel can be only shifted by 0 or {Hossack116 2003} limiting the possible application.
2
Only recently, {Hirvonen117 2014} used a commercially available 1MHz CMOS camera in combination with a singlephoton counting
device in a fluorescence lifetime approach on living cells. They succeeded in reducing the photon intensity to a nontoxic level and were
able to image arrays of 16x64 pixels at 1MHz and 320x256 pixels at 20kHz, still. Although the size of the area would be sufficient for
tracking a particle inside an optical trap, this application and the resulting precision has still to be demonstrated.
3
As a rule of thumb: faster than the corner frequency of a trapped particle.
24
2. Optical tweezers background
T
1
T 0
Feff (x) Fopt (x) dt (2.18)
where Fopt is the optical point force introduced by equation (2.12). In other words, the laser intensity
forming individual traps is shared in time average among different positions of the laser focus. The trajectory
of the focus can be arbitrary. Within this thesis, two different types of timemultiplexed traps are used: line
traps, formed by scanning the focus along a line, and multi traps, formed by a deltalike jumping between
focus positions each kept for a few micro seconds. Details are explained later when they fit in context
(chapters 3.1, 3.2 and 4.1).
The main advantage of timemultiplexed trapping is that the traps are scanned successively allowing
tracking with back focal plane interferometry (see next chapter). The main disadvantage is the frame rate of
data acquisition. AODs, for example, are limited to 100kHz jump frequency (as shown later). The net tracking
101 122
sample rate 1 / fscan per particle i decreases with the number of traps N {Ruh 2011; Guilford 2004}
1 f
f scan AOD (2.19)
on off N
where on is the time the focus is on the particle and off the time spent at the other N  1 particles. The
right hand side of Eq. (2.19) holds if on=const. for every trap. Nevertheless, even for ten traps, this results in
sample rates of 10kHz per trap which is still far above any realistic video microscopy application concerning
biological probes.
25
2. Optical tweezers background
The situation is depicted in Fig. 12. A particle is trapped at position b relative to the center rL = 0 of a
focused laser beam described by the electric field Ei(r). The particle is excited by the incident electric field at
its current position and scatters light coherently, which means that there is a distinct relation between the
phase i and s of the incident and scattered field. Therefore, the scattered field Ei(r, b), or more precisely its
phase s(b), depends directly on the scatterer’s position b. The interference of both fields is evaluated in
Fourier space, i.e., in the back focal plan of a detection lens, where Ei and Es denote the Fourier transformed
fields and I 0 Ei (k x , k y ) , I s b Es (k x , k y , b) the corresponding intensities (for the ease of reading, the
2 2
2 2 2
Ei E s (b) Ei (k x , k y ) E s (k x , k y , b) 2 Re Ei ( k x , k y ) E*s ( k x , k y , b)
(2.20)
I 0 (k x , k y ) I s (k x , k y , b) 2 I 0 ( k x , k y ) I s ( k x , k y , b) cos s (b) i
The incident intensity Io is constant with respect to the scatterer position and can be subtracted
electronically as a constant offset. The intensity of the scattered field is small compared to Is « Io and varies
only slightly with actual displacements caused by thermal motion inside an optical trap and is subtracted, as
well. The only relevant term left is the interference term whose phase dependence (b)=s(b)  i is key in
BFPtracking .
QPD ky
A1 A2
BFP kx
A3 A4
kz=const.
i(kx, ky, kz) s(b, kx, ky, kz)
DL
z
y
b
FP x
s(b, x, y, z)
i(x, y, z)
OL
Fig. 12. Schematic of back focal plane interferometric tracking. A particle
trapped at position b in the FP scatters a portion of the incident, focused
trapping light (red) with phase i (black dashed lines). Due to coherent
scattering, the phase s (green dashed lines) of the scattered light (green)
has a distinct relation to i. This phase relation manifests in the
interferometric intensity distribution according to Eq. (2.20) in the BFP and is
evaluated by the quadrants Aj (j = 1, …, 4) of a QPD in Fourier space (kx, ky).
For the sake of clarity: in the following, denote absolute phases and intrinsic or specific phases. It are
mainly the phases that are of interest but the total phases are needed for calculation.
Lateral phases
In lateral direction a displacement bx or by in the focal plane leads to a tilt in the back focal plane according
to the Fourier shifting theorem E( x bx ) E(k x )eikx bx . Therefore, the total phase of the scattered field can be
written as
26
2. Optical tweezers background
s , x i s , x s , x k x bx
(2.21)
s , y i s , y s , y k y by
Strictly speaking, the phase i = i(bx, by) ≈ const. of the incident field has to be taken at the position bx, by
131
of the scatterer. However, the incident phase of a focused Gaussian beam varies only slowly {Saleh 1991}
over the focal extent in lateral direction (x, y), therefore it is regarded as constant. The phase difference reads
x (bx ) s , x (bx ) i s , x k x bx
(2.22)
y (by ) s , y (by ) i s , y k y by
Axial phase
In axial direction, the principal dependence is the same but its origin is another. Regarding Fourier theory
again, a displacement bz out of the focal plane of a lens with focal length f leads to a parabolic phase
132
dependence in the back focal plane {Goodman 2005} proportional to bz / f which is negligible in the case
1
regarded here . However, the course of the phase of a focused beam exhibits an additional phase shift
133
depending on the axial position. This is called the Gouy effect ({Feng 2001} and references 13 therein)
describing the phase fw(z) of a focused wave in propagation direction according to the Gaussian beam model
2
131
{Saleh 1991}
z 0.61 n 1.22 2 1
2
1
fw ( z ) k0 z tan 1 z0 (2.23)
NA
2
z0 n NA k z a0 kz
z0 is the socalled depth of focus of a beam focused by a lens with numerical aperture NA. kz = 2nm / is
the wave number in the medium with refractive index nm and a0 = NA² / 1.22² a constant. The scattering of a
3
small (Rayleigh) particle is approximately homogeneous in all directions , thus it can be treated as the
diverging part of a focused wave and therewith by the Gaussian beam model, too. However, the phase of the
scattered field depends on the phase of the incident field at the position of the scatterer. The situation is
depicted in Fig. 13, where an additional phase lag of /2 between the incident and the scattered field
134
{Rohrbach 2002} is indicated.
(z) z(bz,1)
Im(E)
j=s(bz,j)
Es(1) Es( 2 )
Es(3)
z(bz,j) ~ bz,j
z(bz,3) 1
3 Ei
Re(E)
z
bz,1 bz,2 bz,3
/2
Incident focused wave i = fw s(bz,1) = i(bz,1) + /2 + s(bz,1) > z,2
s(bz,2) = i(bz,2) + /2  0
Incident plane wave = k0z
s(bz,3) = i(bz,3) + /2 + s(bz,3) < z,2
Fig. 13. Illustration of the Gouy phase effect. (left) Conventional one
dimensional plot and (right) polar plot. See main text for details. Adapted
109
from {Koch 2012}.
1
The exact Fourier transform G (k x , k y , d ) of an object described by g(x, y) at position d in front of a lens with focal length f is
G(k x , k y , d ) A1 exp iA2 (1 df )(k 2x k y2 ) G(k x , k y ) with constant factors A1, A2. For objects in the focal plane, i.e., f = d, the parabolic phase
factor vanishes leaving the conventional Fourier shifting theorem. Here, d = f  bz, f = 2mm and bz < 1µm results in a vanishingly small
phase term.
2
Actually, the Gaussian beam model is not suitable for highly focused beams since it is only a solution of the paraxial Helmholtz equation,
meaning small angels between the beam axis and the wave front normal. Nevertheless, it explains the experimental situation quite well as
shown appendix 6.1 and 6.2.
3
At least, the error made by using this assumption is similar to that taking the Gaussian beam model for the focusing of the laser beam.
Again, this rather simple model describes the experimental behavior quite well.
27
2. Optical tweezers background
The scatterer at different axial positions bz,j (j = 1, 2, 3) is excited by the incident field with phase i(bz,j)
fw(bz,j) at that position. The scatterer responds with a constant /2 phase lag and the spatial dependence
s(bz,j) fw(z  bz,j), thus s(bz,j) = i(bz,j) + s(bz,j) + /2. The phase difference z in the far field (i.e., on the
QPD) then reads
z (bz ) lim s i
z
lim k z bz tan 1 ak z bz k z z bz tan 1 ak z ( z bz ) k z z tan 1 ak z z (2.24)
z 2
s , z ( bz ) i , z
ak z bz 0.5
tan 1 ak z bz ak z bz
2 2
The last simplification holds if bz < 0.5 / akz ≈ 0 / 2 = 532nm (for NA = NAD = 1.2 in water with nm = 1.33)
which is well fulfilled in optical trapping approaches.
Summarizing these findings, the interference term in Eq. (2.20) reads
2 2 2
2 Re Ei E*s (b) Ei E s (c) Ei E s
` const Ei
2
2 Ei E*s (b) cos k x bx k y by ak z bz (2.25)
2
2 Ei E*s (b) sin k x bx k y by ak z bz
This pattern is recorded by the n = 1, …, 4 quadrants of the QPD (see Fig. 12) resulting in four raw signals.
1
Sˆnraw (b) c 0 P(t ) sPD aPA E E (b)
2
i s dk x dk y (2.26)
2 An
P(t) describes a possible time depended modulation of the incident laser intensity, sPD is the sensitivity of
the photo diode in Ampere per Watt and aPA the amplification of the preamplifier in Volt per Ampere.
Assuming constant incident intensity over the range of the particle’s motions in the FP and a flat distribution of
the incident intensity in the BFP, the integral can be further simplified
Sˆnraw (b) P(t ) sPD aPA Pi 1 2
sin(k x bx k y by ak z bz ) dk x dk y
(2.27)
An
where Pi = ½c0Ei² is the total light power of the incident field on the QPD and is the polarizability, i.e.,
the response of the scatterer to the incident field. These signals are linear combined to an orthogonal signal
triplet (Sx, Sy, Sz) according to Fig. 12. The triplet is proportional to the actual bead displacement b = (bx, by, bz)
related by the detector sensitivity g = (gx, gy, gz) for small displacements from the focal center.
S x Sˆ1raw Sˆ3raw Sˆ2raw Sˆ4raw g x bx
Sy Sˆ 1
raw
Sˆ2raw Sˆ raw
3 Sˆ4raw g b y y (2.28)
The corresponding integrals can be straightforwardly calculated as shown in appendix 6.1. Since the lateral
signals Sx and Sy are difference signals, the signal offset Soff ~ Pi(1 + ), still present in the axial signal Sz,
cancels out.
Reflecting this chapter, the power of back focal plane interferometric tracking becomes apparent. Even for
weak scatterers, the vanishingly small total intensity Es² of scattered light is amplified tremendously by the
28
2. Optical tweezers background
1
incident light in the interference term . Further, even tiny nanometer sized position changes in the focal plane
can be detected since they are translated to smooth sinusoidal intensity gradients in the back focal plane.
As already mentioned in chapter 2.1, this theorem relates the thermal energy to the mean energy of any
degree of freedom of a system that has a harmonic potential. In the case of an optical trap with force F = x
and potential V = ½x², this is equivalent to
1 1
x 2 k BT (2.29)
2 2
in every direction. x² = ² is the mean squared particle fluctuation width, i.e., the width of the Gaussian
probability distribution p of the fluctuations.
1 x2
V x2
2
p ( x) ~ e kBT
e kBT
e 2 2
(2.30)
A 40 B 40
Hist(S(b)) (·10³ counts)
Hist(S(b)) (·10³ counts)
30 30
20 2s 20 2p
10 10
²/DOF = 1.84 ²/DOF = 1.84
s = (10.218 ± 0.003) mV p = (20.437 ± 0.007) nm
0 0
Since this distribution has the same form for calibrated and uncalibrated data, the ratio g = s / p of the
widths p and s of the position and signal histogram, respectively, results in the detector sensitivity g. This
1
Even for a weak scatterer with = 1%, the interference or modulation contrast K defined by K 21 20% becomes very pronounced.
2
See {Richly135 2013} and references 1723 therein for a nonexhaustive list of the most commonly used calibration approaches.
29
2. Optical tweezers background
relates the sensitivity to the stiffness g ~ , thus one quantity can be estimated from the other if fluctuation
data are present.
g2
g s k BT (2.31)
k BT s2
This is illustrated in Fig. 14 for simulated fluctuation data with g = 500kV/m (see caption of Fig. 15 for
simulation parameters). Gaussian fits yield p and s and therewith g = (499.976 ± 0.229)kV/m in good
agreement with the input parameter.
In combination with the equipartition theorem, this is the most elegant method to calibrate an optical trap
since it only relies on quantities from the Langevin equation (see chapter 2.1). It only requires uncalibrated
fluctuation data of a trapped particle together with the knowledge of its diameter 2R and the viscosity of the
124 136
immersion medium {Rohrbach 2004; Svoboda 1994}.
PSDmethod
From Eq. (2.4) the power spectral density PSD of the trajectory Sx(t) of the particle’s motion is known.
2 2k BTg 2 1
PSD ( ) S ( ) gx ( )
2
(2.32)
c2
2
S and x denote the Fourier transforms of S and x, respectively. From this, the corner frequency c = /
can be determined and therewith the trap stiffness for spherical particles with radius R and friction
coefficient = 6R in an immersion liquid with viscosity . The detector sensitivity can be estimated
according to 2.4.1, or, additionally, from PSD(0).
This is illustrated in Fig. 15A for simulated data with = 20pN/µm and g = 500kV/m (see caption for
further parameters). From the results of a fit according to Eq. (2.32) = 21.28pN/µm and g = 520kV/m is
obtained which agrees to the input parameters within a limit of 6.4% and 4.0%, respectively. Error treatment is
1
omitted .
Besides analyzing passive fluctuation data, there is an active method, too, where either the trap or the
particle is oscillated sinusoidally at a certain frequency f. From the relative peak heights PSD(f) / PSD(0) in the
spectrum, g and therewith can be determined {TolićNørrelykke 2006}.
137
ACmethod
An alternative method is the autocorrelation (AC) of the signal which can be calculated from the Wiener
Khintchine theorem, relating spectral properties to correlation by a Fourier transform of the PSD.
gx( ) k Tg
2 t
AC ( ) AC ( S (t ))
2 B
e (2.33)
1
This is complicated because error bars cannot be estimated for power spectral densities and autocorrelation functions. Most problematic
is the fitting process, in particular the cutoff frequency until the fit is performed. Additionally, statistics is limited for low frequencies
which has a strong impact on PSD(0). The only way to assess a statistical error is a quantitative analysis of different fitting parameters and
a mean error analysis of multiple data sets.
30
2. Optical tweezers background
Similar to the PSD, the autocorrelation time = 1/ c = / allows determining the trap stiffness.
Consequently, the sensitivity can be determined from AC(0) or according to 2.4.1. The autocorrelation method
has proven to yield less deviating results compared to the PSDmethod because fitting is more robust.
Again, the method is illustrated in Fig. 15B. An exponential fit according to Eq. (2.33) results in =
20.27pN/µm and g = 502kV/m which agrees with the input parameters within the limits of 1.4% and 0.5%,
respectively. Error treatment is again omitted due to the same reason that applied to the PSDmethod.
6
A 7
B 4
10
2
8 5
10 10 8
PSD(S(t)) (V²/Hz)
AC(S(t)) (V²)
9 4
10
2
10 6
10 10 8
6
4
11
10
PSD(t=0) = 0.8mV²/Hz 2 AC(t=0) = 52mV²
fc = c/2 = 423Hz = 1 / c = 395µs
12 7
10 10
1 2 3 4 5
10 10 10 10 10 0 400 800 1200 1600 2000
f (Hz) t (µs)
Fig. 15. Langevin calibration of simulated data (see chapter 0). Simulation
parameters were: = 20pN/µm, g = 500kV/m, T = 303K, R = 531nm, (T) =
0.8mPas, sample rate sR = 1MHz. (A) Power spectral density PSD in red with
fit according to Eq. (2.32)in black. (B) Autocorrelation AC in red (open circles
th
represent every 50 data point) with fit according to Eq. (2.33) in black (only
datapoints fot t < were considered).
Alternatively, the detector sensitivity g can be estimated by a piezo scan. The particle is attached to the
1
coverslip due to a combination of electro static and van der Waals forces and scanned through the laser focus.
The signal S(p) measured by the QPD can be directly linked to the known position p of the piezo. If scanned
along one dimension (x), the gradient at the center position bx,c of the bead given by Sx(px = bx,c) = 0 is the
desired detector sensitivity: g x Sp( p ) . This method is especially useful if Langevin calibration cannot be
x
x
x
b x ,c
applied or as independent reference. The trap stiffness can be determined from PSD(0), AC(0) and 2.4.1.
The method is depicted in Fig. 16A. The overall sinusoidal position signal with linear central region (thick
lines) as a result of experimental scans along x at four different laser powers P is shown in Fig. 16B. The
sensitivities gP are proportional gP ~ P to the incident laser power within 5% as predicted in chapter 2.3, in
particular Eq. (2.27). The extent xl ≈ 350nm of the linear region and xu ≈ 800nm of the unique detection
region is the same for all laser powers. Since the beads (see appendix 6.6.1) used here are the same as in all
measurements on microtubules, these results also serve as a reference and for interpretation of the signals
shown in chapter 1.
Of course, a huge drawback of this method is that the particle has to be immobilized on a surface, thus it
cannot be used for further experiments. Assuming that different particles vary only slightly in diameter, such a
138
scan can be used as a general calibration scan {Ghislain 1994}, too.
1
The relevant potential is called DLVO potential WDLVO(d) = Wvdw(d) + Wel(d) which is the sum of the van der Waals and electro static
potential WvdW and Wel. It depends on the distance d between two interfaces and strongly depends on the ionic strength of the immersion
medium. While WvdW is attractive, glass cover slips and glass or polystyrene beads both have a negative surface charge, thus Wel is
repulsive. The resulting energy barrier of the DLVO potential can be lowered by adding salt (NaCl). A ~1020mM concentration is enough
that beads are able to adhere to the cover slip (salt solutions saturate at 2M). However, this also means that beads stick to a cover slip
even for low ionic strength in a cell medium or buffer, for example. (According to {Friedrich98 2011})
31
2. Optical tweezers background
A z
y Trap 1
C
x saw tooth oscillation
px B 150 P=64mW
P=32mW
P=16mW Trap 2
100 P=8mW
xu y0,1 static
xl
50
Sx(px) (mV)
0
time
px
g8mV = 47 kV/m D
50 20
g16mV = 106 kV/m P=8mW
g32mV = 230 kV/m
Sx(xL) (mV)
10
100 g64mV = 432 kV/m
0
Fig. 16. Schematic of piezo and bead scan. (A) A bead is immobilized on a
coverslip and scanned by a piezo along px in x direction through the focus.
(B) Resulting position signals Sx(px, P) and detector sensitivities gP for four
different laser powers P. Sensitivities of the central linear region (thick lines)
are proportional gP ~ P to the incident laser power within 5%. (C) One trap is
oscillated saw tooth like along xL while a second, static trap holds the bead.
The power of the second trap P2 = 50P1 is much higher than the power P1 of
the first trap preventing falsification of the scan. (D) Result of the laser scan.
The resulting sensitivities gp and gL of the piezo and the laser scan,
respectively, agree within 5% for a comparable laser power P = 8mW.
Neutravidin coated polystyrene beads with 2R = 1062nm were used in B and
D.
To circumvent the drawback of immobilizing the probe during a piezo scan or in cases where this is simply
not possible, the scan can also be carried out for a diffusing particle with a fast laser scan. The laser focus,
forming a stable trap, is rapidly scanned across the trapped particle, thus resulting in a calibration scan similar
139
to the piezo scan {Vermeulen 2006}.
1
However, the particle is free to move during the scan time. If the scan is not carried out fast enough , this
distorts the result. A second issue is the scanning focus exerting optical forces on the particle. This effect is
110
known as ‘kicking effect’ also described in {Faucheux 1995}. To minimize kicking, the laser power can be
reduced. Using multiple optical traps, both issues can be further minimized by confining the particle’s motion
140
with a strong trap while scanning it (slowly) with a soft trap or vice versa {Lang 2002}.
The situation is depicted again in Fig. 16C, which shows the arrangement of a strong (black) and a much
softer scanning trap (red). The resulting position signal is shown in Fig. 16D. The obtained sensitivity g8mW =
53kV/m agrees within 6% with the value g8mW = 47kV/m obtained by the piezo scan. Regarding a variance R
= 3% of the bead size specified by the manufacturer (see appendix 6.6.1), the results overlap well.
1
The mean square displacement MSD(x(t)) = <x²> = 2Dt of the particle can be used as a quantitative measure. D = kBT / is the
diffusion coefficient. If the particle should be restricted to move less than a tenth of its radius R / 10 during a single scan, it has to be
performed faster than t < 3R³/ 100kBT. For a particle with R = 500nm, this results in t < 2.8ms or a scan frequency f > 350Hz.
32
2. Optical tweezers background
LED
L7 L4 BSC L6
DC3 QPD1
Ei
Es BFPDL
DL
L5
Es(b)
FP
F(b)
OL
BFPOL
Ei L2 L1 Laser fiber
DC1
SF BSD BE
WLS
DC2 L3
SF
Camera
Fig. 17: Schematic of the optical setup. BE = beam expander, BSD = beam
steering device, L1L7 = lenses, DC1DC3 = dichroic mirrors, OL = objective lens,
DL = detection lens, BSC = beam splitter cube (50:50), QPD1,2 = quadrant
photo diodes, LED = light emitting diode, SF = spectral filter, WLS = white
light source, Ei = incident light, Es = scattered light, F(b) = force acting on
112
particle at position b. Adapted from {Koch 2014}.
The optical trap is formed by a high numerical aperture (NAOL = 1.2) objective lens (OL) by a = 1064nm
1
laser beam. A twoaxis beamsteering device (BSD) is placed in a plane conjugate to the focal plane (FP) of the
objective lens. A beam expander (BE) expands the initial laser beam with respect to the maximum aperture of
the BSD. The lenses L1 and L2 further expand the beam to match the diameter of the back focal plane (BFPOL)
of the OL and to translate the deflection angle caused by the BSD to a distinct displacement of the laser focus
in the FP. A second objective lens (DL) is used to collect scattered and unscattered light. The light is further
split by a 50:50 beam splitter cube (BSC) and the BFPDL of the DL is imaged by the lens systems L4/L5 and
L4/L6 onto two quadrant photo diodes (QPD) with different magnifications given by the ratio of the lenses’
focal lengths. The signals from the QPDs are electronically preamplified and digitized by DAC cards installed in
a computer (not shown). Another DAC card is used for the output of signals to steer the BSD and to adjust the
current laser power with a feedback mechanism (not shown).
An LED is used together with lens L7 to allow brightfield illumination. A white light source (WLS) can be
used together with spectral filters (SF) for fluorescence illumination. Dichroic (DC1  DC3) mirrors are used to
separate the different light sources. Both, brightfield and fluorescence images are recorded with a camera.
1
The absorption coefficient is the lowest around = 1064nm for most biological samples (see {Svoboda136 1994}). High optical intensities
may cause severe damage to biological probes.
33
3. Spiroplasma melliferum
3. Spiroplasma melliferum
1
Spiroplasma are helicallyshaped, facultative anaerobic bacteria belonging to the class of mollicutes, which
are characterized by the absence of a cell wall. They are active swimmers and selfpropel by a unique zigzag
56
like swimming pattern without, unlike other motile bacteria, any flagella or other appendages {Trachtenberg
55
1998}. All species found until today are associated with eukaryotic hosts {Lo 2013}. While most species are
pathogenic for several arthropods and plants, others are harmless and coexist with their insect hosts –
141
sometimes even in symbiosis {Whitcomb 1981}. They are the causative agent, e.g., of corn stunt and citrus
142
stubborn disease and are, due to the lack of a cell wall, resistant to most antibiotics {Liao 1981}. They are
likely to have the same ancestors as clostridia bacteria and have evolved by genome reduction as a result of
143
the adaption to their parasitic way of life {Razin 1998}. They do not only belong to the smallest, self
replicating cells known, but are also left by evolution with one of the smallest genomes; the smallest only
143 144
double the size of a large virus {Razin 1998; Trachtenberg 2001}. Their relative structural and genetic
simplicity, their pathogenic potential, the wide range of hosts and their adaption to a parasitic lifestyle
141 145
rendered the mollicutes an excessively researched cell type in the past {Whitcomb 1981; Bové 2012}.
Because their membrane can be isolated easily and manipulated in a controlled manner, they have been an
excellent subject of basic research on membrane structure and function. However, only little research has
been carried out regarding their locomotive principles, i.e., the basic biochemical and physical concepts of the
18 58 59 146
molecular machinery generating forces {Kurner 2005; Trachtenberg 2006; Shaevitz 2005; Gilad 2003;
147
Trachtenberg 2003}.
A B
dh
p
dt
Change of
handedness
C D vk E
vk
vk
vc vc
vc
Fig. 18. Illustration of the helical cell. (A) Definition of cell properties as
mentioned in the text for a straight cell. (B) The cell membrane is strongly
bent at the junction between a left and righthanded helix. (C)  (E) As a
result of reducing stress, the cell kinks. A kink traveling down the length of
the cell body with speed vk causes the cell to move in the opposing direction
with the speed vc. Figures are true to scale.
The species investigated here is Spiroplasma melliferum BC3 (ATCC 33219). Their geometric properties
2 60 148
vary {Trachtenberg 2014; Garnier 1981}, however, an average cell has N = 4 to N = 5 windings with a
diameter of the cell tube dt = 190nm, a helical pitch p = 900nm, a diameter of the helix dh = 570nm and a
1
Cells are usually aerobe or anaerobe, meaning that their metabolism either requires oxygen or not. While some organisms even perish
under the wrong conditions, facultative anaerobe cells are capable of switching between an aerobic and anaerobic metabolism.
2
Evidently, there is some statistical deviation and a variation of the geometric properties of different cells is natural. In addition, during
the course of experimental observation, cells were found to appear relatively thin and to possess only few windings (N = 23) when they
were observed soon (<3 hours) after a new culture was prepared. Another 23 hours later, cells appeared much thicker and had more
windings (N = 410), suggesting that the cell diameter dt grows together with the number N of helical windings. Furthermore, older (and
thicker) cells resulted in stronger signals when observed with the BFPtracking technique presented in chapter 3.2. This indicates a
stronger scattering which again is related to cells with an increased diameter (or volume). Interestingly, {Garnier148 1981} found that the
amplitude dh of Spiroplasma citri cells decreases for cells with a high number of windings (N > 7).
35
3. Spiroplasma melliferum
diameter of the body’s helical centerline dcl = dh  dt = 380nm as illustrated in Fig. 18A. They swim by a
consecutive kink traveling down the length of the cell body from one end to the other. The kink pushes against
59
the surrounding fluid causing the cell to swim in the opposing direction {Shaevitz 2005}. On a molecular
basis, the occurrence of that kink is supposed to be related to a conformational switching of cytoskeletal
56
proteins {Trachtenberg 1998}. The cytoskeleton’s main components are the protein MreB which is found
149
among different bacteria and seems to be involved in cell wall synthesis {Olshausen 2013} and overall cell
150 1 2
shape {Doi 1988}, and the protein Fib which has only been found in Spiroplasma so far .
A B C
D E F
Both proteins build several long fibers that are attached in parallel to the membrane as a cytoskeletal
18 151
ribbon spanning from one end of the cell to the other (see Fig. 19 and {Kurner 2005; Trachtenberg 2008}).
In cryoelectron microscopy experiments, the basic elements of the Fibfiber were found to be tetramers that
exist in a continuum of elliptical conformational states when isolated and purified: from nearly circular to
58
nearly flat {Trachtenberg 2006}. The assumption is that there are two distinct conformational states in vivo:
nearly circular and highly elliptical (see Fig. 19E). A change of all elements of one Fibfiber from one
1
Following {Trachtenberg151 2008}, the expression Fib is used as shorthand for ‘Fibril protein’ as product of the fib gene. This must not be
mixed up with the Fib protein as product of the homonymous Fib gene of Drosophila. The official nomenclature seems to be inconsistent.
2
An overview and description of different cytoskeletal associated and Spiroplasma specific proteins and genes can be found in {Bové152
2003}.
36
3. Spiroplasma melliferum
conformation to the other shortens or elongates the whole fiber. If two (or more) fibers undergo a differential
length change, this causes the cell to switch its handedness between lefthanded and righthanded as
144
indicated in Fig. 19F {Trachtenberg 2001}. At the junction between both helices, the cell is bent heavily
resulting in increased stress on the membrane and the cytoskeleton itself as illustrated in Fig. 18B. This state is
energetically balanced by tilting both sides so that they fit together without additional stress, thus causing the
cell to kink at the junction (Fig. 18CE and Fig. 19A). If the underlying differential length change successively
travels down the length of the cytoskeleton, starting at one end, a kink is generated that moves in the same
fashion. This behavior resembles the polymorphic switching of bacterial flagella not only qualitatively, but also
56
on a molecular level {Trachtenberg 1998}. Kinks always start at the same end but it is unclear which
mechanism is responsible for this behavior. However, tips have been identified to be morphological different
from each other and form the remaining cell body. Cells commonly exhibit a blunt or round end and a tapered
153 148
end ({Ammar 2004} and Fig. 19A), but cells with two round or two tapered ends are found, too {Garnier
1981}.
Outline
In this chapter, linescanning optical tweezers are introduced as a novel tool to investigate nonspherical or
noncylindrical cells with optical traps. In particular, chapter 3.1 focusses on the description of optical forces
acting on a trapped, helical cell. Chapter 3.2 describes a novel timemultiplexed shape tracking or imaging
method based on BFP tracking as presented in chapter 2.3. Experiments under different external conditions
are described in chapter 3.3, followed by a new model to describe the molecular motor (chapter 3.4), basing
on the experimental observations.
1
Apparently, line optical traps can be used to guide neuronal growth ({Carnegie160 2008} and references therein).
37
3. Spiroplasma melliferum
1) Description of the force Fpearl of a static focus at position rL = (xL, yL, zL) on a single pearl at position
a(xp) = b + c(xp), where b is the center of mass position of the whole cell and c(xp) = (xp, RHCos(kHxp),
RHSin(kHxp)) describes the actual helical shape (see Fig. 20A+B).
2) Calculation of the total force Fpearls of the static laser focus on all pearls of the cell (Fig. 20C).
3) Accounting for the fast laser movement by timeaveraging Fpearls over one oscillation of the laser
positions rL(t). This leads to an effective force Feff acting on the center of mass position of the cell (Fig.
20D).
F pearl b c( x p ), rL
A B
y
xL
x
xp
Static focus
C
c y F pearls b, rL
xL
x
Static focus
b rL
a
z
D
y F eff
b
y x
x
Oscillating focus
Fig. 20. Pearl model and principle of force generation. (A) The cell with center
of mass position b is modeled as a rope of pearls at position a=b+c, where c
describes the actual helix. The position of the laser focus is rL. (B)(D)
Illustration of the three steps (see main text) to calculate the total force of
112
the oscillating focus on the whole cell. From {Koch 2014}
The intensity distribution of the focus is described by a threedimensional Gaussian with widths i (i = x, y,
z) and an additional Gaussian modulation with width L in scan direction as given by Eq. (3.1) and shown in Fig.
21.
2
r
2
r x
2 ry yL r z
2
x x L z L
L x y z
I (r, rL ) I 0 e e e
e (3.1)
The modulation is necessary to hold the cell in the center of the line trap on the one hand, and to also get a
harmonic potential in scan direction on the other hand. The latter is essential to apply Langevin calibration
techniques as explained in chapter 2.4. The optical force at position r of a point focus at position rL as
described by Eq. (2.13) is proportional to the gradient of the intensity given by Eq. (3.2).
rx 2xL rx2
xr y L
r I (r, rL ) 2 y2y L I (r, rL ) (3.2)
r z
z 2 L
z
The position a(x) of the individual pearls of the cell body are described by
xp
a ( x p ) b c( x p ) c( x p ) RH cos(k H x p ) (3.3)
R sin(k x )
H H p
Together with Eq. (2.13), the force of the static focus on one pearl can be written as
38
3. Spiroplasma melliferum
n
F pearl a( x p ), rL r I a( x p ), rL (3.4)
2c
So far, this is the same as the optical force on an ordinary bead as illustrated in Fig. 9. Now, the force Fpearls
of the static focus on all pearls has to be estimated by integrating Fpearl over all pearls, i.e., integrating over the
1
total extend 0 ≤ xp ≤ LC = Np of the cell. Of course, due to the finite extend of the point focus , not all pearls
experience a force F ≠ 0.
LH
F pearls (b, rL )
1
dt F
pearl
a( x p ), rL dx p (3.5)
0
This integral is highly nontrivial and cannot be solved analytically. However, numerical simulations are
shown in chapter 3.1.1. In the last step, the oscillation of the focus has to be taken into account. This is done
by time averaging Fpearls over half a scan period Ts = 1 / f, i.e., integrating over all focus positions 0 ≤ rL = vLt
≤ LT = vL Ts / 2 during that time
Ts / 2
2
F eff (b) F pearls b, rL (t ) F pearls b, v L t dt
t Ts 0
(3.6)
κ b
The last simplification in Eq. (3.6) is neither trivial nor obvious but holds for (bx, by, bz) <
(L/2,y/2,z/2). Here, is a diagonal matrix with entries x, y ,z as introduced in chapter 2.2.1. The validity
is shown by the simulations in chapter 3.1.1 and has been experimentally proven as shown in chapter 3.1.3.
The linear force dependence leads to a harmonic potential, at least for small displacements b, as it is usually
the case for optical traps. The potential is calculated and the components are defined as shown by Eq (3.7).
b
V eff (b) F eff (b ' )db '
bx by bz
V x
eff
(b) V y
eff
(b) V z
eff
(b )
1
2
x bx2 y by2 z bz2
Numerical simulations of Eq. (3.7) are shown at the end of the next chapter.
3.1.2. Simulation
Due to the dependence of the intensity on a Gaussian function, the integrals (3.5) and (3.6) are nontrivial
and cannot be solved analytically. However, they can be calculated numerically and results are presented in
this chapter.
Simulation parameters
Here, the width of the additional intensity modulation L = 4µm (see Fig. 21) and the length of the line trap
LT = 12µm have been chosen. A cell with N = 4 windings, resulting in a length LC = Np = 3.6µm, has been
simulated. These values reflect the experimental conditions, i.e., the mean length of the cells and empirically
1
In lateral direction, the half e1 width is x =y = 0.61/ NA ≈ 0.5µm and in axial direction z = / (n  nCos()) ≈ 1.3µm.
39
3. Spiroplasma melliferum
found values that allowed stable trapping. The choice of these values can also be directly motivated from the
simulations as shown by a comparison for L = 1µm in appendix 6.7. All prefactors have been set to one since
the polarizability and the exact refractive index of the cell nc are not known. Therefore, all amplitudes are
given in arbitrary units (a.u.). However, the amplitudes of different directions of the same force can be
compared since the principle mathematical operations are the same in every direction.
A I(rx)/I0 B I(rx)/I0
0.8 0.8
0.6 0.6
rx rx
4µm 2µm 2µm 4µm 4µm 2µm 2µm 4µm
Fig. 22 shows the xy and xz planes for each component of the force given by Eq. (3.5) for a helix with fixed
center of mass position b = 0 and varying laser position with L = 4µm. The force components of the yz plane
qualitatively do not differ from the force of a bead in a static point trap.
In principle, the force component Fxpearls in scan direction (Fig. 22A+B) displays two different regions: when
the focus is at an end of the cell and when it is somewhere in between. At the ends, the force is much higher
due to a larger jump of the portion of cell volume that is in the focus comparing different neighboring focus
positions. In contrast, in the central region the change of cell volume, depending on the position of the focus,
is small (yL ≠ 0) or even zero (yL = 0). For yL ≠ 0 the helical form of the cell manifests as shown by the
corresponding line scan in Fig. 22C.
The directions lateral to the scan direction (Fig. 22C+D and G+H) display the same principle behavior,
depending on the direction of laser movement. For displacements along the principal direction, i.e., Fypearls (y L )
and Fzpearls (z L ) , the force resembles a sinusoidal slope (see line scans in Fig. 22F+I) which is typical for the
force of a focus scanning at thin mass density (like a bead, for comparison). In scan direction, again the helical
shape becomes apparent which is especially evident for Fypearls (x L , 0, zL ) and Fzpearls (x L , yL , 0) . Regarding the
amplitudes of the forces, the maximum force in the principal plane, i.e., Fypearls (x L , y L , 0) and Fzpearls (x L , 0, zL ) ,
is much higher than in the other planes (see range of color scale mentioned in the figure caption). A
comparison for L = 1µm is given in appendix 6.7.
40
3. Spiroplasma melliferum
A Fxpearls rL , b 0 B C
3µm xL Fxpearls rL , b 0
3µm xL
yL Fxpearls rL , b 0
zL rL x , 0, 0 1
0µm 0µm
L
rL xL , 0.37 µm, 0
rL xL , 0, 0.37 µm
x
3µm 3µm 3µm 3µmL
1.25µm 4µm
0µm 0µm
1.25µm 4µm 1
D Fypearls rL , b 0 E F
3µm xL Fypearls rL , b 0
3µm xL
yL Fypearls rL , b 0
zL
0µm rL 0, y L , 0
0µm 1
rL 2 µm, y L , 0
rL 0, y L , 0.37 µm
yL
3µm 3µm 1.25µm 1.25µm
4µm
1.25µm
0µm 0µm
1
1.25µm 4µm
G Fz pearls rL , b 0 H I
3µm xL Fz pearls rL , b 0
3µm xL
yL
zL Fz pearls rL , b 0
0µm 0µm rL 0, 0, z L
0.3
rL 2 µm, 0, z L
rL 0, 0.37 µm, z L
3µm 3µm zL
4µm 4µm 4µm
1.25µm
0µm 0µm
1.25µm 4µm 0.3
The effective forces Fxeff (bx ) , Fyeff (by ) , Fzeff (bz ) in the principal directions (Fig. 23A,D,H and C,F,I) resemble
those of a bead scanned through a static point trap (see Fig. 9). In y and zdirection, this is not surprising since
a line optical trap is not elementary different from an optical point trap in these directions. In scan direction,
this behavior is due to the additional Gaussian intensity modulation. At least for small displacements, the
situation of a helix described by the effective force is equal to a bead in a static point trap.
The line scans shown in Fig. 23C,F,I display nicely the linear force dependence for small displacements bx,
by, bz as postulated by Eq. (3.6). This was the initial intention in order to be able to use Langevin calibration, for
example. As shown by the line scans in Fig. 23C,F,I, the variation of x(bx, bz), y(bx, bz) and y(bx, by) (green
and orange lines) is small in directions lateral to the scan direction due to the smooth variation according to
41
3. Spiroplasma melliferum
L1. In scan direction, the linear force constant varies more rapidly due to the sharper intensity distribution
according to y, z.
A Fxeff b B C
9µm bx
Fxeff b
by 9µm bx
bz Fxeff b
0µm b b x , 0, 0
0µm 4
b b , 0.37 µm, 0
x
bx
9µm 9µm 9µm 9µm
1µm 4µm
0µm 0µm
4
1µm 4µm
D Fyeff b E F
9µm bx
Fyeff b
by 9µm bx
bz Fyeff b
0µm b 0, b y , 0
0µm 40
b 2 µm, b y , 0
by
9µm 9µm 1µm 1µm
1µm 4µm
0µm 0µm
4µm 40
1µm
G Fz
eff
b H I
9µm bx
Fzeff b
by 9µm bx
bz Fzeff b
0µm 0µm b 0, 0, b z
b 0, 0.37 µm, b z 10
bz
9µm 4µm 4µm
9µm 4µm
1µm
0µm 0µm 10
1µm 4µm
Fig. 23 Total force of the oscillating laser focus on the whole cell for L =
4µm. (A), (B) Force profiles Fxeff in the xy and xz plane. Force amplitudes 
5/5a.u. for both Figs. (C) Line scans as indicated in A. (D), (E) Force profiles
Fyeff in the xy and xz plane. Force amplitudes 50/50a.u. for Fig. D and 
3/0a.u. for Fig. E. (F) Line scans as indicated in D. (G), (H) Force profiles Fzeff in
xy and xz plane. Force amplitudes 0.04/0.04a.u. in Fig. G and 13/13a.u. in
Fig H. (I) Line scans as indicated in G. Color scales range from negative (blue)
to positive (red) values.
Regarding the maximum amplitudes of the forces in the principal directions (see caption of Fig. 23), one
finds: Fxeff (bx ) Fzeff (bz ) Fyeff (by ) as observed experimentally, too (see chapter 3.1.3 and 3.2.2). This order
can be explained by the effective widths of the intensity distribution of the line focus: L < z < y. Since the
optical force is a gradient force which is mainly influenced by the widths of the focus, this is not very surprising
2
but should be noted .
Fyeff (b x , 0, b z ) , as shown in Fig. 23E, displays a force profile that appears rather strange since it should be
zero in the whole plane but has a broad negative dip with a maximum at b=(0, 0, 0). This is due to the cosine
dependence of the helix in that direction and the intensity modulation according to L. Pearls in the center of
the trap experience a slightly higher force than the remaining pearls. During the integration (see Eq. (3.5) and
1
This fact is very practical and used during the analysis of experimental data. The forces Fyeff(bx) and Fzeff(bx) are assumed to be constant
with respect to bx, or in other words, y(bx) = const. and z(bx) = const.
2
In the experiments, if a cell was able to escape the trap, it either swam out in scan direction or in axial direction during it turned itself
inside the trap, something that can happen if the trap is not strong enough.
42
3. Spiroplasma melliferum
(3.6)), this leads to an asymmetry that manifests in a negative force. However, the amplitude is small
compared to the maximum amplitude of Fyeff (b x , b y , 0) , i.e., 3a.u. to 50a.u..
Fzeff (b x , b y , 0) should again be zero in the whole plane but instead displays a profile that resembles one of
the principal directions. The reason is similar to Fyeff (b x , 0, b z ) as explained in the last paragraph, although, in
contrast, Fzeff (b) 0 is zero for b = (0, 0, 0). Here, the asymmetry during the integration appears for b ≠ 0 due
to the sine dependence of the helix in axial direction. A comparison for L = 1µm is given in appendix 6.7.
The effective potentials are parabolic for small displacements b in all directions as shown in Fig. 24. Again,
this is expected in y and z direction since these directions are determined by the properties of a point focus.
In scan direction, the harmonic shape is caused by the smooth Gaussian intensity modulation according to L =
4µm. For a sharper modulation width as shown appendix 6.7, (L = 1µm), this is not the case anymore. Here,
the effective force Fxeff (bx ) already displays a saddle point at bx = 0 instead of a linear slope.
A Vxeff bx , by , 0 B C
9µm bx
9µm
Vx
eff
bx , 0, bz
by bx
Vxeff b
bz b
0µm 9µm 0 9µm x
0µm
9µm 9µm
1µm 4µm b b x , 0, 0
0µm 0µm b b x , 0.37 µm, 0
1µm 4µm 25
D Vyeff bx , by , 0 E F
9µm bx
by
Vyeff b
0µm 1µm Vyeff 0, by , bz b
0µm 1µm 0 1µm y
by
1µm bz
4µm
9µm 0µm
1µm b 0, b y , 0
0µm 4µm b 2 µm, b y , 0
1µm 25
G V z
eff
bx , 0, bz H I
9µm bx
bz Vzeff b
0µm 1µm Vzeff 0, by , bz b
0µm by 4µm 0 4µm z
1µm bz
4µm
9µm 0µm
4µm
b 0, 0, b z
0µm 4µm
b 0, 0.37 µm, b z
4µm
25
Fig. 24 Effective potentials for L = 4µm. (A), (B) xy and xz plane of V . x
eff
Amplitude of potential 25/0a.u. for both Figs. (C) Line scans as indicated in A.
eff
(D), (E) xy and yz plane of Vy . Amplitude of potential 25/0a.u. for Fig. D
and 45/0a.u. for Fig. E. (F) Line scans as indicated in D. (G), (H) xz and yz
plane of Vzeff . Amplitude of potential 25/0a.u. for Fig. G and 45/0a.u. for
Fig. H. (I) Line scans as indicated in G. Color scales range from negative (blue)
to positive (red) values.
43
3. Spiroplasma melliferum
The potential depths in the principal directions Vxeff (bx ) , Vyeff (by ) , Vzeff (bz ) are identical. This may seem
irritating at first glance, since the maximum amplitudes of the corresponding effective forces were completely
different (max(Fxeff(bx) = 5, max(Fyeff(by) = 50, max(Fzeff(bz) = 13). However, during numerical integration, the
amplitudes are weighted with the widths of the forces in the corresponding directions which are bx = ±9µm, by
= ±1µm, bz = ±4µm, resulting in nearly the same amplitudes of the integration result.
The potentials lateral to the scan direction, Vyeff (0, by .bz ) and Vzeff (0, by .bz ) , are again governed by the
behavior of a point trap since the scanning laser has no effect in time average.
Regarding the line scans of the principal direction shown in Fig. 24C,E,I, the variation of the curvature given
by i (i = x, y, z) for different positions can be analyzed. Even for large displacements (bx = 2µm) in scan
direction, the force constant y and z do not vary significantly due to the smooth intensity modulation L. The
variation of x is much more pronounced due to the much smaller focal width y and z. These observations
reflect the similar behavior of the effective forces.
For stable trapping of an object inside a line optical trap, the extend of the potential has to be larger than
the extend of the object itself. In the case of the helical bacterium, two dimensions (diameters dt of the cross
section) are much smaller than the width of a point focus, the third (length Lc) is much larger. A stable trap can
be formed by rapidly scanning the laser along a line with a length of LT > LC + x. To form a harmonic
potential, the intensity of the laser has to be further modulated by a Gaussian with a characteristic width of L
which has to be large enough to cover the length LC = Np of the cell.
3.1.3. Measurement
For an experimental verification of the harmonic shape of the potential, Boltzmann statistics can be used.
In thermal equilibrium, the motion of a particle in a potential V(x) is given by the probability distribution p(x)
according to
V ( x)
p ( x) p0 e kBT
(3.8)
p0 normalizes the distribution: p ( x)dx 1 . In trapping experiments, the positions b = (bx, by, bz) are
measured and the histograms px(bx), py(by) and pz(bz) can be calculated. The potentials are then obtained from
p( x)
V ( x) k BT ln (3.9)
p0
This is shown in Fig. 25 for data of the representative measurement presented in chapter 3.2.2. The
measured potentials (open circles) are fitted by parabolas f(bi) = ½ibi2 (solid lines). The fits agree well with
the experimental data points justifying the theory. In all three directions, the passively diffusing cell was
exposed to peak energies of ~9kBT which is similar to a bead diffusing in a point trap.
44
3. Spiroplasma melliferum
2
4
V(b) (kBT)
6
8 Vx(bx) / fit
Vy(by) / fit
Vz(bz) / fit
10
3.2. 3D imaging and force sensing of helical cells by line optical tweezers
A general description on the formation of the signal triplet (Sx, Sy, Sz) = (gxbx, gyby, gzbz) of a bead displaced
by b = (bx, by, bz) from the center of a point focus by evaluating the interference pattern of scattered and
unscattered light in the back focal plane of a detection lens was shown in chapter 2.3. Using the pearl model
again, the signal of the scanned helix can be described similar to the force calculation in the last chapter.
Without loss of generality, the laser is assumed to oscillate along the x direction in the following.
a(xL)
y
xL
Fig. 26. Detection schema. The cell tube is modeled as a chain of pearls. At
every position xL of the laser focus, the displacement a(xL) of the bead at
position xL is measured.
During laser oscillation, data from the QPD is recorded at a temporal sampling rate sR. For every
acquisition at time t, the signal triplet S(xL(t)) from the QPD according to Eq. (2.28) is proportional to the
displacement of the pearl at position a(xL). However, due to the strong modulation of the laser intensity in
order to form a harmonic trapping potential in all direction (see chapter 3.1.1), the signal has a large and time
dependent offset. This affects especially the axial direction, since here the signal is the sum of all quadrants
1
and thus directly proportional to the laser intensity. In lateral direction, the background cancels out .
1
This is only true in theory. In practice, either a twoaxis galvanometric scan mirror or a twoaxis AOD is used. Both scanners consist of
two independent scan units (one for x, one for y) which are displaced axially by ~1cm. The QPD is in a conjugate plane to the center of the
scanner, thus differently deflected beams are not exactly matched at the same position on the QPD. This causes a slight offset also in
lateral signals.
45
3. Spiroplasma melliferum
A B
xL(t) (µm) xL(t) (µm)
0 2 4 6 8 10 12 0 2 4 6 8 10 12
xL(t) (µm)
4 5 6 7 8
empty
Empty scan: Sy 1.0 1.6
y signal (mV)
raw
z signal (V)
50 Raw data: Sy 1.4
z signal (V)
0.0 1.2
1.0
0 empty
1.0 Empty scan: Sz 0.8
raw 0.6
Raw data: Sz
50 2.0 400 600 800
t (ms)
0.0 0.2 0.4 0.6 0.8 1.0 1.2 0.0 0.2 0.4 0.6 0.8 1.0 1.2
t (ms) t (ms)
C
40 Subtract background: S (t ) S raw (t ) S empty (t)
Difference signals (mV)
20
E xL(t) (µm) F xL(t) (µm)
0 5 10 0 5 10
0.0
0.0
0
20
Sy(xL)
1.0
1.0
40 Sz(xL)
0 2 4 6 8 10 12
xL(t) (µm)
2.0
2.0
Calibrate
D cy(xL)
t (s)
t (s)
200 cz(xL)
Amplitude (nm)
400 400
3.0
3.0
100
cz(xL,t) (nm)
200 cy(xL,t) (nm)
0 0 0
200 p
/2
3 4 5 6 7 8
5.0
5.0
xL(t) (µm)
3D plot: c( xL , t ) xL , c y ( xL , t ), c z ( xL , t )
G c( x , t ) t = 0ms
L
t = 2.5ms
c( xL , t )
y
x z
z y x
200nm
Fig. 27. Signal processing and image creation. (A) + (B) QPD raw signals
Syraw(t), Szraw(t) and empty scan data Syempty(t), Szempty(t) of directions lateral (y,
z) to the cell axis. (C) Signals Sy(xL), Sz(xL) with subtracted background/empty
scan. (D) Actual pearl position c(xL) after calibration. The signals in C have
1
been Fourier filtered to eliminate high frequency noise and the offset b has
been subtracted. (E) + (F) Kymographs of cy(xL, t) and cz(xL, t). Data of
consecutive line scans (as in D) are plotted as lines of an image. Amplitudes
are encoded in gray values. (G) 3D representation of the data in two different
perspectives for two successive line scans (t = 0ms and t = 2.5ms).
1
The Fourier spectrum Ϝ(Sy(xL, t) is calculated and high frequency noise (f ≈ 50kHz) is cut out (set to zero). The inverse Fourier transform
Ϝ1 is then applied to get the filtered signal.
46
3. Spiroplasma melliferum
To address this problem, an empty scan Sempty(t) is performed after each measurement and subtracted from
the actual raw signal Sraw(t) recorded with the cell. As shown in Fig. 27AC, this results in a signal S(t) that is
directly proportional to the shape of the cell body:
xL (t )
S(t ) S raw
(t ) S empty
(t) ~ a x L (t ) RH cos k H xL (t )
(3.10)
R sin k x (t )
H H L
The estimation of the detector sensitivity gi (i = x, y, z) for each direction, relating the signal Si = gici to the
actual position of the cell ci is explained in the next chapters. However, since it fits well here, the results are
taken in advance. The calibrated slope for one scan is shown in Fig. 27D. Since the current laser position is
known, the time axis can be mapped to the laser position. The /2 phase shift between the y and zsignal can
be seen nicely as well as the helical pitch p. In order to properly asses the temporal course of the signals
(usually 800 frames or line scans per second), the data is plotted in kymographs as shown in Fig. 27E+F. Here,
data from consecutive line scans are plotted as lines of a picture, amplitudes are shown in gray values. The
helicity, encoded in an alternating black/white pattern can be seen. Using an edge detection algorithm, the
ends of the cell can be traced over time (red lines) and the Brownian motion of the cell inside the trap
becomes visible.
Plotting the y and zdata of a single scan vs. the position xL of the laser, 3D images are obtained as shown
109
in Fig. 27G. According movies can be found in the supplementary information of {Koch 2012}.
Different calibration schemes have already been mentioned in chapter 2.4. However, trap and detector
calibration for a helical object in a linescanning optical trap is not straight forward and needs combinations of
different techniques. Besides this, calibration is different in each direction as described in the following.
Experimental parameters for the result shown in the following are: LT = 12µm, f = 2kHz, sR = 500kHz, L
= 3µm, P0 = 180mW. The length LC of the cell was LC = RE – LE ≈ 4µm equivalent to N ≈ 4.5 windings at a
pitch p ≈ 900nm. The total measurement time was T = 110s.
This is the scan direction. According to Eq. (3.10), cx(xL) = xL and thus the signals Sy(t) and Sz(t) recorded at
discrete times t can be mapped to the laser position xL(t). The discretization xL = LT / (sR / 2f) is given by the
length LT of the trap, the scanning frequency f and the sample rate sR of the data acquisition.
The main difficulty is the determination of the center of mass position bx of the cell in this direction. One
approach is to use an edge detection algorithm to find the beginning (left edge – LE) and end (right edge –
RE) of the cell in a kymograph of Sy(xL, t) or Sz(xL, t). The result of such edge detection was already shown in
Fig. 27E+F. From the edges, the center of mass position can be determined according to bx(t) = (LE(t) + RE(t))
/ 2 (Fig. 28A not the same data as in Fig. 27). The width x of the histogram Hist(bx) (Fig. 28B) obtained by
fitting can be used together with the equipartition theorem (chapter 2.4.1) to obtain the trap stiffness x
k BT
x (3.11)
x2
47
3. Spiroplasma melliferum
AC(bx) (µm²)
Exponential fit
Hist(bx) (10 )
9
3
6.0 8 8
bx (µm)
7
6
x=307ms
5.0 4
²/DOF=14.5 5
x=502±11nm 4
4.0 0
0 20 40 60 80 100 1.0 0.5 0.0 0.5 1.0 0 100 200 300 400
t (s) bx (µm) t (ms)
Fig. 28. Calibration in x direction. (A) Center of mass position bx(t) in scan
direction. (B) Histogram Hist(bx) of bx together with Gaussian fit of width x.
(C) Autocorrelation AC(bx) of bx with exponential fit and time constant x.
The main calibration constants are determined now (gx = 1 since no positional calibration is necessary);
however, the viscous drag coefficient x can be determined by fitting the exponential decay of the
autocorrelation AC(bx). From the resulting time constant x, x can be calculated
x x x (3.12)
This is of particular interest since, in contrast to a sphere or a cylinder, there is no analytical expression to
calculate the drag on a helix.
ydirection
If the viscous drag coefficient was known, the detector sensitivity gy could be determined from a Langevin
calibration of the signal b’y = Sy(by) corresponding to the center of mass positions. However, as previously
mentioned, is not known here. Instead, another parameter has to be determined to enable calibration. This
is done by a calibration scan that directly results in the detector sensitivity gy as illustrated in Fig. 29. The cell is
caught by a line trap and instantly the direction of the laser is turned by 90°, thus scanning perpendicularly to
the cell long axis. The signal Sycal(yL) of this scan can be mapped to the distance of the laser position yL to the
cell center b’y = Sycal(yL) = 0 since, similar to the actual scan direction (x), the position of the laser focus yL(t) is
known at any time. The detector sensitivity gy is the gradient of the signal at Sycal(yL) = 0 as shown in Fig. 29B
(green line).
cal
gy S y ( yL ) (3.13)
y S 0
After the laser direction is turned, the cell is free to drift and especially to turn into the current direction of
112 162
the line trap. As discussed in {Koch 2014; Koch 2010}, gy(t) for successive scans at time t is remarkably
stable for at least t = 50ms (=40 scans at f = 400Hz). On the other hand, gy(xL) obtained by calibration scans
at different positions xL of the cell differ only by max ~10% indicating that this method is not very prone to
systematic errors. To further increase the accuracy of gy, the mean value of the first 40 scans can be taken.
48
3. Spiroplasma melliferum
yL (µm)
A B 3.5 4.0 4.5 5.0 5.5 6.0
Calibration scan
Sy (yL) (mV)
100 Fit
gy
0
cal
x 100
y z
400 500 600 700 800
C t (ms)
250
by (nm)
0
250
In general, gy obtained by this method can be used to scale the signals Sy(xL). However, gy can only be used
to get accurately scaled amplitudes as long as the signal Sy(xL) is in the linear range yl ≈ ±150nm of the
detector response Sycal(yL) (compare Fig. 29B+C). Unfortunately, already the diameter D = 2RH = 370nm of the
cell centerline is larger than this value. The problem further increases since the cell diffuses by some ten to
hundred (depending on the trap stiffness y) nanometer and the kink amplitude of actively selfpropelling cells
are much larger than the cell diameter. To overcome the limitation of the linear detection range, the recorded
signals Sy(xL) can also be calibrated by fitting Sycal(yL) with ∂/∂y exp(y2/y2) between the extremes (min(Sy)
1
2
and max(Sy)) and inverting the resulting function: ay(xL) = S1ycal(Sy(xl)). This method is limited by the unique
detection region yu ≈ 600nm  800nm (depending on the properties of the optical detection path, compared
to Fig. 16) which is much larger than yl. Data calibrated by the inverse of the calibration scan has already
been shown in Fig. 27 of the last chapter.
A B by (nm) C
200 0 200
Hist(b'y), Hist(by) (10 )
20 5
3
15 Exponential fit
3
200 6
b'y (mV)
by (nm)
10 2
0 4 y=1.7ms
5 200 ²/DOF=2.0
2
1
400 'y=2.96±0.03mV
0 0
1
See Appendix 6.1 and {Pralle123 1999} for explanation.
2
Especially for long cells, kinks rarely escape this limit. These cases can be identified by a slight dip in the single of the kink in opposing
direction and are rejected if necessary.
49
3. Spiroplasma melliferum
The remaining calibration, i.e., finding y and gy, is similar to the x direction. The unscaled center of mass
position b’y(t) = gy/qy by(t) has to be determined first. This is done by calculating the mean value b’y(t) =
<Sy(xL,t)>xL (see Fig. 30A) between LE and RE , i.e., LE(t) ≤ xL ≤ RE(t). Again, a Gaussian fit to Hist(b’y) (Fig.
1
30B) in order to determine the width y of the signal histogram is used to estimate the trap stiffness y
’
2
k BT g y
y (3.14)
y' 2 q y2
Here, an additional correction factor qy explained in detail in the next chapter is already introduced. The
viscous drag coefficient y is determined by fitting the exponential decay (Fig. 30C) of AC(by(t)) or AC(b’y(t))
resulting in the autocorrelation time y.
y y y (3.15)
2
Here, a calibration scan as in y direction cannot be performed due to experimental limitations . However,
due to the symmetry of the cell, z = y is an assumption that suggests itself. The center of mass position b’z(t)
= <Sz(xL,t)>xL can again be determined by the mean of Sz between LE and RE (see Fig. 31A). A fit to the
exponential decay of AC(b’y) results in the autocorrelation time z and thus in the trap stiffness z
z y y
z y (3.16)
z z z
A Gaussian fit to the histogram Hist(b’z) is used to determine the width z of the histogram resulting in the
detector sensitivity gz
gz z ' y gy
z' z' (3.17)
qz k BT y z qy
Here, similar to the y direction, a correction factor qz explained in the next chapter has been introduced.
A B bz (nm) C
1000 500 0 500 1000
30
Hist(b'z), Hist(bz) (10 )
8
3
20 9
6 Exponential fit
600 8
b'z (mV)
bz (nm)
7
10 0 4
6
z=19.4ms
600
0 2 ²/DOF=4.28 5
1200 'z=6.62±0.05mV
10 0 4
0 20 40 60 80 100 10 0 10 0 10 20 30 40
t (s) b'z (mV) t (ms)
’
Fig. 31. Calibration in z direction. (A) Signal b z(t) corresponding to center of
mass position bz(t) obtained from calibration. (B) Histogram Hist(b’z) and
Hist(bz) of b’z and bz together with a Gaussian fit to Hist(b’z) and its width ’z.
(C) Autocorrelation AC(b’z) of b’z with exponential fit and time constant z.
1
The laser needs the time t = LH / vL = LH / (2LTf) ≈ 8µs to travel from LE to RE. Comparing the time y = 1.7ms (see Fig. 30C), the cell
needs in average to explore the potential of width y ≈ 180nm (see Fig. 30B), this leads to a broadening y ≈ y t/y ≈ 1nm of the
histogram and to a relative systematic error y / y ≈ t / y ≈ 0.5%.
2
Either a fast piezo to move the coverslip with the cell as a whole, a precisely moveable or focal tunable lens {Tanaka163 2014} in order to
perform a calibration scan would be needed.
50
3. Spiroplasma melliferum
Results
Calibration results for different scans/cells are comparable for identical trap parameters (laser power P0,
modulation width L, etc.). Results for the data shown above are given in the following as representatives:
gx 1 g y 67115 584 Vm g z 16794 1349 Vm
q y 3.8 qz 1.46
x 0.032 0.002 µm
pN
y 0.294 0.008 µm
pN
z 0.025 0.001 µm
pN
As already mentioned at the beginning of this chapter, experimental parameters were: LT = 12µm, f =
2kHz, sR = 500kHz, L = 3µm, P0 = 180mW. The length LC of the cell was LC = RE – LE ≈ 4µm equivalent to
N ≈ 4.5 windings at a pitch p = 900nm. The measured and corrected (see next chapter) diameter of the cell is
D = 276nm and the total measurement time was T = 110s.
Remarkably, the viscous drag coefficients par = x and per = y parallel (par) and perpendicular (per) to the
long axis of the helix differ from those of a cylinder with equivalent size, i.e., length Lcyl = LC and Dcyl = D + dt =
466nm.
2 Lcyl 4 Lcyl
cyl
par 10.3 fNµms cyperl 13.4 fN s
(3.18)
ln 0.2
Lcyl
Dcyl ln 0.84
Lcyl
Dcyl
µm
(T = 303K) = 0.8mPas is the viscosity of the surrounding medium. Especially surprising, the coefficient for
the helix is larger in direction parallel (x) to the long axis compared the perpendicular (y) direction, which is
contrary to the cylinder. From a biophysical point of view, this facilitates kinking the cell and promotes pushing
the cell forward on a helical trajectory by pressing the largest kink free portion of the cell against the
surrounding fluid  the prevailing swimming pattern of Spiroplasma. Thus, the kinking swimming behavior of
Spiroplasma might be a consequence of hydrodynamic adaption of the helical cell body to its environment.
51
3. Spiroplasma melliferum
A possible explanation for the large deviation (factor of 30) of the drag coefficient in perpendicular
direction to that of a cylinder is hydrodynamic constriction and turbulence. For a helix moving parallel to its
long axis, fluid has to flow through the narrow tubular inside of the cell with a diameter of DH – 2dt ≈ dt =
190nm as illustrated in Fig. 32A+B. Similarly to the friction coefficient close to a wall, flow is hindered
tremendously inside such a small tubular structure. If, in contrast, the helix is moved perpendicular to its long
axis, fluid can pass more easily as indicated in Fig. 32D. Turbulence, as indicated in Fig. 32C, can be excluded at
164
Reynolds numbers Re ˂ 2000 which is the case here (see footnote 2 on page 19) {Zamir 2000}. As a result,
the kinking swimming behavior of Spiroplasma might be a consequence of hydrodynamic adaption of the cell
body to its environment.
Following the three step procedure to obtain the effective trapping force Feff (chapter 3.1.1), the force
constants eff,i (i = x, y, z) can be estimated theoretically: a linear approximation of the gradient force on a
single bead (or pearl) given by Eq. (2.13) results in the trap stiffness prl of a single pearl
12
1.13
nm I 0V 1
x
pN
κ prl 2y 1.13 (3.19)
c µm
12 0.17
z
To recapitulate the quantities: x2, y2, z2 are the focal width of the trapping laser spot, c the speed of
light, nm = 1.33 the refractive index of the immersion medium (water) and = 3(m²1)/(m²+1) the polarizability
of the scatterer described by the ClausiusMossotti relation, where m = ns / nm and ns = 1.4 is assumed for the
refractive index of the scatterer. The volume V of the scatterer is given by the diameter dt = 0.2µm of a pearl
and I0 = P0 / (x2) with P0 = 180mW.
The force constant prls of all N ≈ 4 pearls inside the focal volume increases by accordingly: prls = Nprl. The
oscillating laser focus distributes the optical power over an increased area Alt = 2xLT instead of Apt = x2 for
a point focus. This leads to a further reduction of the effective trap stiffnesses eff by A = Apt / Alt = 1/15. In
addition, the focal width in scan direction is given by the intensity modulation L of the laser thus further
decreasing eff by x / L = 0.18. Taken together:
Phenomenologically, when using the calibration method described above without the correction factors qy,
qz, the resulting cell diameters D’ = 2c’y ≈ 2c’z ≈ 60nm were found to be much smaller than the expected value
60
D = (380 ± 70) nm as obtained by Trachtenberg {Trachtenberg 2014} by thorough analysis of darkfield
measurements. The distribution of D is very broad (see Fig. 36C, grey Gaussian), however, this cannot explain
such a strong deviation.
52
3. Spiroplasma melliferum
To check the validity of the calibration method, a second way of measuring the cell’s diameter can be
carried out as illustrated in Fig. 33A. The cell is rapidly scanned meanderlike by the laser which is equal to
consecutive calibration scans (as shown in the last chapter) in y direction, each slightly displaced in x
direction. The characteristic points of the sinusoidallike (detector response) curve Sy(yL) of individual scans
follow the helical geometry of the cell, i.e., a sinus in the projection to the xyplane (Fig. 33A+B). The
characteristic points are the position of the minimum (minpos), the zero crossing point (x0pos) and the
maximum’s position (maxpos).
This second method (M2) has been tested by simulations of the interferometric QPDsignal (Fig. 33B and
appendix 6.2) and experimentally (Fig. 33C), which is tricky to perform and described in the following. To
suppress Brownian motion, the cell is trapped by a line trap while scanned meanderlike by a second,
1
independent focus as illustrated in Fig. 33A. The meander scan is carried out consecutively several (N ≈ 100)
times over a distance of xm = 3µm in x direction and with a spacing of x,m = 100nm. Individual meander scans
2
A B 2.0 C 6.0
M1
5.5
1.5 M2 20 60
yL(t) (µm)
yL(t) (µm)
Sy (a.u.)
10
Sy (mV)
5.0 40
1.0 0 20
10 4.5 0
y 0.5 20
20
4.0 40
x
0.0 3.5
0 1 2 3 6 7 8 9 10 11 12 13 14 15
xL(t) (µm) xL(t) (µm)
Fig. 33. Meander scans. (A) Illustration of a meander scan (dashed line) which
presents a second method (method 2) of determining the cell diameter D.
The cell is caught by a line trap (red ellipse) while the meander scan is
performed. (B) Simulation of the QPD ysignal (Sy) of a meander scan as
shown in A. (C) Experimental realization of a meander scan as illustrated in A.
The cell is repeatedly scanned in x direction over a distance of xm=3µm
(individual scans are separated by black/white dashed lines) with a
discretization of x,m=100nm.
For every line minpos(xL), x0pos(xL) and maxpos(xL) are determined from Sy(yL, xL) as shown in Fig. 34A.
Again, individual meander scans are separated by black dashed lines. As it can be expected, all three curves are
nearly identical, thus only one needs to be analyzed in detail. To estimate the helix’s diameter D, every scan is
fit by a sine function. The result is shown for maxpos(xL) of three successive scans in Fig. 34B (the individual
offsets have already been subtracted). The first two fits result in nice sinusoidal curves whereas the third does
not. This is mainly due to Brownian motion of the cell, which is reduced by the line trap but not completely
eliminated. Bad fits can be identified by the spatial frequency k of the sinus, which is also a fit parameter (Fig.
34D). In order to select only proper fits, a power spectral density (PSD) of maxpos(xL) is calculated as shown in
Fig. 34C. Here, the spatial frequency kH = 1/p = 0.94 ± 0.17µm1 (Fig. 34C, grey box) of the cell can be clearly
identified. This interval is used to filter the fit results (Fig. 34D, grey box). The cell diameters D’y = 2Afit (Afit is
the amplitude of the fit) of the remaining fit results in shown in Fig. 34E together with the mean diameter
D’mean = (95.5 ± 4.5)nm. Again, this result is much smaller than the expect value which manifests a systematic
error.
1
Both beams are generated by using the first and zeroed diffraction order of an AOD. The angle of the first order (and thus the
displacement in the focal plane) can be modulated by the AOD and is used to generate the line trap. The zeroed order has a fixed angle.
Here, the displacement in the focal plane is done by scan mirrors. The setup is shown in Appendix 0.
2
Individual lines in y direction were performed with a frequency f = 400Hz over a distance of ym = 10µm. This results in a repetition rate
rm = 2f / (xm / x,m) = 27Hz of the meander scan.
53
3. Spiroplasma melliferum
A 5.4
B
minpos
5.2
yL(t) (µm)
100
yL(t) (µm)
5.0 50
x0pos
0
4.8 50
maxpos 100
4.6
6 8 10 12 14 6 8 10 12 14
xL(t) (µm) xL(t) (µm)
C Helix D E
1.5
PSD(maxpos) (a.u.)
4 Meander 120
2
scan
1.0
k (1/µm)
D'y (nm)
1 80
6 0.5
4
40 D'Mean = (95.5 4.5)nm
2 0.0
0.1 0
2 3 4 5 6 7 8 2
1 0 10 20 30 40 50 60 0 2 4 6 8
k (1/µm) scan no. no.
Fig. 34. Experimental result of method 2. (A) Positions of the minima
(minpos), zero crossing point (x0pos, Sy=0) and maximum (maxpos) of Sy(xL,
yL(t)) for every line at xL. Individual meander scans are separated by black
dashed lines. (B) Sinus fits to maxpos (blue lines, also shown in Fig. 33A). The
constant offset for every scan has been subtracted. (C) Power spectral
density (PSD) of maxpos clearly showing the spatial frequency km = 0.33µm1
of the meander scan and kH = (0.94±0.17)µm1 of the helix. (D) Frequencies
from fits to individual meander scans and allowed frequency band (grey box)
from the PSD. (E) Resulting apparent diameters D’y after filtering the fit
results by the allowed frequency band. Error bars are the standard deviation
of a single value obtained by averaging. The mean value D’mean and its
uncertainty is indicated by the red box.
Using the pearl model again, this systematic error can be explained. Due to the physical dimensions of the
cell dt = 190nm and p = 900nm compared to the focal widths x = y ≈ 500nm and z ≈ 1300nm three
problems arise:
1) Every data point of the recorded signal Sy(xL), Sz(xL) does not represent the displacement c(xL) of a
single pearl at position xL to the focus center rL but the average of the displacements of several pearls
inside the focal volume (see Fig. 35A).
1
2) Every pearl at location c(xL) is exposed to a different intensity . As a result, every pearl has a different
weight during this averaging (see Fig. 35A+B).
3) Since the focal widths y, z are different in lateral and axial direction, pearls at the same distance to
the focus center have different weights in different directions (see Fig. 35B).
1
This effect is not incorporated by calibrating with the total detector response as done here, since the characteristic sinusoidal slope of
that function is purely characterized by the phase dependence between scattered and unscattered light as explained in chapter 2.3.
Within the linear detection region, this effect is negligible because the intensity can be assumed to be constant. Here, depending on the
cell diameter D, individual pearls can be far out of this zone. Due to the smooth intensity distribution in z direction, a pearl at cx = xL and
cy = 0 (> cz = D) is exposed to the maximum focal intensity I0. This is not the case for a pearl at cx = xL but cz = 0 (> cy = D), which is
exposed to a reduced intensity I < I0 due to step intensity gradient in y direction. See Fig. 35B.
54
3. Spiroplasma melliferum
Iy
A Ix B
z
y
x cy=D/2
x z y
z
p
cz=D/2
xL Iz
x
y
dt
S y ( xL ) z
S (x ) gy
c y ( xL ) q y y L
gy dh=D+dt
Fig. 35. Signal averaging. (A) Due to the lateral extend x of the focus, the
positions of all pearls inside the focus are averaged to one center position
Sy(xL)/gy instead of the true position cy(xL) of the pearl at position xL. (B)
Different focal widths y and z and therewith different intensities Iy(cy),
Iz(cz) result in a different weight for pearls with the same displacement cy =
cz from the focus center.
As a consequence, the measured cell body amplitudes c’y(xL) and c’z(xL), calibrated with a calibration scan
or determined by a meander scan, are smaller than the true amplitudes cy(xL) and cz(xL). The factors qy = cy/c’y
and qz = cz/c’z needed to scale the measured amplitudes and get a correct result are determined by simulations
of the interferometric scattering signal (see appendix 6.2). Here, the QPD signals of a meander scan as
illustrated in Fig. 33A were simulated for actual diameters 40nm ≤ D ≤ 600nm and pitches 400nm ≤ p ≤
2200nm with discretization D = 20nm and p = 100nm. The resulting, apparent diameters D’ are shown for
both methods in Fig. 36A+B. The dependency of the measured, apparent diameter D’ on the geometry of the
helix becomes evident. For large pitches p > 1000nm, all pearls inside the focal volume have nearly the same
displacement to the focus in directions lateral to the long axis of the cell; therefore, averaging has a minor
influence on the measured diameter. The simulations also show the limits of the scanning technique presented
here: for pitches p < x = y, the diameter of the cell cannot be measured at all .
1
Fig. 36C shows the dependency of the apparent diameter D’y to the actual diameter D, as well as the
correction factor qy, for a pitch p = 900nm. By comparing the measured cell diameter D’ with the simulations,
the actual diameter D of the cell can be estimated. Further, the correction factor qy(D’) can be directly
determined. This is indicated exemplarily for the data of method 1 shown in the last chapter and method 2 for
the experiment shown above by the fine dotted lines (red and green, respectively) in Fig. 36C. Method one
resulted in an apparent diameter D’1 = 57nm which, according to the simulations, is equivalent to qy = 4.0 and
D1 = 230nm. With method two, the apparent diameter was D’2 = 95nm and thus, qy = 3.4 and D2 = 330nm.
144
The grey Gaussian in Fig. 36C shows a fit to measurements of the cell diameter presented in {Trachtenberg
2001}. This distribution has a mean cell diameter D = (360 ± 90)nm. Comparing D1 and D2 to this distribution,
both values are within 1.0 and 1.4 standard deviations D=90nm. Thus, the results of both methods together
with the correction factors qy derived by simulations are in good agreement with values obtained by
complementary techniques.
1
Since the width x = 0.61/ NA depends directly on the wavelength , shorter wavelengths increase the spectrum of helical pitches that
can be measured.
55
3. Spiroplasma melliferum
A Method 1 B Method 2 C
600 600
pmax=2200nm pmax=2200nm
500 500
200 200
100 100
pmin=400nm
pmin=400nm
0 0
0 100 200 300 400 500 600 0 100 200 300 400 500 600
Actual diameter Dy (nm) Actual diameter Dy (nm)
Fig. 36. Simulation of QPD signals and apparent cell diameters. (A)+(B)
Resulting diameters D’ for method 1 and 2 applied to simulation data for cell
diameters 40nm ≤ D ≤ 600nm and pitches 400nm ≤ p ≤ 2200nm with a
discretization of D = 20nm and p = 100nm (for visualization, only p =
200nm is shown). (C) Actual diameter (solid lines) and calibration factor qy
(thick dashed lines) as a function of the apparent diameter obtained by
simulation for p = 900nm. Experimental results are shown by thin dashed
lines. A reference distribution of the cell diameter D obtained by a Gaussian
144
fit to data from {Trachtenberg 2001} is shown in grey.
The factor qz is determined by a theoretical consideration: according to the calibration method explained in
the last chapter, the detector sensitivity gz depends on the factor qy. In other words, the averaging over all
pearls inside the focus is already taken into account. The only thing left that has to be addressed are the
differing focal widths in y and zdirection. Thus, qz = y/z = 1/2.6 is assumed which results in the same
diameters Dy ≈ Dz ≈ 230nm in both directions.
The factor qz cannot be determined experimentally for both methods since an axial scanning of the focus is
1
not possible .
Since the next chapter concentrates on analyzing the kink behavior of Spiroplasma, the corresponding
signals are illustrated briefly in the following. The kymograph in Fig. 37A shows one kink cycle consisting of two
individual kinks. Kinks traverse the cell body from right to left with respect to time. This can be seen by the
strong white (positive amplitude) and black (negative amplitude) peaks of the signal. Arrows are placed beside
kinks as a guide to the eye. During analysis, the durations of kinks TK1 and TK2 as well as the interkink time Tik,
i.e., the time between both kinks, are estimated as illustrated in the figure (also see chapter 3.3).
A single line scan during the kink cycle is shown in Fig. 37B. The first and second kink separated by
approximately 1.3µm can be seen. The amplitude difference of ~1700nm is much larger than the unique
detection region (UDR, see chapter 3.2.2 and Fig. 29). This is founded in the correction factor qy ≈ 4.0 and the
signal averaging of several pearls inside the focus as explained in the last chapter. Positions cy of pearls outside
the UDR are averaged with those inside leading to a position signal S(cy(xL)) smaller than that corresponding to
the true position cy(xL) of the cell at the position xL of the laser focus. The correction by qy reveals the true
amplitude. While this fact seemed to be a drawback of the technique in the last chapter, it is the key why kink
amplitudes can be imaged uniquely. However, since pearls of a kink overlap tremendously in directions lateral
to the scan axis, information about the helicity gets lost during averaging.
1
In principle, spatial light modulators, tunable optical lenses or fast piezo actuated lenses could be used to create focal shifts in the image
plane. However, none of these tools is available at one of the setups used here.
56
3. Spiroplasma melliferum
For comparison, the corrected positions cy(xL) and cz(xL) of a unkinked cell are shown in Fig. 37C. The
shortening (~900nm) of the cell length Lc projected on the scan axis (xaxis) during kink propagation is clearly
visible. Axes and markers of both Figs. are true to scale.
xL (µm) 1000
A B
2 4 6 8 10
5.55
cy(xL) (µm)
5.60 1 500
1st kink
Amplitude (nm)
5.65 0
5.70 1
0 1st kink
5.75 2nd kink
t (s)
TK2 Tik
5.80
TK1
5.85 500
C
200
0
200 cy(xL)
cz(xL)
3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0
xL(t) (µm)
Fig. 37. Signal of a kinked cell. (A) Kymograph of a whole kink cycle consisting
of two individual kinks highlighted by black and white arrows aside kinks as a
guide to the eye. (B) Single line scan as indicated by the blue line in A. Axes
and markers true to scale. (C) Signals of an unkinked cell (same as Fig. 27D).
Axes and markers are true to scale.
In this chapter, it has been shown how the center line c of a helical bacterium can be tracked and imaged in
3D at high frame rates. The interferometric position signal S(c(xL)) from a QPD was mapped to the current
position xL of an oscillating laser focus which was used to scan the cell. Due to a modulation of the laser
intensity (which is necessary to form a stable, harmonic trap), the subtraction of an empty scan as background
signal was necessary. A calibration method to obtain the true position c from S(c) was shown and an
important systematic error was presented. This systematic error was explained theoretically and it has been
shown by a combination of simulations and theoretical considerations how it can be compensated. Since the
complete measurement and calibration is complex, a short summary, which can be seen as recipe, is given in
the following:
Record QPD data of the cell in a line trap.
Record QPD data of a scan perpendicular to the axis of the cell for a dead cell > calibration scan.
Record empty scan of both directions.
Subtract empty scan from actual data.
Use calibration scan as lookup table to scale the measured ysignal Sy of the cell.
Get the apparent mean cell diameter D’y.
Get the correction factor qy(D’y) from the simulation data (compare Fig. 36C).
Calibrate the detector using eqs. (3.14)  (3.17) to get gz.
Scale Sz by gz.
57
3. Spiroplasma melliferum
For the ease of comparison, graphs of identical quantities are colorcoded for different experiments.
As mentioned in the motivation of this work, an analysis of freeswimming Spiroplasma cells is difficult
because their locomotive pattern is threedimensional and they usually move quickly out of the microscope’s
59
plane of focus. Shaevitz et al. {Shaevitz 2005} analyzed their swimming pattern by pressing cells between two
coverslips, thus creating a thin probe chamber. They verified that this configuration reproduces statistics of the
freeswimming cell under nonconstricted circumstances but an impact of the chamber cannot be completely
1
excluded .
However, using DIC microscopy, they found a mean velocity of individual kinks of vK ≈ 10µm/s at a mean
cell length of Lc = 4.4µm. This results in a time Tk = 440ms a single kink needs to propagate through the entire
cell. Furthermore, they found a mean time between kinks within a cycle of Tik = (260 ± 70) ms (ik = inter kink).
These values can be used as a reference for data obtained from trapped cells since the trap itself has an
impact on the swimming behavior.
The imaging method by linescanning optical tweezers as explained in chapter 3.2 can be used for precise
temporal analysis of kink statistics over a long time. The duration of whole cycles as well as of individual kinks
of a cycle, and therewith the kink velocity, can be measured. Regarding the kymographs shown in chapter
3.2.4, this can either be done by determining the start and end of a kink/cycle (in the following referred to as
2
method 1) or by determining the slope of a kink to obtain the kink velocity directly (method 2) . To get a
measure for the significance of the results, errors are estimated for both methods. In the first case, the
accuracy for estimating start and end of a kink/cycle by eye in a kymograph is used as measure. In the second
case, different lines to fit the slope of a kink were painted onto a kymograph to get a maximum and minimum
of the slope. From this, the mean slope with deviation was determined. If not otherwise stated, method 1 has
been used in the following.
The result of such an analysis is shown in Fig. 38. The duration Tcycle of whole cycles has been determined
by method 1 whereas the velocities vK1 and vK2 of single kinks within a cycle were determined by method 2.
Every parameter f was fitted by a linear function f(t) = f(t)t + f0 to see if f changes with respect to time.
3
In the experiment shown here, the cell died after approx. 40s due to the formation of reactive oxygen
species ROS (see Appendix 6.5). Interestingly, Tcycle increases significantly by Tcycle = (3.2 ± 0.4) ms/s with
time. Tcycle depends on the velocity vK1 and vK2 of the kinks within a cycle and the time between Tik (ik =
interkink) both kinks. A closer look at these parameters reveals that only the kink velocities vK1 and vK2 change
1
Personal communication.
2
Method 1 is the preferred way to analyze kink data because start and end of kinks are clearly defined. However, sometimes start and
ending of a kink cannot be identified clearly and method 2 is applied.
3
The choice of a linear dependence is based on observation.
58
3. Spiroplasma melliferum
with respect to time. The changes vK1 = (0.25 ± 0.02) (µm/s)/s and vK2 =  (0.16 ± 0.02) (µm/s)/s differ
significantly. This might also be addressed to a systematical error during estimation of the kink velocity since
the second kink within a cycle is often more difficult to identify and analyze than the first one. Connected to
this problem, the most problematic parameter to estimate is Tik which cannot be determined properly for each
cycle. This is why the number of data points differs in Fig. 38 A and B. In contrast to Tcycle, Tik = (52 ± 79)
µs/s does not indicate a significant change of Tik. Last, the number of cycles per second Ncycles/s can be
regarded. This number is obtained by counting all cycles nc(t) during a time frame of T = 5s, where nc(t) is the
number of complete cylces that were observed during the time t.
nc ( t T / 2)
N cycles
(t ) 1 1 tT (3.21)
s T nc (t T/ 2) 2
Similar to vK1 and vK2, this frequency also strongly decreases with respect to time. Thus, the molecular
motor driving single kinks does not only become slower, it also initiates kinks less frequently. This might be not
surprising since humans or animals, when getting old, also cannot walk that fast anymore and carry out single
steps less frequent.
A 500 B 80
450 70
400 60
Tcycle (ms)
Tik (ms)
350 50
300 40
250 Tcycle = (3.2 ± 0.4) ms/s 30 Tik = (52 ± 79) µs/s
²/DOF = 3.44
200 20
0 5 10 15 20 25 30 35 40 0 5 10 15 20 25 30 35 40
t (s) t (s)
C 40 D 40
vk1 = (0.25 ± 0.02) (µm/s)/s vk2 = (0.16 ± 0.02) (µm/s)/s
35 ²/DOF = 2.56 35 ²/DOF = 2.04
30 30
vk1 (µm/s)
vk2 (µm/s)
25 25
20 20
15 15
10 10
0 5 10 15 20 25 30 35 40 0 5 10 15 20 25 30 35 40
t (s) t (s)
E 5
4
Ncycles/s (Hz)
0 5 10 15 20 25 30 35 40
t (s)
Fig. 38. Kink analysis of a dying cell. (A) Time of overall kink cycles Tcycle. (B)
Interkink time Tik, i.e., time between kinks of a cycle. (C) Velocity vK1 of first
kink. (D) Velocity vK2 of second kink. (E) Number Ncycle/s of cycles per second.
Another way of analyzing the temporal motor characteristics is a timeresolved power spectral density
(PSD) of the energy Wopt(t) the cell exerts against the optical trap. Wopt is obtained after calibration with the
trap stiffness y, z and the true slope position ay(xL) = qy Sy(xL) / gy, az(xL) = qz Sz(xL) / gz for every line scan:
59
3. Spiroplasma melliferum
RE ( t )
RE (t ) LE (t )
Wopt 1
Np
xL LE ( t )
y a y2 ( xL ) z az2 ( xL ) Np
xLT
(3.22)
LE(t) and RE(t) were the beginning (LE = left edge) and end (RE = right edge) of the cell obtained by an
edge detection algorithm applied to a kymograph (see Fig. 27). xLT = 96nm (typically) is the discretization of
the line scan. The latter has to be taken into account since y and z are the effective force constants of the
center of mass position b. The result is shown in Fig. 39C+D for a cell before (red) and after (blue) its death due
to laser irradiation. Wopt ≈ 10kBT (blue line in Fig. 39D) for the dead cell is due to Brownian motion in all
directions (compare to chapter 3.1.3). The active cell can raise additional ≈ 50kBT against optical forces and
internal strain.
2
B 10
Sz
8
xL (µm)
2
C T=200ms T=200ms T=240ms
75
living cell
dead cell
50
W (kBT)
25
0
4.0 4.1 4.2 4.3 4.4 4.5 4.6 4.7 4.8 4.9 5.0
t (s)
D
100
living cell
80 dead cell
W (kBT)
60
40
20
0
0 1 2 3 4 5 6 7 8 9 10
t (s)
Fig. 39. Energy raised by the cell against the optical trap. (A) + (B)
Kymographs of signals Sy and Sz showing four kink cycles (purple) in different
projections. (C) Energy against optical trap corresponding to cycles shown in
A and B. (D) Optical energy for a long time frame.
The time resolved power spectral density PSD( , t, Twin) of Wopt is obtained by subsequently calculating
PSD Wopt (t ')  Wopt ( , t ) 2 for a time window t ≤ t’ ≤ t + Twin which is shifted by Twin. Typically, Twin = 6s
and Twin = 0.1s is chosen.
PSD( , t , Twin , Twin ) PSDt ,Twin , Twin Wopt (t ') FTt ,Twin , Twin Wopt (t ')
2
(3.23)
60
3. Spiroplasma melliferum
105
100
95
t=2.85s t=2.90s t=2.95s t=3.00s
.90s t=2.95s t=2.85s
t=3.00s90 Noise limit
t=2.90s
t=3.05s t=2.95s t=3.00s t=3.05s
85
45
≈
40
Low 1.3 3.0 6.5
PSD(W) (a.u.)
0.05
35 deformation 1.3
2.0
30 Strong 3.0
PSD (Hz)
25 deformation 4.5
20 6.5
15
10
5
0
0
10
20
30
40
50
60
70
80
90
100
110
120
130
140
150
t (s)
Fig. 40. Time resolved power spectral density plot with amplitudes in
arbitrary units (a.u.).
The result is plotted in Fig. 40. The amplitudes (black/white and colored isolines) are given in arbitrary units
(a.u.) since no quantitative analysis is performed. To recall the situation: the data represent a cell during death
struggle. Strong peaks at PSD ≥ 6.5a.u. only occur at low frequencies f < 5 Hz corresponding to the frequency
of kink cycles Ncycles/s. Remarkably, all isolines shift to lower frequencies with respect to time, except for PSD ≤
0.05a.u. which represents the background noise.
A B C D
59.2s
59.3s
39.9s
40.1s
40.3s
80.5s
59.4s
59.5s
80.0s
59.6s
80.3s
40.2s
80.4s
39.8s
80.1s
5.5s
5.2s
40.0s
80.2s
5.6s
5.7s
59.7s
5.3s
5.4s
x x x x
y y y y
E F G H
10 10 10 10
Sy Sy Sy Sy
8 8 8 8
xL (µm)
xL (µm)
xL (µm)
xL (µm)
6 6 6 6
4 4 4 4
2 2 2 2
5.5
5.6
5.7
5.2
5.3
5.4
40.1
40.2
40.3
59.5
59.6
59.7
80.3
80.4
80.5
39.8
39.9
40.0
59.2
59.3
59.4
80.0
80.1
80.2
W (kBT)
W (kBT)
W (kBT)
40 40 40 40
20 20 20 20
0 0 0 0
40.2
40.3
59.6
59.7
80.4
80.5
39.8
39.9
40.0
40.1
59.2
59.3
59.4
59.5
80.0
80.1
80.2
80.3
5.6
5.7
5.2
5.3
5.4
5.5
M
t=2.85s t=2.90s t=2.95s t=3.00s 45
t=3.05s
40
Low 1.5
40
1.5
PSD(W) (a.u.)
8
2.0 35
deformation 6
3.0
30 5.0 4 30
PSD (Hz)
25
0
20 deformation 20
15
10 10
0 0
0
10
20
30
40
50
60
70
80
90
100
110
120
130
140
150
t (s)
Fig. 41. Complementary information of a dying cell. (A)(D) Different time
frames showing successive images of a trapped cell captured by brightfield
microscopy. (E)(H) Corresponding kymographs of lateral signal Sy(xL, t). (I)
(L) Corresponding optical energy Wopt. (M) Kymograph of PSD(, t).
61
3. Spiroplasma melliferum
To better interpret these findings and for comparison to other data, Fig. 41 shows brightfield images,
kymographs, Wopt and PSD(, t) for the same measurement. At the start of the measurement (t < 10 s), a lot of
consecutive cycles can be seen corresponding to PSD > 6.5a.u. These amplitudes vanish after t ≈ 40s at the
same time when no cycles can be seen in the brightfield images anymore and one might define the cell being
dead when examined in a conventional microscope. For t > 40s there are still (until t ≈ 70s) some pseudo kinks
visible in kymographs, i.e., kinks that start but do not propagate along the entire cell (Fig. 41 G). These pseudo
kinks can be linked to PSD ≈ 3a.u. and can be hardly identified in brightfield images. For t > 70s some small
tilts hardly visible in the kymograph are still present that might be addressed to Brownian motion and rotation.
However, at this time, the amplitude of the PSD is still above the background (PSD = 0.05a.u., red line in Fig.
40) and decreases with time indicating that the cell might not already be dead in a sense that there is only
passive motion (diffusion) left.
The production of ROS, their impact on the molecular machinery driving kinks and finally the rapid cell
death impair linescanning optical tweezers as a tool for long term studies of single cells under different
experimental conditions. The production of ROS is a wide spread problem in biosciences with the maybe most
prominent example of rapid photo bleaching during fluorescence experiments. ROS can be scavenged by
1
oxygen scavenging chemicals (see Appendix 6.5). In rare cases simple scavengers such as DTT already work
quite well.
A 700
Tcycle = (2.0 ± 0.1) ms/s
600 Tcycle = (0.08 ± 0.06) ms/s
²/DOF = 2.08
²/DOF = 1.76
Tcycle (ms)
500
400
300 320 340 360 380 400 420 440 460 480 500 520 540 560 580 600
t (s)
B 4
3
Ncycles/s (Hz)
300 320 340 360 380 400 420 440 460 480 500 520 540 560 580 600
t (s)
C 40
PSD(W) (a.u.)
35 0.5
1.0
30 2.0
3.0
25
PSD (Hz)
4.0
20
15
10
5
0
300 320 340 360 380 400 420 440 460 480 500 520 540 560 580 600
t (s)
Fig. 42. Influence of DTT as oxygen scavenger. (A) Duration of whole cycles.
(B) Frequency of cycles. (C) Time resolved PSD. As a guide to the eye, the
dashed white line indicates the maximum frequency at PSD() = 0.5a.u.
1
Dithiothreitol or Cleland's reagent.
62
3. Spiroplasma melliferum
An experiment using DTT is shown in Fig. 42. The cell was kept alive for roughly ten minutes. Regarding kink
statistics such as Tcylce, the motor seems not to be influenced during the first 400s of the experiment. The
change of Tcycle, Tcycle(300s ≤ t ≤ 400s) = (0.08 ± 0.06)ms/s does not differ significantly from zero. For t > 400s,
two periods, each lasting ≈ 100s, were observed resembling the behavior of a dying cell shown in the last
chapter, indicating that DTT loses its scavenging capabilities. Remarkably, the cell somehow seems to recover
at t ≈ 490s since Tcycle drops to its initial value Tcycle(t < 400s) ≈ 350ms. Regarding the frequency Ncycles/s of kink
cycles, at the time when Tcycle starts to increase the first time (t ≈ 420s) Ncycles/s starts to decrease
monotonically without recovering until the cell does not kink anymore (t ≈ 600s).
The timeresolved PSD plot draws a similar picture but it is different in detail. Here, amplitudes/isolines
undergo a stepwise behavior. Isolines are constant, but can drop abruptly (within ≈ 15s) drop to another level.
The times corresponding to these drops (t ≈ 405s and t ≈ 525s) do not coincide with the characteristic drops of
Tcycle or Ncycles/s.
Hist(Tcycle) Hist(Tik)
A 0 20 40 60 80 B 0 20 40 60
300 200
<Tcycle> = (200 ± 1) ms <Tik> = (101 ± 2) ms
Tcycle = (29 ± 2) ms Tik = (36 ± 4) ms
²/DOF = 1.75 160 ²/DOF = 1.55
250
Tcycle (ms)
Tik (ms)
120
200
80
150
40
TK2 (ms)
100 100
80 80
60 60
vK2 (µm/s)
50 50
40 40
30 30
0.5
PSD (Hz)
1.0
2 20 2.0
3.5
5.0
1 10
0 0
0 20 40 60 80 100 120 140 0 20 40 60 80 100 120 140
t (s) t (s)
Fig. 43. Influence of GOC as oxygen scavenger and distribution (Hist) of
quantities describing kinks. (A) Duration Tcycle of whole cycles. (B) Time Tik
between kinks of a cycle. (C)+(D) Duration TK1 and TK2 of first and second
kink within each cycle. (E)+(F) Velocity vK1 and vK2 of first and second kink
within each cycle. (G) Frequency Ncycles/s of cycles per second. (H) Time
resolved PSD.
63
3. Spiroplasma melliferum
DTT was found to have a negative impact on the interferometric signals. Therefore, a different oxygen
scavenger has been tested. Glucose oxidase and catalase (GOC) were mixed similar to the prominent
scavenger called GoCat (see Appendix 6.5) used for fluorescence experiments. Using GOC, cells were found to
survive more than 10min and no observable impact on the signals. Therefore, GOC seems to be superior to
DTT. Kink statistics for GOCtreated cells are shown in Fig. 43. All quantities stay constant in mean which is also
reflected by the histograms (Hist) and Gaussian fits with acceptable reduced chisquares.
An elegant way of directly manipulating the molecular motor showed to be the linetrap itself. The trapping
laser power, and thus the optical force acting on the cell, was changed reversibly in a stepwise manner from a
base level of Pmin = 150mW to Pmax = 450mW and back in 100mW steps every 10s. To check the reproducibility,
this was repeated at least two times for each cell. Cells were treated with the GOC scavenger to prevent
effects of ROS.
t (s) B t (s)
A
105
115
125
105
115
125
15
25
35
45
55
65
75
85
95
15
25
35
45
55
65
75
85
95
5
5
220 120
200 110
Tcycle (ms)
Tik (ms)
180 100
160 90
140 80
120 70
150
250
350
450
350
250
150
250
350
450
350
250
150
150
250
350
450
350
250
150
250
350
450
350
250
150
P (mW) P (mW)
t (s) t (s)
C D
105
115
125
105
115
125
15
25
35
45
55
65
75
85
95
15
25
35
45
55
65
75
85
95
5
100 50
TK1, TK2 (ms)
45
vK1, vK2 (µm/s)
90
40
80
35
70
30
150
250
350
450
350
250
150
250
350
450
350
250
150
150
250
350
450
350
250
150
250
350
450
350
250
150
P (mW) P (mW)
t (s) t (s)
E F
105
115
125
105
115
125
15
25
35
45
55
65
75
85
95
15
25
35
45
55
65
75
85
95
5
4.4 40
PSD(Wopt) (a.u.)
Ncycles/s (Hz)
30 0.25
PSD (Hz)
4.0 0.5
1.0
3.6 20 2.0
4.0
3.2 10
0
150
250
350
450
350
250
150
250
350
450
350
250
150
150
250
350
450
350
250
150
250
350
450
350
250
150
P (mw) P (mW)
Fig. 44. Kink statistics during power ramp experiment. (A) Duration Tcycle of
kink cycles. (B) Time Tik between two kinks within a cycle. (C) Duration TK1
and TK2 of first and second kink within a cycle. (D) Velocities vK1 and vK2 of
kinks within each cycle. (E) Frequency Ncylces/s of cycles per time. (F) PSD plot.
Different gray levels indicate different laser powers.
64
3. Spiroplasma melliferum
Analysis of the kink times and velocities is shown in Fig. 44. The duration of whole cycles Tcycle ≈ Tik + TK2 as
well as the duration TK1, TK2 of individual kinks within cycles decreases with increasing laser power. The
reduced kink times TK1, TK2 for higher laser powers directly result in a higher kink velocity vK1 and vK2.
Interestingly and in contrast to all other experiments shown so far, also the time Tik between two kinks within
a cycle decreases with increasing laser power. When kinks propagate faster down the length of the cell body
and, in addition, the time between kinks decreases, the kink frequency Ncycles/s can increase theoretically.
Indeed, this behavior is observed as shown in Fig. 44E. The fact that Ncycles/s is significantly larger during the
second ramp could not be observed in every other experiment.
The PSD plot shows a smooth transition of all isolines to higher frequencies with increasing laser power.
This is much more pronounced during the first power ramp than during the second. However, the behavior can
be addressed to the fact that every process of the cell seems to be faster with increased laser power as
described in detail above.
A remarkable behavior shall be pointed out here: all the changes of Tcycle, Tik, TK1, TK2, vK1, vK2 and Ncycles/s
mentioned above are reversible and can be directly changed by the incident laser power instantly. In other
words, the bacterial behavior can be directly steered and manipulated by the linescanning optical tweezers,
thus rendering it a unique and special tool for biophysical experiments. The principle shown here might also be
useful for experiments with other types of traps such as point traps (depending on the type and shape of the
object under investigation).
Besides the direct, optical manipulation of the cell, it can also be manipulated by drugs and chemicals.
165
Sodium azide (NaN3) is known to inhibit or impair the cytochrome oxidase complex {Stannard 1948}
166
involved in the ATP synthesis complex {Haddock 1977}. The treatment with NaN3 is therefore a good
complementary experiment to the induced cell death, since again the production of ATP is influenced which
should result in the same principle behavior (increase of Tcycle and decrease of vk upon addition of NaN3).
1
The drug was applied by a pipet and a nanoliter syringe pump as it is used in patch clamp experiments
167
{Sakmann 1976}. The pipet with a diameter of ~100µm was placed close to the field of view of the camera
chip (~100µm – 200µm away from the trap) and remained in the probe the whole time. Initially, the cell was
trapped for a while (~50s) before the application of the drug was started. In brightfield pictures, the moment
2
when the NaN3 flow reached the cell could be clearly identified since it was nearly flushed out of the trap. This
short time period (~10s) is indicated by the transparent grey boxes in Fig. 45. All parameters extracted from a
kymograph were fitted by a linear function to the data points before and after the addition of NaN3 to see if
there is any temporal change of each parameter similar to Fig. 38 in chapter 3.3.2. However, none of the
parameters changes significantly with time, neither before nor after the drug was applied. Therefore, the
mean value of data points before and after the addition is estimated to see changes caused by NaN3. GOC was
used to suppress the effect of ROS.
After drug application, Tcylce (Fig. 45A) increases significantly in a stepwise manner by T = (87 ± 8)ms.
However, since the number of data points is low especially after the addition of NaN3, the relatively small
standard deviation of the mean might not be statistically relevant. The standard deviation of a single data
point has the same principal statistical problem; however, it is much larger (by the factor N if N is the
number of data points) and can be used as a more coarse measure of significance. Here, the standard
deviations are Tcycle = 31ms and Tcycle = 29ms of data points before and after the addition, respectively. Even
if both standard deviations are summed up, the result is smaller than the jump T = 87ms of Tcycle indicating
that it is not just an artifact. For Tik, a step of T = (32 ± 8)ms can be identified, too, but compared to the
standard deviations Tik = 25ms and Tik = 33ms of a single data point, the significance is not as pronounced as
for Tcycle. The situation becomes more pronounced again when looking at single kinks: Tk1 (Fig. 45C) increases
1
Eppendorf CellTram vario. Smallest adjustable volume: 2nl. Volume release per turn: 960nl.
2
Experimentally, due the finite volume change that can be realized with the pump, a minima fluid flow of 510µm/s can be realized.
65
3. Spiroplasma melliferum
by T = (53 ± 8)ms with TK1 = 22ms and TK1 = 33ms, TK2 (Fig. 45D) by T = (55 ± 10)ms with TK2 = 35ms and
TK2 = 44ms, the velocity of the first kink vK1 (Fig. 45E) drops by v = (5.4 ± 0.8) µm/s with VK1 = 3.0µm/s and
vK1 = 3.1µm/s and vK2 (Fig. 45F) drops by v = (4.5 ± 0.9)µm/s with vK2 = 3.6µm/s and vK2 = 3.3µm/s.
Regarding the kink frequency Ncycles/s (Fig. 45G), a difference cannot be clearly identified. Although there
seems to be a drop to lower frequencies of isolines in the PSD plot (Fig. 45H) after the addition of NaN3, the
measurement time was too short to draw a definite conclusion.
Taken together, NaN3 has indeed the same effect as the production of ROS underlining that ROS affects
ATP production here. However, only a single dataset for this type of disturbance is present because the
application of a drug by the syringe pump is difficult (cells often get flushed out of the trap due to a high flow).
A B 350
Tcycle = (0.2 ± 0.8) ms/s Tik = (0.07 ± 0.4) ms/s Tik = (0.6 ± 0.8) ms/s
600 300
²/DOF = 0.7 ²/DOF = 0.5 ²/DOF = 0.9
Tcycle = (0.4 ± 0.4) ms/s 250
Tcycle (ms)
Tik (ms)
200
150
400
NaN3 100 NaN3
<Tcycle> = (402 ± 5) ms <Tcycle> = (489 ± 6) ms 50 <Tik> = (155 ± 4) ms <Tik> = (187 ± 7) ms
300
Tcycle = 31ms Tcycle = 29ms Tik = 25ms Tik = 33ms
0
0 10 20 30 40 50 60 70 80 90 0 10 20 30 40 50 60 70 80 90
t (s) t (s)
C D TK2 = (0.8 ± 0.8) ms/s
TK2 = (0.4 ± 0.4) ms/s
400 TK1 = (0.04 ± 0.4) ms/s TK1 = (0.8 ± 0.8) ms/s 400 ²/DOF = 1.6
²/DOF = 1.0
²/DOF = 0.4 ²/DOF = 1,0
TK2 (ms)
300
TK1 (ms)
300
NaN3
200 200
NaN3
<TK1> = (213 ± 3) ms <TK1> = (266 ± 7) ms <TK2> = (247 ± 5) ms <TK2> = (302 ± 9) ms
100 100
TK1 = 22ms TK1 = 36ms TK2 = 35ms TK2 = 44ms
0 10 20 30 40 50 60 70 80 90 0 10 20 30 40 50 60 70 80 90
t (s) F t (s)
E
vK1 = (0.01 ± 0.6) (µm/s)/s vK1 = (0.10 ± 0.08) (µm/s)/s vK2 = (0.06 ± 0.04) (µm/s)/s vK2 = (0.01 ± 0.07) (µm/s)/s
40 ²/DOF = 0.6 ²/DOF = 0.7 40 ²/DOF = 0.6 ²/DOF = 0.9
NaN3
vK1 (µm/s)
vK2 (µm/s)
30 30
20 20
NaN3
<vK1> = (28.4 ± 0.4) µm/s <vK1> = (23.0 ± 0.7) µm/s <vK2> = (24.8 ± 0.5) µm/s <vK2> = (20.3 ± 0.7) µm/s
10 10
vK1 = 3.0µm/s vK1 = 3.1µm/s vK2 = 3.6µm/s vK2 = 3.3µm/s
0 10 20 30 40 50 60 70 80 90 0 10 20 30 40 50 60 70 80 90
G t (s) H t (s)
2.0 40
Tpl NaN3 Tpl
1.5 30
PSD(W) (a.u.)
Ncycles/s (Hz)
1.0
NaN3
PSD (Hz)
2.0
1.0 20 3.5
5.0
7.5
0.5 10
0.0 0
0 10 20 30 40 50 60 70 80 90 0 10 20 30 40 50 60 70 80 90
t (s) t (s)
Fig. 45. Kink statistics of sodium azide (NaN3) treated cells. (A) Duration Tcycle
of whole cycles. (B) Time Tik between kinks within a cycle. (C)+(D) Duration
TK1 and TK2 of first and second kink within a cycle. (E)+(F) Velocities vK1 and
vK2 of first and second kink in a cycle. (G) Frequency Ncycles/s of cycles per
time. Grey boxes mark the time when NaN3 is applied to the cell. Colored
transparent boxes indicate the mean values and standard deviation of a
single value. (H) PSD plot. The dark grey box marks when NaN3 is applied and
light grey boxes indicate the time Tpl = 6s used to calculate the PSD.
66
3. Spiroplasma melliferum
During dozens of experiments, different smaller observations and findings were made which are
summarized in the following. They all further characterize the molecular motor driving kinks.
525.0
530.0
535.0
540.0
Sy Sy Sy Sy Sy
520.5
525.5
530.5
535.5
540.5
521.0
526.0
531.0
536.0
541.0
521.5
526.5
531.5
536.5
541.5
522.0
527.0
532.0
537.0
542.0
522.5
527.5
532.5
537.5
542.5
t (s)
t (s)
t (s)
t (s)
t (s)
523.0
528.0
533.0
538.0
543.0
523.5
528.5
533.5
538.5
543.5
524.0
529.0
534.0
539.0
544.0
524.5
529.5
534.5
539.5
544.5
525.0
530.0
535.0
540.0
545.0
F 7
200
6
Tic (ms)
100
5
0
Tic (s)
4
3 100
310 315 320 325 330
2
1
0
300 320 340 360 380 400 420 440 460 480 500 520 540 560 580 600
G 4 t (s)
3
Ncycles/s (Hz)
300 320 340 360 380 400 420 440 460 480 500 520 540 560 580 600
t (s)
Fig. 46. Side observations. (A)(E) Kymographs of Sy of consecutive time
frames (5s) of the DTT experiment (compare chapter 3.3.2). Individual kink
cycles are marked in purple. (F) Time Tic between two consecutive cycles. (G)
Frequency Ncycles/s of cycles.
67
3. Spiroplasma melliferum
This can be best seen when approaching the death of a cell when Ncycles/s decreases and there are
sometimes periods lasting up to a couple of seconds without cycles. Fig. 46AE shows five kymographs of
consecutive 5s time intervals of the Sy signal during the DTT experiment as already shown in chapter 3.3.3.
Short periods without activity are most often followed by two or more cycles (purple) in series. Note that the
helical ground frequency can hardly be resolved in Fig. 46. This is an unsolved problem in some measurements.
However, kinks are clearly visible.
This can be seen especially for long cells. The time between two cycles Tic (ic = intercycle) as shown in Fig.
46F (inset) is then negative. In Fig. 46A, individual cycles are marked by purple boxes. Already the first two
boxes (520.5 ≤ t ≤ 521.5s) overlap indicating that the second cycle has started before the first has ended.
Regarding Fig. 46G, there is a clear oscillation of ~1/5s = 0.2Hz of Ncycles/s visible. This is the same in all
other experiments as shown in the other subchapters of chapter 3.3. The oscillation is also visible in PSD plots
for the highest amplitudes (purple isolines).
Partial kinks
Phenomenologically, shortly before cell death, i.e., when there are no complete cycles anymore, single
partial kinks that do not travel the whole length of the cell body were still observed. A closer look reveals that
these pseudo kinks sometimes also occur between complete cycles. This can be nicely seen in a kymograph of
the DTT experiment shown in Fig. 47, where three pseudo kinks (light blue boxes) are always followed again by
complete cycles (purple boxes).
10
Sy
8
xL (µm)
2
570.5 571.0 571.5 572.0 572.5 573.0 573.5 574.0 574.5 575.0 575.5
t(s)
Fig. 47. Pseudo kinks. Partial kinks (light blue) do not travel the whole length
of the cell body but are followed by complete cycles (purple) again.
During the NaN3 experiment as well as during power ramp experiments, abnormal behavior of single kinks
and cycles has been observed, although only in rare cases. Fig. 48 shows selected signals during power ramp
68
3. Spiroplasma melliferum
experiments. In Fig. 48 A, four cycles can be identified, where the individual kinks of the first two cycles travel
in opposite directions. If the first kink of a cycle reaches the cell’s end, it is reflected back. A behavior that has
59
been reported by {Shaevitz 2005}, too, but for cells with one end tethered to a surface. The kinks of the two
cycles following travel in the same direction, the commonly observed behavior. Interestingly, the time
between kinks Tik increases by a factor of two when kinks of a cycle travel in opposite directions. However,
since the second kink starts at the same end where the first kink ended, the time between the end of kink 1
and the start of kink 2 is in good agreement with Tik of normal cycles.
Fig. 48 B+C shows the Sy and Sz signal of a different cell. Here, individual kinks within a cycle travel in the
same direction but the kinks of consecutive cycles travel in opposite directions. Both observations are only
made at high laser power or when NaN3 is applied to the cell.
0 ≈0.28s ≈0.32s
0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0
t (s)
B C
Sz
10 Sy 10
≈0.10s ≈0.10s
xL (µm)
xL (µm)
5 5
≈0.08s ≈0.08s
5.00 5.10 5.20 5.30 5.40 5.00 5.10 5.20 5.30 5.40
t (s) t (s)
Fig. 48. Abnormal kink behavior during power ramp experiment. (A) Sy signal
at P = 300mW. (B) + (C) Sy and Sz signal at P = 450mW. Graphs A and B+C
show signals of different cells.
For short cells (N = 23 windings), the interkink time Tik > Lc / vK can be much longer than the time a single
kink needs to travel the cell body as shown in Fig. 49. This implies that the start of the second kink is triggered
by a time dependent process rather than by internal, mechanical strain of the cell that switched handedness.
10
TK1 TK2
xL (µm)
5
Tik
0
Fig. 49. Kink cycle of a short cell with N = 3 windings and Lc = 3.5µm.
69
3. Spiroplasma melliferum
Another interesting observation is the dependence of the interkink time Tik on the projected length Lc of
1
the cell. This is shown in Fig. 50 for 10 representative cells of different length Lc. A linear fit reveals a
significant decrease Tik = 16.5 ms/µm of Tik(Lc). The most likely explanation is a mechanical effect of the
optical trap. Optical forces exert a torque on the kinks which might facilitate kinking (see chapter 3.4 for a
more detailed discussion). Torque increases with cell length because they reach further out of the trap center
during kinking. In this context, Tik(Lc = 0) = (177 ± 10) ms can be regarded as the interkink time in absence of
the optical trap. Indeed, the value agrees within the standard deviation to the value Tik = (260 ± 70) ms
presented in chapter 3.3.1 for the freeswimming cell.
150
100
Tik (ms)
50
Tik(Lc = 0) = (177 ± 10) ms
Tik = (16.5 ± 1.7) ms/µm
0
3 4 5 6 7 8 9
Lc (µm)
1
Phenomenologically, a steplike behavior is possible, as well, but cannot be explained physically.
2
Shaevitz and Gitai {Shaevitz168 2010} mention the possibility that MreB helices are the mechanical consequence of forcing a linear
polymer into a cylindrical container. Since both cytoskeletal ribbons (MreB and Fib) align on a helical path along the cell membrane, this is
another indication that MreB might serve as a substrate for Fib.
70
3. Spiroplasma melliferum
A B C vk
Change of
handedness vc
D E
Another system closely related to the kinking behavior of Spiroplasma is the bacterial flagellum. E. Coli
cells, for example, possess several (typically 45) helical, filamentous appendages called flagella that align into
a bundle (see Fig. 52A). Each flagellum is driven by a rotary motor (see Fig. 52C+D) generating torque on the
attached filament. The bundle rotates and generates a propulsion force moving the cell forward. Individual
flagella leave the bundle from time to time and undergo a polymorphic transition between states (see Fig.
52A) of different handedness caused by a change of the rotational sense between clockwise (CW) and counter
clockwise (CCW). Related to this change of handedness is a kink similar to that in Spiroplasma. This mechanism
169
is used to tumble the cell and steer the swimming direction during chemotaxis. Vogel and Stark {Vogel 2010
170 171
and 2012 and 2013} explained the polymorphic transition theoretically by varying the torque exerted by
the motor. Individual flagella consist of eleven protein filaments arranged in a tubular symmetry (see Fig. 52B).
Each protein can adapt two different conformational states. If the motor torque is altered, twelve different
helical states can occur depending on the number of filaments in the same conformation. Their model purely
uses mechanical and hydrodynamic arguments on the scale of the whole filament and gets by without a
molecular description. Despite the elegance of this model, it relies on a torque generating motor which is
absent in Spiroplasma and thus this model cannot be applied.
71
3. Spiroplasma melliferum
A B C D
Fig. 52. Flagellar system of E.Coli. (A) Top: Flagella bundle during normal
swimming mode (left) which undergo polymorphic transitions during
172 173
tumbling (right). Adapted from {Bray 2004; Macnab 1977}. Bottom:
Possible polymorphic states (nR) depending on the conformational state of
169
the protofilaments of the flagellum. Adapted from {Vogel 2010}. (B) Left.
Crosssection of a flagellum showing four domains (D0D3) of each flagellin
subunit. Right: Sideview of a filament showing the helical stacking of
174
subunits. Adapted from {Speier 2011}. (C) Electronmicrograph of the
62
rotary motor. Adapted from {Berg 2000}. (D) Schematic composition of the
62
flagellum and its motor. Adapted from {Berg 2000}.
The subsequent switching of individual Fibproteins excludes a process based on purely random switching
of single filament subunits. Instead, it necessitates a cooperative switching of neighboring subunits, i.e., the
preference of switching if the nearest neighbor has switched already. Such a model has been proposed in the
175 176
1960s {Koshland 1966; Monod 1965} on the basis of allosteric regulation. Here, binding of a ligand
molecule to a subunit causes its conformational switch and conformations are coupled between different
subunits of an allosteric structure. However, this model has been also used to describe the rotational flagella
motor itself. In particular, it has been applied to the switching mechanism that steers the rotational sense of
the motor, i.e., CW and CCW. This complex consists of 34 identical proteins (FliM) assembled in a ring
structure. Individual proteins (a protomer) can, again, exist in two different conformations. Switching between
both is triggered by ligand (CheY) binding. Classically, the allosteric model of cooperative subunit switching has
been used to describe the overall switching of the ring in a binary manner, i.e., as a concerted switching of all
177
subunits at the same time. However, experiments at millisecond temporal resolution {Sowa 2005} revealed
a noninstantaneous switching between CW and CCW rotation as well as transient speed fluctuations of
178
flagella which cannot be explained by the classical model. To address this behavior, Bai et al. {Bai 2010} used
172 179
an extension of the classical allosteric model called conformational spread {Bray 2004; Duke 2001}. It
facilitates a conformational change of a protomer even without binding of a ligand depending on the
conformational state of the nearest neighbors. After determining the correct choice of the parameters of the
model (e.g., coupling strength, ligand concentration), they were able to qualitatively reproduce their
experimental data by simulations and found a very good agreement between the analysis of both.
72
3. Spiroplasma melliferum
vK1(t) and vK2(t) All parameters vK1(t) and vK2(t) vK1,K2(ILaser) ~ ILaser Kinks of same cycle
decrease. constant. decrease when can run in different
NaN3 is applied. Tik(ILaser) ~ 1/ILaser directions
Production of O2 O2 radicals scavan
radicals impairing ged → const. ATP NaN3 impairs ATP Ncycles/s(ILaser) ~ ILaser Kinks of consecutive
ATP production production production cycles can run in
All parameters different directions
Switching in Tik(t) shows weak depend on external,
both directions optical force Caused by high
dependence on NaN3 optical forces in
depends on ATP
concentration Switching power ramp exp.
depends on
Tik(t) = const. mechanical Escape strategy in
tension on unfavored
Both kinks are cytoskeleton conditions
correlated, i.e.,
have a fixed Kink velocity
time dependence Basic model for sequential higher compared to
subunit switching free swimming cells
Initiation of
second kink Kink velocity
does not depend depends on optical
on ATP. Extension to basic model force
incorporating tension on Tik(Lc) depends on
Ncycles/s(t) decreases the cytoskeleton length of cell Lc
Initiation of first Increased optical
kink depends on torque on long cells
ATP.
Cycles come in
series
Ncycles/s(t) is
periodic
Mean ATP
consumption >
production
The experimental observations characterizing the molecular motor of Spiroplasma are summarized in Fig.
53 together with possible explanations and conclusions regarding the motor. An analysis of this Fig. suggests a
basic model depending on ATP concentrations and an extension to incorporate an external, mechanical
influence. The relevance of the experimental findings and conclusions for the basic model are indicated by the
grey level of the background.
73
3. Spiroplasma melliferum
The theory of conformational spread seems to have a great potential to explain the molecular motor of
1 178
Spiroplasma . Based on the application to the flagella switch {Bai 2010}, it seems to be a promising
candidate to explain the sequential switching of subunits, ATPdependent kink velocities (ligand concentration
based switching rates) as well as partial kinks. Therefore, a theoretical model for the linear motor of
Spiroplasma based on the theory of conformational spread and its application to the bacterial flagella switch is
178
given in the following. As also mentioned in {Bai 2010}, a qualitative analysis of the model crucially depends
on the choice of input parameters (e.g., coupling energy, ligand concentration). To address this problem,
thorough simulations are necessary that have to be compared carefully to experimental observations. This is
not within the scope of this thesis. However, predictions for the parameters of the model are made which
should serve as a good basis for this purpose.
For simplicity, only one Fibfilament is considered in the following. A coupling between different filaments
is discussed at the end of this chapter.
Ecb
E JL Ecub
Free energy
kub↔b
kub↔b
E JL
ell
cir
Ecub E JL E JR
E JR Ecub
EL
Ecub
Ecb EL
E JR E b
c
EL
Switch
3 E EL
J
R
J EL
L
2 E J
EL
Ligand (ATP)
EL (kBT)
L b
1 E J E c
EL
0 State 1 (circular) E EL
J
R
J E b
c
EL
R
1 E J EL
State 2 (elliptical)
0.0 0.5 1.0 1.5 2.0
cL / c0.5 EL
Fig. 54. Schematic of the model. (A) Energy diagram of a single protomer.
Thick arrows denote the straightforward pathway for consecutive protomer
switching triggered by ligand binding. Bottom left: dependence of ligand
binding energy EL on ligand concentration. (B) Table summarizing all possible
states and corresponding energies of a protomer depending on the
conformation of nearest neighbors and presence of a bound ligand.
The basic assumptions of the model are (see Fig. 54 for assistance) as follows:
1) Each subunit (protomer) of the filament undergoes rapid transitions at a rate ,c between two
different conformations in thermodynamic equilibrium. These states are denoted ‘ell’ and ‘cir’ for
elliptical and circular in the following.
1
This has already been argued in {Koch162 2010}.
74
3. Spiroplasma melliferum
2) Conformational switching is regulated by binding of a ligand molecule at rate 0,lb. The ligand
concentration cL shifts the conformational equilibrium, i.e., the energy gap Ec between both
1
conformational states in the bound Ecb and unbound Ecub situation . In absence of conformational
change, EL(cL) = kBT ln(cL/c0.5) represents the concentration dependent ligand binding energy with
2
the ligand concentration c0.5 at Ec = 0 {Ma180 2012}. Since the experimental findings suggest an ATP
dependent switching model, the regulating ligand is likely ATP itself.
3) There is a nonvanishing interaction or coupling between adjacent protomers lowering their
combined energy if they are in the same state. To account for boundary conditions of the linear chain,
different coupling energies EJL > EJR for left and right neighbors are assigned.
The situation is depicted in Fig. 54A for conformational switching and ligand binding of an individual
protomer neglecting coupling. Based on this picture, Fig. 54B summarizes the total energy Gi of protomer i
including coupling, relative to the ground state set as all protomers in the circular state without bound ligands.
The ligand binding and unbinding rate in the circular and elliptical conformation are denoted by kubcir b and
ell
k , respectively
ub b
lb lb 1
c Ecb EL Ecb EL
kubcirb 0,lb e kBT kbcirub 0,lb e kBT
c0.5
lb lb 1
(3.24)
c Ecub EL Ecub EL
k ell
ub b 0,lb e kBT k ell
b ub 0,lb e kBT
c0.5
lb is an additional bias between forward and backward rates corresponding to ligand binding or unbinding.
For lb = ½, ligand binding is independent of conformation, i.e., kubcirb kubellb . Further, the rates of
conformational change depending on the presence of a bound (b) or unbound (ub) ligand are kubcir ell and
kbcir ell :
c c 1
Ecub Ecub
kubcir ell 0,c e kBT
kubell cir 0,c e kBT
c c 1
(3.25)
Ecb Ecub
kbcir ell 0,c e kB T
kbell cir 0,c e kB T
c is again an additional symmetry factor favoring either forward or backward rates of conformational
switching. Since c = ½ causes a neutral bias, this might be a good starting point for simulations
The conformational transition rate 0,c = N / Tk ≈ 6kHz can be estimated by the amount N = Lcyt / dFib ≈
600 of Fibprotomers that have to switch conformation during the time Tk ≈ 100ms a single kink needs in
average to travel the entire cell. Lcyt = 6µm is the length of the cytoskeletal ribbon3 and dFib = 10nm the
diameter of a single subunit {Trachtenberg182 2003}. This value agrees well with the switching rate = 10kHz
178
of the FliM model ({Bai 2010 and references therein} and references therein). Hence:
0,c 6kHz (3.26)
1
Here, the general asymmetric situation is treated because this gives another degree of freedom. For simplicity of illustration, the
symmetric case with Ecub = Ecb = Ec can be used.
2
The metabolism of mollicutes is described in {Lo55 2013; Bové152 2003}. One possible pathway is the production of ATP from glucose and
arginine {Shirazi181 1995}.
3
The length of an average cell is Lc = 4µm. However, this is the projected length and the cytoskeletal ribbon is attached to the cell
membrane. It spans through the cell on the shortest possible helical trajectory. This leads to a length of the cytoskeleton of Lcyt ≈ 1.5Lc.
75
3. Spiroplasma melliferum
Based on the assumption that protomers also switch at the rate 0,c in thermodynamic equilibrium in the
absence of ligands, 0.5kBT ≤ Ecub ≈ Ecb ≤ 2kBT seems to be reasonable but has to be explored by simulations,
too:
0.5kBT Ecub Ecb 2kBT (3.27)
The ligand in the FliM model, as well as the FliM complex itself, only has a regulating function on the
overall motor complex. In the Spiroplasma model, it is likely that the ligand is ATP itself and serves as a
regulator and fuel for the motor at the same time. Not only that this would be an efficient strategy, another
regulator associated with the cytoskeleton is not known at present. In addition, the finding of ATP dependent
kink velocities is little compatible to a model based on conformational switching triggered by a ligand different
172
to the energy supply. Bray and Duke {Bray 2004} derive the critical coupling energy Ej = 2kBT ln(N) for a
chain of N protomers that is needed for a completely coupled allornothing switching behavior. This results in
Ej ≈ 12.8 kBT ≈ GATP which is approximately the energy GATP = 12.5kBT gained by hydrolysis of ATP to ADP
and serves as upper boarder for the coupling energy. As shown in appendix 6.9, different coupling energies EJL
and EJR result in a side preference for starting the sequential conformational switching of the entire filament.
This is the experimentally observed case and displays a profound feature of the model since it gets by without
any mechanism that could be regarded as a head which initiates kinks. Hence:
EJR EJL GATP 12.5kBT (3.28)
Upper and lower boarders for the ligand binding rate 0,lb can be estimated as shown in the following. As
derived in appendix 6.9, the total energy of a subsequent switching filament composes of a constant and a
linear growing contribution. The constant contribution is the sum of coupling energies EJL + EJR of nearest
neighbors while the growing part reflects the energetic cost n’Ecub of n’ switched protomers without bound
ligand. In this picture, two ATPs are needed to account for coupling and additional ATPs every n’ = GATP / Ecub
≈ 10 protomers to account for the growing energy term . Thus, based on Eq. (3.26) 0,lb ≈ 0,c / n’ ≈ 600Hz
1
2
seems to be a reasonable upper boarder. On the basis of experimental findings, the authors of the FliM model
use = 10Hz ({Bai 2010 and references therein} and references therein) for their ligand binding rate, which
178
is much smaller than 0,lb estimated here. However, the ligand binding rate depends on the ligand
concentration which likely is different for ATP in Spiroplasma and CheY (the ligand for the FliM motor) in E.Coli.
An indication for a low binding rate 0,lb is the time Tik ≈ 50ms between two kinks within a cycle, which should
be directly linked to conformational back switching of the first protomer of the filament. Since 0,c has to be
high to explain the observed kink velocities (spread of conformational change), the only mechanism to explain
Tik is an unbinding rate 0,lb ≈ 1 / Tik = 20Hz serving as lower boarder. This is further discussed in appendix 6.9.
20 Hz 0,lb 600 Hz (3.29)
The broad range for 0,lb seems to be a contradiction but is consistent with different binding and unbinding
rates kub b and kb ub biased by the factor lb in Eq. (3.24). Hence:
1
0 lb (3.30)
2
Since kink velocities are the same for both kinks, a bias of conformational switching between circular to
elliptical and elliptical to circular is unlikely:
1
c (3.31)
2
1
In other words, two ATPs are needed to initiate conformational switching of the entire filament and to compensate the costs of coupling
and N / 10 ≈ 60 ATPs are needed to account for the switching of all protomers.
2
To trigger a conformational switch of a protomer upon ligand binding one prerequisite is 0,lb < 0,c because otherwise it would be
equally likely to unbind the ligand instead of switching the conformation as it can be seen by comparing Eqs. (3.24) and (3.25) or from Fig.
54A.
76
3. Spiroplasma melliferum
Thereby, estimates for all parameters of the model are derived serving as good starting point for
simulations (also see further below).
A manifest explanation for the coupling energy is a mechanical tension between neighboring subunits in
different conformations, i.e., with a different diameter in direction of the filament. A variation of this tension
thus should result in a variation of the coupling energy and further in different switching rates k cir ell . This
would ultimately result in different spread velocities or durations for switch cycles of the whole filament. The
optical trap used in the experiments shown in chapters 3.3.2  3.3.6 exerts external, optical forces Fext on the
cell, which, on the other hand, result in a torque Mext on the kink. This torque causes an additional stress in the
cell membrane and the cytoskeleton attached to it. Thus, trapped cells should display a significant change of
kink statistics compared to freeswimming cells. Comparing kink velocities of vK ≈ 10µm/s to vk ≈ 25µm/s and
the durations between kinks within a cycle of Tik ≈ 250ms to Tik ≈ 50ms for freeswimming (chapter 3.3.1) and
trapped (chapter 3.3.2) cells, respectively, this is exactly the observed case. These kink statistics have also
shown to be directly proportional (or antiproportional) to the intensity of the laser trap (see chapter 3.3.4)
and thus the force acting on the cell. This is again consistent with a force or tension dependent coupling
energy with coupling energies E JL,0 and E JR,0 in the force free case:
EJL ( Fext ) EJL,0 M ext ( Fext ) EJR ( Fext ) EJR,0 M ext ( Fext ) (3.32)
One observation that is particular striking and a profound test of any model is the start of individual kinks
within a cycle at different ends and the start of consecutive cycles at different ends during trapping
experiments at high power, i.e., high optical force (see chapter 3.3.6). This, again, can be explained by a
dramatically shift of the coupling energies causing an inversion of the initial boundary condition, i.e., EJL > EJR
> EJL < EJR. Additionally, a mechanical effect on the ratio between Ecb and Ecub is reasonable, too, and would
also explain the experimental observations mentioned above:
Ecb Ecb ( Fext ) Ecub Ecub ( Fext ) (3.33)
59
Shaevitz et al. {Shaevitz 2005} found different pitch angles for the left and right handed helix giving rise to
different mechanical properties of both conformational states.
Simulation strategy
Due to the complexity of the model, founded in the concerted switching of 600 proteins, analytical
conclusions are hardly possible and demand for a simulation strategy. The probability of conformational
switching and ligand binding have to be ruled out for every protomer in the filament based on the
corresponding rates k si s 'i and kub b . si and s’i denote different states of the ith protomer associated with an
energy difference Gi = G’i – Gi given by Fig. 54B.
c c 1
Gi Gi
ksi s 'i 0,c e kBT
ks 'i si 0, c e kBT
(3.34)
A crucial role take the model parameters such as 0,c, 0,lb and all the involved energy differences.
Reasonable sizes have been derived during the course of this chapter making up a good starting point. Bai and
178 180
coworkers {Bai 2010; Ma 2012} used a MonteCarlo simulation to address the stochastic nature of the
model. This seems to be a good strategy.
When determined by simulations, the parameters of the model might be relatable to physical properties
such as groundstate tension in the cytoskeleton or an intrinsic friction during the propagation of
77
3. Spiroplasma melliferum
conformational switching. Especially the rate constant 0,lc and the bias factor c are related to the curvature
of the potential in both states, which is related to the abovementioned physical properties of the motor.
So far, the fact that multiple Fibfilaments are present in a typical cell which seem to be laterally connected
to a flat sheet has been ignored. However, this can be addressed by introducing an additional coupling term
between protomers of different filaments. It is an interesting question how this influences the rates of the
model during simulation or if it is another prerequisite for the observed functioning of the motor.
Furthermore, the roles of the MreB ribbon and the differential switching mentioned at the beginning of this
chapter have to be addressed.
Theoretical description: based on a common description of optical gradient forces Fopt ~ I (Eq. (2.13))
acting on trapped spheres, an analytical description of the effective optical force Feff the oscillating laser focus
exerts on a helical body has been formulated. The body has been modeled as a chain of interconnected
spherical pearls (the pearl model) with a high resistance to deformation. This assumption bases on the
experimental observation that cells are not squeezed inside the trap. The optical force Fopt(a) on each pearl at
position a has been integrated over all pearls of the body and the time average has been calculated to account
for the oscillation of the focus. Since the resulting equations cannot be solved analytically, numerical
simulations have been performed to study the form of the effective optical force Feff (Eq. (3.6)) and potential
Veff (Eq. (3.7)) acting on the cell body for different displacements b of its center of mass position to the trap
center. Forces Feff,x = xbx, Feff,y = yby, Feff,z = zbz in all directions x, y, z showed to be linear for small
displacements bx, by, bz; similar to the case of single beads in point traps.
Experimental verification: experimentally derived calibration factors x = 0.032pN/µm, y = 0.294pN/µm
and z = 0.025pN/µm (chapter 3.2.2) for an exemplary cell, expressing this linear relation, agreed well to
theoretical approximations x = 0.041pN/µm, y = 0.300pN/µm and z = 0.045pN/µm (chapter 3.2.3).
Experimentally obtained positions histograms p(b) have been used to estimate the form of the potential Veff(b)
= kBTln(p) ~ b2 which showed to be harmonic in agreement to the theory and simulations. Fits to the
potential revealed similar force constant x = 0.035pN/µm, y = 0.302pN/µm and z = 0.024pN/µm (chapter
3.1.3) as derived by calibration.
Estimation of deformation energy: on the basis of the experimentally derived trap stiffnesses, the optical
energy Wopt(b) according to Eq. (3.22)) has been estimated, which the bacterium needs to exert during kink
propagation against the optical trap. For a dead but structurally intact cell, Wopt < 10kBT has been determined
corresponding to the peak energy of a bead diffusing inside an optical trap. The actively selfpropelling cell
raised additionally Wopt ≈ 40kBT in mean and Wopt ≈ 80kBT maximal against the trap (chapter 3.3.2). However,
to determine the maximum deformation energy the cell is able to exert, the trapping laser power needs to be
78
3. Spiroplasma melliferum
further increased until the trap clearly hinders kink amplitudes which has not been observed so far. See
discussion of the motor model further down for more information.
Active deformations visible after cell death: the timeresolved power spectral density PSD(Wopt(b)) of the
optical energy Wopt(b) has been introduced (chapter 3.3.2) as an analytical method to analyze the statistics of
cellular processes related to locomotion, i.e., deformations of the cell body in the present research. This
evaluation reveals bacterial activity long (≈ 120s) after the cell is presumed to be dead (≈ 45s) (i.e., does not
kink anymore) when observed in conventional brightfield microscopy. This kind of activity is distinct from
noise or thermal motion and displays a remarkable feature of the force measurement method shown here
combined with advanced analysis methods. The origin of the deformations corresponding to this activity is
unknown. A possible explanation is shortlived kinks, similar to the pseudokinks described in chapter 3.3.6.
Comparison to other techniques: the estimation of forces and energies involved in the locomotion of
bacteria and other micronsized cells is essential to understand the underlying molecular machineries and
basic principles. However, most publications only address forces involved in static mechanical properties such
53 183
as resistance to deformation {Wang 2010; Dombrowski 2009}. Techniques addressing force generation
37
during propulsion of large eukaryotic cells, such as the measurement of substrate deformations {Legant
184
2010}, cannot be applied to single, freeswimming bacteria. Experimental approaches {Stellamanns 2014;
185 186
Xin 2014; Chattopadhyay 2006} addressing power or energy estimation during active or dynamic whole
cell processes of bacteria or other small unicellular organisms are rare and only capable of extracting forces
exerted on/by the whole cell, highlighting the importance of developing new techniques. The method shown
here is able to measure deformation forces of individual body elements as well as of the whole cell at high
temporal precision (≤ 2kHz).
With only dt = 200nm in diameter and a helical amplitude of the centerline of approximately dh – dt =
190nm, the cell’s physical dimensions are at the edge of the classical diffraction limit x ≈ ½. Additionally, its
rapid deformations in all directions demands for a fast threedimensional imaging or tracking technique to
analyze its locomotion. The detection method shown here relies on interference of scattered and unscattered
light which, in principle, allows tracking position changes at precisions down to the angstrom scale and with
54 128
MHz temporal resolution {Abbondanzieri 2005; Kress 2007}. The pearl model has been used again to
derive a theoretical formulation of detection signals S(a) = ga (Eq. (3.10)) for the elements/pearls at position a
= b + (xL, RHcos(kHxL), RHsin(kHxL)) of a linescanned helix.
Force and position calibration: a calibration approach to determine the detector sensitivity g and trap
stiffness has been introduced based on a combination of fast calibration scans and the commonly used
Langevin calibration method (chapter 3.2.2). However, experimentally obtained diameters dh of the cell were
approximately a factor of four smaller than reference values dh = 360nm obtained by complementary
60
techniques {Trachtenberg 2014}. This necessitated the introduction of correction factors qy = 3.8 and qz = 1.5
which were explained and derived by numerical simulations (chapter 3.2.3) of the analytical description of
detection signals. These factors clearly depend on the overall geometry of the helix, i.e., pitch p and diameter
1
dh, and originate from a position averaging of different pearls inside the extended focal volume .
Technical limits, resolution and tracking precision: the fundamental limit of the technique are helical
pitches p > 400nm ≈ x/2 larger than half the focal width and cell diameters dh < 600nm ≈ x smaller than the
focal width. For p < x/2, the positions of all pearls inside the focal volume are averaged to a constant value
similar to the averaging inside a static point focus. This limit corresponds to the classical resolution limit of
conventional light microscopy approaches. However, the tracking precision tp of individual body elements is
much higher and can be estimated to tp ≤ 5nm {Koch 2012}. The temporal resolution is fundamentally
109
limited only by the speed of the beam steering device. Here, maximum scan rates of the linetrap were f =
1
Which is larger in axial direction, thus the correction factor qz < qy is smaller than in lateral direction.
79
3. Spiroplasma melliferum
2kHz. High frame rates are necessary to distinguish temporal changes of kink statistics in different
experimental conditions. The shortest change of any quantity determined here is the course of the duration Tk
of individual kinks on the millisecond time scale. High frame rates are also needed for trap calibration to
increase statistics, needed for Langevin calibration, and to reduce measurement time.
Comparison to other techniques: conventional superresolution techniques such as STED, PALM or STORM
typically suffer from slow frame rates below 10Hz and rely on fluorescence staining which has not been
reported for Spiroplasma so far. Thorough analysis of geometrical properties of individual cells has been
60
reported using darkfield microscopy {Trachtenberg 2014}. However, the authors did not demonstrate its
potential applicability to the analysis of rapidly deforming cells which might be possible using a fast camera.
59
Swimming speeds and kink statistics of Spiroplasma have been investigated by DIC microscopy {Shaevitz
2005} which, equipped with a fast camera, would be an alternative method, too. However, both techniques
suffer from their need of advanced (video) tracking algorithms which are laborious, especially in axial
direction, but achieve similar tracking precisions as back focal plane interferometry, in principle. Another
benefit of the interferometric tracking method shown here is the possibility of measuring deformations and
related forces at the same time.
The interferometric imaging method described here has been used to study kink statistics (e.g., kink
velocity vk and interkink time Tik) in different experimental situations. These experiments serve as
characterization of the underlying molecular motor which was then modeled on the basis of the experimental
findings.
Comparison of trapped and freeswimming cells: the swimming pattern of Spiroplasma is characterized by
a pair of kinks running down the length of the cell body, always starting at one end. In mean, a high kink
velocity vK ≈ 25µm/s and time between kinks of a pair Tik ≈ 50ms of a trapped cell results in N ≈ 4 cycles per
second (chapter 3.3.2). Freeswimming cells exhibit a much lower kink velocity of vk ≈ 10µm/s and larger time
59
between kinks of a cycle Tik ≈ 260ms resulting in N ≈ 1 cycles per second (chapter 3.3.1 and {Shaevitz 2005}).
This clearly indicates an effect of the optical trap on the molecular motor of Spiroplasma, likely of mechanical
origin due to optical forces.
ATP dependent molecular motor: Oxygen scavengers were used to suppress the formation of reactive
oxygen species (ROS) caused by the optical trap. ROS inhibit ATP production and ultimately cause the cell to
die within ≈60s. This allows investigating single cells for ten minutes or longer and revealed a Gaussian
distribution of vK and Tik (chapter 3.3.3). Furthermore, a periodicity of T ≈ 5s of the number of kink pairs per
time Ncylces/s was found (chapter 3.3.6). Without scavenger, kink velocities vK and the frequency of kink pairs
Ncylces/s decrease when approaching cell death (chapter 3.3.2). This indicates an ATP dependent process of kink
propagation. The time between kinks Tik ≈ const. remained constant, indicating an ATP independent process.
This is further supported by similar observations made when applying sodium azide (NaN3), an ATPinhibitor,
to trapped cells (chapter 3.3.5). Here, the kink velocity vk ≈ 28.4µm/s decreased significantly to vK ≈ 23.0µm/s.
The time between kinks Tik ≈ 155ms increased to Tik ≈ 187ms, but the statistical significance was not distinct.
Influence of external mechanical properties on the molecular motor: cells exposed to an alternating, step
wise increase and decrease of optical forces in a pyramidal fashion displayed immediate and reversible
response by distinct changes of the kink velocity vK, time between kinks Tik and frequency of kink pairs Ncylces/s
(chapter 3.3.4). This further supports the indication that the molecular motor is sensitive to external
mechanical influences, such as the rigidity of its local microenvironment. The observation that the time
between both kinks of a cycle Tik(Lc) depends on the length of the cell (chapter 3.3.6) also fits to this
assumption and can be linked to an optical torque exerted on the kinked cell by optical forces.
Direction of kink propagation: in extreme conditions such as high laser power (optical force) or poisonous
chemical conditions (sodium azide), successive kinks have been observed that start at different ends (chapter
80
3. Spiroplasma melliferum
59
3.3.6), an observation that is unusual and has been reported so far only for cells fixed at one end {Shaevitz
2005}.
The molecular motor of Spiroplasma relies on a polymeric chain or filament composed of identical protein
subunits spanning the entire length of the cell. Individual protomers can exist in different conformational
states ranging between nearly circular to elliptical. A subsequent switch between the conformational extremes
(circular <> elliptical) of each protomer, starting at one end of the cell and traveling towards the other, results
in a net length change of the chain. A differential length change of this filament, relative to other elements
such as a supporting cytoskeletal ribbon, causes the cell to change handedness of its helical cell body. This
ultimately results in a kink at the position of handedness change which travels down the cell body from one
end to the other.
Motor model: the subsequent spread of conformational change was modeled theoretically basing on an
allosterically triggered spread of conformation. The model relies on a double well (see Fig. 54) potential
Kramer’s rate theory for conformational switching of single protomers at rates kbcir ell and kubcir ell (Eq. (3.25))
depending on the presence of a bound (‘b’) or unbound (‘ub’) ligand. The rates of ligand binding or unbinding
kubcir b and kubell b (Eq. (3.24)) depend on the ligand concentration c and the conformational state (‘cir’ for
circular, ‘ell’ for elliptical) of the protomer. Additionally and essential for the model, interaction or coupling
energies EJL ≠ EJR between adjacent protomers favor the cooperative, conformational switching of nearest
neighbors by lowering their combined energy. Without coupling, each protomer switches conformation
randomly and a concerted, sequential switching, as it is needed to explain the experimentally observed kink
propagation, is hardly possible. The influence of an external force Fext is accounted by introducing force
dependent coupling and conformational switching energies EJ ( Fext ) and Ec ( Fext ) .
Estimation of model parameters: based on the experimental observations, all relevant parameters of the
model were estimated. In particular, these are the conformational switching rate 0,c ≈ 6kHz and the ligand
binding rate 20Hz ≤ 0,lb ≤ 600Hz in thermodynamic equilibrium, the energy gaps ½kBT ≤ Ecb ≈ Ecub ≤ 2kBT
between different conformational states in the doublewell potential for a bound or unbound ligand, the
coupling energies EJR < EJL < GATP = 12.5kBT and the bias factors 0 < lb < ½ and c ≈ ½ for the ligand
binding rate kub b and switching rate k cir ell .
Predictions of the model: the abovementioned parameters are only estimates. The true values have to be
determined by computer simulations of the model and can be used to determine the curvature of the
potential and the intrinsic friction during conformational switching afterwards. However, some predictions can
be made anyway.
Prediction 1: it has been shown (appendix 6.9) that the total energy Gtot needed for a complete kink cycle
splits into two parts: a constant term EJR + EJL ≈ GATP related to coupling and a linearly growing term ≈ n’Ecub
≥ 60GATP compensating the energetic coast of n’ conformational changes. In other words, initiation of a cycle
1
is triggered by two ATP molecules releasing their energy at the same time which is a process that depends on
the ATP concentration. Thus, the model predicts that cycles are less frequently initiated when ATP is depleted.
This has been observed during experiments.
Prediction 2: in the model, the speed of the conformational spread depends on the rate of ligand binding.
The later depends on the ligand concentration. Here, the ligand is assumed to be ATP itself, thus the model
predicts ATP concentration dependent kink velocities which were observed experimentally.
Prediction 3: it has been shown (appendix 6.9) that the introduction of different coupling energies EJL > EJR
predicts kinks which always travel in the same direction, which start at the same end and that the start of a
cycle somewhere in the middle is excluded. This, again, is the experimentally observed case.
1
One molecule to compensate energetic costs of coupling and one to compensate switching of the first protomers.
81
3. Spiroplasma melliferum
Prediction 4: based on the estimates for the model parameters, the energetic equivalent of ≈60ATP per
kink cycle is required at minimum to explain kink propagation. This results in a power P = 60GATP / Tcycle ≈
85GATP/s ≈ 1070kBT/s of a freeswimming cell with Tcylce ≈ 700ms (chapter 3.3.1). For a trapped cell, Tcylce ≈
300ms decreases resulting in an increase of power consumption to P ≈ 200GATP/s. The power exerted against
the optical trap can be estimated from the mean optical energy Wopt ≈ 40kBT during kinking, resulting in P =
Wopt / s ≈ 40kBT/s ≈ 3GATP/s. This value is much smaller than the prediction of the model but does not reflect
the total exerted deformation energy as discussed above. This prediction is further discussed in the following
part.
Comparison to hydrodynamic models: following the discussion of estimating the minimum and maximum
power consumption involved in the locomotion of Spiroplasma {Koch 2010}, a consumption of 50GATP/s ≤
162
P ≤ 250GATP/s determined by slenderbody and resistiveforce theory has been estimated {Yang 2009}.
187
The authors used mainly hydrodynamic arguments to determine the swimming efficiency of the kinking, helical
cell body depending on the number of kinks per cycle. Interestingly, the authors found that a double kink cycle
is only preferred if the molecular mechanism requires at least 40ATPs per cycle. For lower values, a cycle
consisting of four kinks in total is more efficient. Although the work of Yang et al. does not regard the motor on
a molecular level, their theoretical findings agree well to the prediction of a minimal requirement of 60ATPs
per cycle made above.
188
Comparison to a purely Kramer’s rate based molecular model: Roth {Roth 2013} used a similar
description based on a doublewell potential Kramer’s rate theory of protomer switching as introduced here.
However, there are two principle differences. First, the Roth model tried to include the curvatures of the
potential and the intrinsic friction during switching explicitly. These quantities are summarized to the single
parameter 0,c in the present model. Secondly, a ligand concentration dependent allosteric triggering of
conformational change was not included. Instead, the model assumed the release and distribution of GATP
over all protomers without addressing its origin. However, the Roth model already considers a coupling
between nearest neighbors. The conformational switching rates were directly related to the experimentally
accessible variables such as the kink velocity which is a good strategy, but did not allow any prediction since
the input parameters of the model are too specific and diverse. To overcome this drawback, the author tried
to include the influence of external forces, such as the optical trap, and to vary this force by attaching beads to
the cell which did not work properly. In contrast to the detailed, analytic approach of the Roth model, the
present model is a minimalistic description which has still enough degrees of freedom to explain all
experimental observations and to make some principle predictions for the behavior of the motor, even
without thorough simulations of the stochastic but also concerted switching of all ≈600 protomers
Comparison to related systems: the present description of the molecular motor bases on the model of the
switchmechanism of the rotary flagella motor in E. Coli, which regulates the sense of rotation of flagella
178 180
{Bai 2010; Ma 2012}. This switch consists of 34 identical protomers (FliM) assembled to a ring. Each
protomer is able to switch between two conformations, similar to the Fibril protein of Spiroplasma. However,
the FliM complex has only a regulating function concerning the motor, contrary to the Fibril complex which is
172
the motor itself. The basic theory of cooperative interaction between nearest neighbor introduced by {Bray
189
2004; Duke 1999} has also been used to model the polymorphic transformation of helically flagella
190
{Srigiriraju 2006}. If the direction of rotation is reversed in the flagellar motor, the flagellum undergoes a
traveling transition of handedness. Similar to Spiroplasma, this transition is connected to a kink in the
171
flagellum which propagates to its end. Vogel and Stark {Vogel 2013} were able to model this behavior by a
theory based on the global elastic and hydrodynamic properties of the whole flagellum. However, this model
relies on a torque generating motor at the attachment of the filament to the cell body which is not present in
Spiroplasma.
Conclusions: a great power and remarkable feature of the allosterically regulated spread of conformations
is that only a fraction of protomers need to bind and hydrolyze ATP to switch the conformation of the entire
filament. Additionally, since the experimental findings suggest a process depending on the concentration of
ATP, the ligand of the model is likely the energy carrier ATP itself. Both properties are very efficient and might
187
have been favored during evolution, an argumentation similarly made by {Yang 2009} who found that the
geometry and kinematics of Spiroplasma are optimized concerning hydrodynamic efficiency. This also fits well
82
3. Spiroplasma melliferum
to the overall reductionism and simplicity of this kind of bacteria which evolved by genome reduction from
56
more complex cells {Trachtenberg 1998}. In contrast to any other present model, the theory introduced here
explicitly favors kinks starting always at the same end and traveling in the same direction by introducing
different coupling between nearest neighbors. A bias between both ends for externally applied forces or
strong shifts in ligand concentration allows spontaneously inversion of kink direction. This is a remarkable
quality and excellent escape strategy, enabling an immediate change of the swimming direction by 180° in the
case of nonfavored chemical or mechanical situations. A conventional ‘Uturn’ by rotational fluctuation needs
much longer or might not be possible in narrow, spatial constrictions. Such a behavior, which has also been
observed experimentally, would not be possible by a model based on any kind of head mechanism initiating
kinks, such as a torque generating rotating motor.
Outlook
The theory on optical trapping and tracking of helical cells by lineoptical tweezers is established and
exhaustively tested by experiments. The transfer of the shape adaption method to other cells has to be
proven, but might be a promising approach in other cases. However, the simulation of the molecular motor
model introduced here is clearly the next step and might unravel interesting new experiments and further
tests. If determined by simulations, the rather abstract parameters of the model have to be linked to more
physical quantities such as the stiffness of the cell membrane, the cytoskeleton or the viscous drag of the
entire cell. Connected to that, determining the mechanical properties of the cell, such as its flexural rigidity,
might give further insights in the connection of these properties to the parameters of the model. A complete
understanding of the motor and the biomechanical properties related to that would ultimately result in the
possibility of constructing an artificial microswimmer that is able to move and transport cargo, independently
of external influences or human control.
Remark
The experimentally demonstrated and theoretically modeled sensitivity of the cell’s swimming dynamics to
external forces displays a clear mechanotransduction pathway, related to the research on microtubules
presented in the second part of this thesis.
83
4. Microtubules
4. Microtubules
Microtubules are hollow tubes with a diameter of DMT ≈ 25nm, a typical length in the range of 1µm ≤ LMT ≤
1
20µm and a thickness of the tube wall of dMT ≈ 4.5nm. Tubulin, the basic building block, is a polar heterodimer
made of  and tubulin, two nearly spherical proteins. Dimers assemble into larger oligomer chains, socalled
protofilaments, by alternating  and tubulin as illustrated in Fig. 55. At a critical length of ~12 dimers
{Fygenson 1995}, individual protofilaments are laterally linked by and contacts, forming a sheet.
191
192
Sheets are inward curved {Pampaloni 2008}, thus closing to form hollow tubes – predominantly for 13
protofilaments. Interestingly, adjacent protofilaments within a sheet are slightly displaced (~0.9nm) in
longitudinal direction resulting in a helical lattice and a discontinuity at the seam when the sheet is closed. The
helical pitch matches exactly three basic tubulin units for 13 protofilaments, thus the lattice is called three
start helix. Microtubules with 817 protofilaments and a two to fivestart helix have been found in vitro
193 194 195
{Hawkins 2010}, giving rise to different and distinct physiological functions {Savage 1989; Wade 1990}.
Once assembled, microtubules can grow and shrink by polymerization and depolymerization of tubulin
dimers at the microtubule ends. Without mechanical load, microtubules rarely break and individual dimers
would never depolymerize somewhere from the center. The number of lateral and longitudinal contacts of a
196
single dimer renders the overall structure very stable {Lewin 2007}. However, the number of dimer contacts
at the ends is much less, thus depolymerization is more likely here. The probability to polymerize and
depolymerize strongly depends on the number of free tubulin dimers in solution leading to the socalled
dynamic instability in vivo. In vitro, microtubules can be stabilized and prevented from depolymerization by
2 3 198
the addition of stabilizing agents such as Taxol or GMPCPP . Yu et al. {Yu 2013} argued that microtubule
stabilizers may also modulate the structure and thus its function and mechanics. Indeed, different stabilizing
197
agents bind at different sites to the tubulin dimer ({Jordan 2004} and references therein) and mechanical
193
properties have been shown to differ for differently stabilized microtubules (see {Hawkins 2010}).
1
The intrinsic polarity of the tubulin dimers is present throughout the whole microtubule, a feature that is used for directional motor
transport for example.
2
Taxol (also Paclitaxel), besides various other drugs, is used as a common anticancer agent. By impairing the dynamics of filament
polymerization, mitosis is blocked and tumor cells are killed. Microtubules are one of the most prominent targets in clinical cancer therapy
{Jordan197 2004}.
3
GMPCPP (also GPCPP or sodium salt) is a nonhydrolizable GTP analog. GTP is used to polymerize microtubules (see appendix 6.6.1).
85
4. Microtubules
A B
Fig. 56. Different lateral and longitudinal dimer contacts render microtubules
a certain flexibility. Depending on the number of protofilaments different
192
helical lattices arise. Adapted from {Pampaloni 2008}.
As mentioned in the introduction, the project corresponding to this part of the thesis aims at investigating
cellular force transduction by the microtubule cytoskeleton under welldefined geometrical conditions.
213
Microtubules are used in mechanosensing systems {Nick 2008} and have been demonstrated to conduct
214
mechanical forces in fly campaniform mechanoreceptors {Liang 2014}. A defined assembly of cytoskeletal
215
structures has been achieved by various techniques {Vignaud 2012} such as micropillars and holographic
216 217
optical tweezers {Mohrdieck 2007; Uhrig 2009}. All these techniques rely on stochastic attachment or
growth of filaments to a substrate such as beads. A controlled and targeted alignment comprising more than a
single filament has not been demonstrated yet. Forces acting on single filaments have been measured for
205 218 51
stabilized {Kurachi 1995; van Mameren 2009} and polymerizing {Kerssemakers 2003} filaments as well
219
as bundles {Streichfuss 2011}, but defined networks of more than one filament have not been tested so far.
Typical experiments on mechanical properties of the cytoskeleton reflecting the capability of conducting forces
220 221
and external stimuli rely mainly on bulk rheological measurements in vitro {Gardel 2004; Xu 1998} or in
222 223 224
vivo {Guo 2014; Hoffman 2006; Chen 2015}. Such measurements hardly allow drawing specific
conclusions on individual filaments or components, especially in vivo since here, a mixture of various cellular
components is measured and conclusions on a single constituent are not possible.
During the course of this chapter, it is demonstrated how cytoskeletal networks with a welldefined
topology can be created, probed and analyzed employing optical tweezers. Starting with single filament
experiments as basis, general laws and conclusions on the conduction of mechanical stimuli are established
and their transfer to larger networks is analyzed.
1
See chapter 4.1.1.
2
Apparently, there are some errors in the published version of Table 1 in that article and the authors published a corrigendum.
86
4. Microtubules
In particular, the chapter is organized in three parts. It starts with chapter 4.1 by introducing a mechanical
concept used to describe microtubules, an introduction to microrheology, a method which is used to analyze
the frequency response of materials, and the essential background on the timemultiplexed optical tweezers
configuration used here.
Chapter 4.2 summarizes different experiments on the mechanics and rheological properties of single
microtubule filaments, which are needed as a solid background to understand small microtubule networks
shown and analyzed in chapter 4.3.
A straight beam or filament buckles under a mechanical load, i.e., compressing forces in longitudinal
1
direction of the beam. The situation is depicted in Fig. 57A. The most commonly used theory to describe the
deflection w(x) at position x of a buckled beam of length L compressed by the length L in longitudinal
direction bases on the Euler beam approach. The deflection for a filament with hinged ends can be described
227 228
by a sine function {Gross 2009; Gross 2007}.
w(x, L ) A( L ) sin x (4.1)
L L
The amplitude A(L) of the buckle is derived in appendix 6.10. However, under a steadily increasing force, a
227 228
perfect beam does not buckle until a critical force Fcrit is reached {Gross 2009; Gross 2007}.
L’
A(L)
A
F x
L L
Fig. 57. Buckling beam. An initially straight beam with length L buckles under
a compression force F. The buckling amplitude A(L) depends on the
longitudinal compression L.
For forces F > Fcrit, the beam bends to the side with spring constant and depending on the longitudinal
compression L
F ( L ) Fcrit 1 L Fcrit L (4.2)
2L
Both spring constant = Fcrit / 2L and critical force Fcrit = ²EI / L² depend on the properties of the
material. EI is the bending modulus or flexural rigidity combining the beams elastic properties described by
2
the Young’s modulus E and its geometry by the area moment of inertia I. In a small angle approximation, the
deflection angle A can also be expressed in terms of the applied force:
F
A2 8 1 4 L (4.3)
Fc L
1
Most reports on the bending stiffness of microtubules are based on the Euler beam theory, too. However, there are several other
approaches that account for the molecular structure of microtubule filaments. These more sophisticated theories find their niche mainly
for very short filaments (L < 5µm) and often do not allow simple calculations rather than demanding thorough simulations. A good
overview and discussion can be found in {Zhang225 2014; Zhang226 2014}.
2
For a hollow cylinder with inner diameter Ri ≈ 8nm and outer diameter Ro ≈ 12.5nm, this is I 4 Ro4 Ri4 ≈ 1.6·1032m4.
87
4. Microtubules
This description is used in chapter 4.2.1 to estimate the flexural rigidity of single microtubules and in appendix
6.10 to derive the buckling amplitude A(L), needed to calculate the viscous drag acting on a rapidly
compressing and relaxing microtubule filament.
4.1.2. Microrheology
Rheology, in general, describes the deformation a or flow of a material when exposed to a force F.
Depending on the material properties and the rate of applied stress s(F) as a function of force, the response
can be elastic, plastic and/or viscous. A viscous deformation is related to energy dissipation, typically in form of
heat, while the energy is stored as elastic or deformation energy in the other two cases, respectively. The
1
behavior of a material can differ tremendously for different rates of applied stress . Geophysicist and
climatologists, for example, describe the flow of lava, stone, glaciers and tectonic plates in terms of rheology.
Microrheology, on the other side of the length scale, is used to describe the predominantly viscoelastic
deformation of polymer solutions, for example.
229 230
The following theoretical description bases on the work presented by {Atakhorrami 2006; Mizuno
231 232
2008} and has been used to describe the response of passive {Gittes 1997} and active {Mizuno 2007}
233 234
cytoskeletal actin solutions and complete cells {Nijenhuis 2012; Mizuno 2009}. Similar approaches have
235
been used to study the frequencydependent response of collagen networks {Münster 2013}, the
236 237
mechanical role of intermediate filaments in living cells {Guo 2013} and single DNA strands {Meiners
238 239
2000}. A good introduction and overview of microrheology is given in {Pullarkat 2007; Squires 2010}.
a() d(1,2)
(2pPMR, 2pAMR)
Two particles
F()
Since optical tweezers can be used to exert forces on particles, causing displacements, and to measure
both quantities simultaneously, it is an ideal technique for microrheology. Indeed, most of the above
mentioned publications employ optical tweezers. As depicted in Fig. 58, different methods can be used to
obtain the frequencydependent particle displacement function a of an optically trapped bead placed in a
viscoelastic environment, when exposed to a force F (see Fig. 58). In passive microrheology (PMR), F = Fth is
defined by the random, thermal force Fth as described in chapter 2.1. In active (AMR) approaches, the trap is
1
Simple Newtonian fluids, like water for example, are purely viscous and can be described by a constant viscosity. NonNewtonian fluids,
like a solution of starch (pudding), behave completely different. They are fluidlike for gently applied forces but act like a solid if a force is
applied rapidly. This finally leads to the strange phenomenon that a person can run on a solution of starch filled into a swimming pool
without sinking but it cannot walk slowly on it. In terms of rheology, such a material has a frequencydependent elastic storage modulus:
vanishingly small at low frequencies and large at high frequencies.
88
4. Microtubules
displaced sinusoidally at a frequency a, thus, F = Fopt(a). Either one (1p) or two (2p) particle rheology can be
used to obtain the local material properties, i.e., on the length scale of the fluctuations of the particle (1p), or
the response at a distant site, i.e., at the length scale of the particle separation d(i,j) (2p). In Fourier space, the
(in general) complex response function A(i,, j ) ( ) A '(i ,, j ) iA ''(i ,, j ) relating the force spectrum F(j), ( ) , applied
to particle j, to the resulting spectrum of displacements a(i,) ( ) of particle i is given by
a(i,) ( ) A(i,, j ) ( ) F(j), ( ) (4.4)
Here, , denote the direction perpendicular and parallel to both particles in 2p experiments, which
normally is x and y. The latter convention also applies to 1p experiments. For the ease of reading, these
subscripts are omitted if dispensable. However, fluctuations of a trapped bead are always governed by the
optical trap and do not directly reflect the intrinsic properties of the surrounding medium alone. Thus, A is
only an apparent response function reflecting the trap, the probe and the probed material and the true
response function has to be determined, reflecting the probe and the properties of the material itself. In
single particle measurements, the corresponding relation reads
A( )
( w) or A( w) (4.5)
1 A( ) 1 ( w)
denotes the trap stiffness. When using two particles, this relation becomes more complicated and
diverse, since the response A(1), , A(2), of single particles as well as the interparticle response A(1,2)
, has to be
estimated for both directions , :
(i)
(4.6)
,
1 (i) A(i), (j) A(j), (i) (j) A(i), A(j,) A(i,, j)
2
A(i,, j)
(i,, j) (4.7)
1 (i) A(i), (j) A(j), (i) (j) A(i), A(j), A(i,, j)
2
(i), (j) (i), (j), (j) (i,, j)
2
(i)
(4.8)
A ,
1 (i) (i), (j) (j), (i) (j) (i), (j), (i,, j)
2
(i,, j)
A(i,, j) (4.9)
1 (i) (i), (j) (j), (i) (j) (i), (j), (i,, j)
2
For an isotropic and homogenous material (e.g., water), (i), (j), are equal in both directions. The true
response function is independent of the properties of the trap but still depends on the probe, i.e., the size and
shape of the bead. To provide for a better comparison of different materials, the true material properties are
expressed by the viscoelastic response function G = G’ + iG’’. For spherical beads, for example, the influence
of the probe can be ‘subtracted’:
1 1 1
G(i), G (i, j) G(i, j) (4.10)
6 R (i) (i), 4 d
(i, j) (i, j)
8 d (i, j)
(i, j)
From the definition of Eq. (4.4), it is clear that only linear materials can be investigated this way. The
linearity has to hold at any fixed frequency 1 but can be nonlinear for a varying frequency , i.e., ( 1) =
A(1)F( 1) but A( ) . Since pure water is isotropic and homogenous, it is a linear material and its
response function GH 2O only depends on its viscosity H 2O
GH 2O i H 2O (4.11)
89
4. Microtubules
Especially, it does not depend on the direction ( , ), the distance d(i,j) between two probes or the radius
(i)
R of the probe (bead). Different, but related, approaches to determine the apparent response function A
(which depends on the properties of the trap, the probe and the material), the true response function
(which depends on the properties of the probe and the material) and the pure material response function G
(which solely depends on the properties of the material) are given in the following. These approaches were
used here to analyze the frequency response of microtubules and to test the method and its principle
limitations with beads suspended in the water.
In equilibrium, the fluctuationdissipation theorem relates the imaginary part of the response function
A’’() to the power spectral density PSD( )  a( ) 2 (see chapter 2.1) of the particle fluctuations a(t).
1
A ''( ) PSD ( ) (4.12)
2k BT c2
2
Real and imaginary parts of any complex function A = A’ + iA’’ are related by the KramersKronigrelation
240
({Schnurr 1997} and reference therein):
2
A '( ) cos( t ) A ''( ) sin( t ) d dt (4.13)
0 0
This integral can be solved analytically for a particle immersed in a purely viscous fluid like water.
1 1
A( ) c i (4.14)
2 c2
The apparent response function is dominated by the optical trap with c = / . From Eq. (4.5), the purely
229
imaginary true response function follows which agrees to the stationary Stokes result ({Atakhorrami 2006}
and reference 28 therein).
1
( w) i (4.15)
Using Eq. (4.10) results in the pure material response function G given by Eq. (4.11)
In active approaches, the Fourier transformed particle displacements a() and apparently applied force
F() = xL( = a) have to be calculated resulting in the complex apparent response function A() when
evaluated at the driving frequency a of the actuating trap.
a( )
A( ) (4.16)
F ( ) a
The true response function and the material response function G can be obtained according to Eqs. (4.5)
and (4.10).
The theoretical slopes derived from passive microrheology before are plotted together with the results of
a Brownian dynamics simulation (see appendix 6.12) of the active method for different trap stiffness in Fig.
59. The apparent response function A (Fig. 59A) is governed by the properties of the trap as described by Eq.
90
4. Microtubules
(4.14). The position of the maximum of the imaginary part (blue) corresponds to the corner frequency c = /
of the trap. The same holds for the decay A’( c) = ½A’(0) of the real part (red). The imaginary part (blue) of
the material response function G (Fig. 59B) depends linearly on the frequency and is indigent of the
properties of the trap (and probe) according to Eq. (4.11). In theory, the real part G’ = 0 (red) of the material
response function (of water) is zero. However, the denominator of Eq. (4.5) is approximately zero causing a
nonvanishing G’ and ’ during analysis. This effect can be of relevance when interpreting microrheology
results.
2
100 Solid line: analytical solution of 10
Solid line: analytical solution of
passive microrheology passive microrheology
90 Symbols: BD simulation of active Symbols: BD simulation of active
microrheology microrheology
1
80 10
= 10 pN/µm A', A'' = 10 pN/µm G' / G''
= 20 pN/µm A', A'' = 20 pN/µm G' / G''
70 = 100 pN/µm A', A'' = 100 pN/µm G' / G''
0
A', A'' (10 m/N)
60 10
50
1
40 10
30
2
20 10
10
3
0 10
1 Hz 10 Hz 100 Hz 1 kHz 10 kHz 100 kHz 1 Hz 10 Hz 100 Hz 1 kHz 10 kHz 100 kHz
f = /2 (Hz) f = /2 (Hz)
Fig. 59. Theoretical and simulated complex apparent response function A
(left) and material response function G (right) for a spherical bead in water.
Similar to 1pAMR, the Fourier transformed particle displacements a(i)( ) and apparently applied forces
F () = xL( = a) yield the complex apparent single particle and inter particle response functions A(i,) ( )
(i)
and A(i,, j) ( ) when evaluated at the driving frequency a of the actuating trap.
a(i), ( ) a(i), ( )
A(i), ( ) A(i,, j) ( ) (4.17)
F(i), ( ) F(j), ( )
a a
The true response functions then follow again from Eqs. (4.6) and (4.7). For comparison of experimental
results and for testing purpose, the known material response function G = i can be used to calculate the
true response functions according to Eq. (4.10) and therefore the apparent response functions A using Eqs.
(4.8) and (4.9). This is shown in Fig. 60 together with simulated data (see appendix 6.12) for comparison.
Interestingly, the imaginary part of the apparent interparticle response function is bipolar with a negative and
230
a positive slope, in contrast to the single particle response function (compare to Fig. 59 and see {Mizuno
2008}). The position of the zero crossing point as well as the position of the maximum of the real part A’ is
determined again by the properties of the traps. The amplitudes of both parts are sensitive to the distance d(i,j)
between both traps and larger for smaller d because the hydrodynamic coupling is stronger.
91
4. Microtubules
3
d = 20µm 0
2
1
1
2
0
1 Hz 10 Hz 100 Hz 1 kHz 10 kHz 100 kHz 1 Hz 10 Hz 100 Hz 1 kHz 10 kHz 100 kHz
f = /2 (Hz) f = /2 (Hz)
Fig. 60. Real part (left) and imaginary part (right) of the apparent inter
particle response function for beads with a radius R = 531nm in water. Solid
lines are theoretical curves as explained in the main text. Markers represent
simulation results.
Experimental results for the material response function G are shown in Fig. 61. Within the experimental
accuracy, the single particle, as well as the interparticle, response function are equal and independent from
the probe and results collapse onto the theoretical curve according to Eq. (4.11). The deviations at low
frequencies (f < 10Hz) is due to the sensitivity of the true response function to the distance d(i,j) » R(i)
between beads. Results for the true response function and an analysis of the reproducibility of individual
measurements is shown in appendix 6.12.
= =
Fig. 61. Experimentally obtained single particle (left) and interparticle (right)
material response function G for different distances d(i,j) between beads.
Individual data points are the average of five successive measurements and
error bars represent the standard deviation of the mean.
In general, passive microrheology techniques suffer from limitations of the KramersKronigrelation. The
highest frequency decade typically scatters tremendously for results of this transformation. Active techniques,
on the other hand, are often limited by the maximal (or minimal) oscillation period that can be reached by the
corresponding beam steering device. At low frequencies, one prerequisite is a stable system because any drift
would falsify the result.
92
4. Microtubules
In order to create a static grid of multiple optical traps, each able to hold a bead acting as anchor point of a
polymer network, the laser focus is rapidly scanned between different positions using an AOD. The traps are
called timemultiplexed. Here, a theoretical description of such an array and different methods how it can be
created in practice is given.
Each position is held by the focus for a distinct time on = 1 / fAOD = 20µs (typically) before the laser jumps
to the next position. fAOD is the maximum deflection rate of the AOD limited by the velocity of the ultrasonic
1
wave propagating through the optical crystal and the width of the laser beam . If a grid of N point traps is
created, every trap is visited again by the laser after a period scan. The right hand side of Eq. (4.18) holds if all
traps are identical, i.e., on = const.
N
scan on , j N on (4.18)
j 1
This period cannot be chosen arbitrarily. If there are too many traps or if on,j (j = 1, …, N) is too large for at
2 101
least one of the traps, individual trapped beads may escape . As shown in {Ruh 2011}, the effective trap
stiffness eff(N) of a single trap within a grid of identical traps decreases by the factor N compared to the
stiffness of a single trap.
eff (4.19)
N
Interestingly, this is not true for the detector sensitivity g(N) = const. which is invariant concerning the
number of traps.
z
x
y
+Aa y0,1 Aa y0,2
Fig. 62. Schematic of a two trap optical grid. Trap 1 (red) is actuated on a
sinusoidal trajectory while the position of trap 2 (blue) is kept constant. This
is the basic trap arrangement used for active rheology experiments later on.
A big advantage of using an AOD is that different types of traps can be generated simultaneously (in time
average) and altered rapidly. An example is shown in Fig. 62. Here, a sinusoidally oscillated trap (trap 1) is
combined with a point trap (trap 2). Both traps are multiplexed. The frequency fa as well as the amplitude Aa of
the oscillation of trap 1 can be changed within scan. However, there are two principally different ways how N
traps, each moving in time average on a trajectory rL,j(t) = (xL,j, yL,j, zL,j) can be multiplexed. Mathematically,
the different multiplexing can be expressed by the functions rL,j while the complete trajectory rL of the laser
focus can be expressed as the sum of all trajectories rL,j as shown in the following.
N
yL (t ) yL , j (t ) (4.20)
j 1
For the ease of simplicity, only one dimension is shown and treated mathematically in the following. Other
dimensions are deduced accordingly.
1
See chapter 4.1.4 for further details.
2
Depending on the stiffnesses of individual traps, tscan > 1kHz is a realistic, general lower limit.
93
4. Microtubules
Trapmultiplexing
In this mode, the traps are multiplexed consecutively, meaning that the laser focus first moves along the
complete trajectory of trap 1, followed by the complete trajectory of trap 2 and so forth. This is shown in Fig.
63. The trajectory for each trap j (j = 1, …, N) can be modeled as
j 1
yL , j (t ) f j (t ) t i scan s , j t i scan s , j on , j s , j on, k (4.21)
i k 1
Here, fj(t) is the function describing the actual trap movement and (t) is the Heaviside or unit step
function , which is responsible for the correct timemultiplexing. In the example: fj(t) = Aasin(2fat)+y0,1.
1
In general, on,j (j = 1, …, N) is different for each trap now. In the example shown here, on,1 = 1000µs and
on,2 = 20µs add up to scan = 1020µs. The big drawback of this mode is, if one of the traps is modulated slowly,
i.e., has a large on, that beads of other traps can escape because the laser focus is absent for too long. In the
current example: if trap 1 would be modulated by fa = 10Hz, this result in scan ≈ 100ms, i.e., scan ≈ 100ac of a
point trap and thus all other beads would escape.
Thus, this mode cannot be used to probe a polymer network at frequencies fa ≤ 100Hz.
fa = 1000Hz
1.5
1.0
Focus position : yL(t)
0.5 Trap1 (actor) : yL,1(t)
Trap2 (sensor) : yL,2(t)
0.0
0 200 400 600 800 1000 1200 1400 1600 1800 2000
t (µs)
Fig. 63. Laser position for two traps generated by trap multiplexing. Trap 1 is
actuated sinusoidally at fa = 1000Hz, Aa = 1µm and y0,1 = 2µm and
multiplexed consecutively with a static trap (trap 2) at y0,2 = 0µm. The
position of the static trap is held for on,2 = 20µs.
True timemultiplexing
Here, the laser jumps between individual traps after on = 20µs = const. which is constant for every trap.
Individual trajectories of different traps are carved up and multiplexed piecewise. This is shown in Fig. 64 for
the forgoing example, to have a comparison between both modes.
j 1 j 1
yL , j (t ) f j (t , i ) t i scan t i N scan on
i N (4.22)
f j (t , i ) t iN j 1 on t iN j on
i
fj(t, i) is again the function describing the actual trap movement and the step function (t) accounting for
the temporal multiplexing. However, due to the intricate splitting, fj also depends on the current cycle number
i within a period. The temporal absence of the focus during the visit of other trap locations results in a more
1
By definition: (t) = 0 for t < 0 and (t) = 1 for t ≥ 0.
94
4. Microtubules
complicated function in order to get a continuous transition of yL,i after each jump to another trap location. In
the example: f1 (t , i ) A sin 2 f a t i ( N 1) on y0,1 .
In this mode, the scan frequency scan = Non is again given by the constant time on = const. and the
number of traps N. If every time varying trajectory yL,j is assigned the time constant per,j (per,1 = 1 / fa and per,2
= 20µs in the example), the least common multiple (LCM) of the constants of all traps can be used to define
per = LCM(per,j) describing the periodicity of the overall laser trajectory yL(t). In this way, it is guaranteed that
no bead can escape its trap since it is visited again by the laser every Non, independent from per,j. Individual
trajectories rL,j are cut into Nj pieces given by
per , j
Nj (4.23)
N on
In the present example, N1 = 25 and 250 ≤ Nj ≤ 250,000 for the frequency range 100Hz ≥ f ≥ 0.1Hz as it
was typically used in the experiments shown in chapter 4.2 and 4.3.
i=0 i=1 i=2
scan scan scan Aa
on on on on on on 3.0 Focus position : yL(t)
Trap1 (actor) : yL,1(t)
per
2.5
2.5 Trap2 (sensor) : yL,2(t)
2.0
yL(t) (µm)
2.0 fa = 1000Hz
1.5
yL(t) (µm)
1.5 1.0
1.0 0.5
0.0
0.5
0 100 200 300 400 500 600 700 800 900 1000
0.0 t (µs)
0 20 40 60 80 100
t (µs)
Fig. 64. Laser position for two traps generated by true timemultiplexing.
Trap 1 (red) is actuated sinusoidally at fa = 1000Hz, Aa = 1µm and y0,1 = 2µm
and multiplexed in alternating pieces with length on = 20µs with a static trap
at y0,2 = 0µm (trap 2, blue).
In the light of the experimental objective, trap 1 is also called actor trap (since it is used to actuate a bead
later on) and all further traps are called sensor traps (since they are purely used to sense the response of the
actuation). However, all traps could be moved on nearly arbitrary 2D trajectories simultaneously. It shall be
also noted here that only the true timemultiplexing mode is used for the following experiments. Despite this,
both modes have their experimental importance depending on the actual task.
The (true) multiplexing nature is also present in the interferometric tracking signal obtained from the QPD.
The signals Sraw = (Sxraw, Syraw, Szraw) have to be split in segments of on = 20µs and sorted to obtain a signal Sj =
(Sx,j, Sy,j, Sz,j) proportional to the movement of every single bead with index j (j = 1, .., N). The principle is
1
shown in Fig. 65 for the axial signal Szraw of an array of N = 4 empty traps . Data acquisition is started per
software trigger simultaneously to the data output responsible for steering the trap array. Nevertheless, there
is an electronic delay of d ≈ 4µs between input and output which has to be cut off of the signal. Furthermore,
2
every change of the frequency or amplitude of the ultrasonic wave in the optical crystal of the AOD needs acc
3
≈ 7µs to propagate through the crystal and to fully establish the new optical grating . The grating is in an
1
This example has been chosen because, on the one hand, the sum signal Sz depends on the nonconstant diffraction efficiency of the
AOD and, on the other hand, changes between different trap positions are better visible without a trapped bead.
2
Very likely caused by the electronics of the AOD driver.
3
According to the operation manual of the AOD, the access time acc = D / v ≈ 7µs is given by the beam diameter D ≈ 5mm and the
velocity of the acoustic wave v = 650m/s.
95
4. Microtubules
undefined state during this time and thus the first 7µs of the signal corresponding to every trap have to be cut
off, too.
scan scan
Period 1 Period 2
on on on on on
d Trap 1 Trap 2 Trap 3 Trap 4 Trap 1
0.12 acc acc acc acc acc
Run 1
0.11 Sz,3=<Sz> Sz,4=<Sz> Run 2
Run 3
Sz (V)
0.10 Run 4
Sz,1=<Sz> Run 5
Sz,2=<Sz>
0.09
0.08
0 20 40 60 80 100
t (µs)
Fig. 65. Illustration of signal processing for an array of N = 4 empty traps. The
axial raw signal Szraw of the QPD is split into pieces of identical length on =
20µs. Data of each piece corresponds to the position signal Sz,j of another
trap with number j = 1, …, N. To illustrate the reproducibility of this method,
the measurement is performed five times (Run 1, …, Run 5).
In order to suppress high frequency noise caused by the AOD and to get equidistant sample points, the
remaining signal is averaged over every period. Mathematically, this can be expressed by
t ' j ( t ) on t mod(t , scan )
S j (t ) S raw (t ') j (t ) scan on (4.24)
t ' j ( t ) acc d
scan on
is the floor function, mod(a, b) the modulus of a / b and j marks the first data point corresponding to
trap j during the scan period i. Here, the effective sample rate sReff = 1 / scan = 1 / Non of every trap depends
on the number of traps N. This also reduces the amount of data by a factor of sR · scan = 20 for a sample rate
sR = 1MHz typically used for data acquisition.
A B 30 30
x signals (mV)
40
Sx,1, Sx,2 (mV)
50 40
50
50 60
75
raw Sx,1 Data trap 1 70
Sx 60
Sx,2 Data trap 2
100 80
2.0 3.0 4.0
0 50 100 150 200 0 5 10 15 20
t (µs) t (ms)
C D 80 100
100
60
y signals (mV)
50
50 40
20 0
0
0
raw Sy,1 Data trap 1
50 Sy 20 50
Sy,2 Data trap 2
2.0 3.0 4.0
0 50 100 150 200 0 5 10 15 20
t (µs) t (ms)
Fig. 66. Signal processing for an array of two traps with beads. Trap 1 is
actuated sinusoidally with yL,1(t) = Aasin(2fat), Aa = 800nm and fa = 100Hz.
(A), (C) The raw signal Sxraw and Syraw, respectively, is shown together with the
data pieces in color corresponding to each trap (red = trap1, blue = trap2).
(B), (D) Resulting position signals Sx1,2 and Sy1,2 for two periods of the
sinusoidal oscillation.
Fig. 66 depicts the situation for an arrangement of two traps displaced by ≈11µm in y direction. Each trap
holds a single bead trapped in a solution of pure water. Trap 1 (the actor) is moved sinusoidally at fa = 100Hz
96
4. Microtubules
and Aa = 800nm (see 4.1.6 for more details). The raw signals Sxraw and Syraw (Fig. 66A+C) are processed as
described above to obtain the position signals Sx,j and Sy,j (j = 1, 2) as shown in Fig. 66B+D. The sinusoidal
oscillation of bead 1 caused by the oscillating actor trap can be clearly seen in the position signal Sy,1 (Fig. 66D,
red data points) of the actor bead. The position signals Sy,2 of the second bead is only influenced by Brownian
motion (see next chapter for the corresponding calibrated signal). The same applies to Sx,1 and Sx,2. As
discussed in 6.11, axial signals are not relevant during the experiments on microtubules shown in this thesis,
thus they are not shown for the sake of simplicity and clarity.
To calibrate an array of beads, the method of choice would be the Langevin method (see chapter 2.4.2)
because it is fast and can be flexibly applied to obtain the trap sensitivities gi and stiffness i (i = 1, …, N) of an
101
array of traps with arbitrary geometry and number of traps N {Ruh 2011}. Unfortunately, it relies on the
fluctuation of single beads inside the optical trap in the vicinity of any disturbance which would falsify the
1
result . The experiments on microtubules shown later are critical in a sense that coated beads do not stick to a
microtubule on first contact. Usually dozens of trials are needed until stable binding happens. However,
sometimes single filaments can be found with one bead already attached to one of its ends. These filaments
are used preferably for experiments but they necessitate a different calibration approach. In the light of the
method used to calibrate the detector during linetrapped Spiroplasma bacteria shown in chapter 3.2.2, a laser
scanning approach has been chosen to find the detector sensitivity g of an array of traps holding beads. The
situation is depicted in Fig. 67.
Trap 1 Trap 3
static static
z
x
y
y0,1 = y0,2 y0,3 = y0,4
Als Als
Trap 2 Trap 4
sawtooth oscillation sawtooth oscillation
Fig. 67. Schematic of the laserscanning calibration approach. Each bead is
held by a strong, static point trap (trap 1 and 3 in black). In addition, each
bead is scanned by a sawtoothlike movement of the focus similar to a line
trap (trap 2 and 4, red and blue, respectively). All traps are timemultiplexed.
Each bead is scanned by a weak, short and fast line trap which results in consecutive calibration scans
similar to those for Spiroplasma calibration. In order to restrict bead diffusion which falsifies the result, each
bead is held by a strong and static point trap in addition. All traps are (true) timemultiplexed resulting in 2N
traps for an array of N beads. In order to find the optimal scanning frequency fls, amplitude Als and laser power
2
Pls, these parameters have been systematically varied and the resulting value of gls has been compared to the
result gfix of a bead immobilized on the cover slip. The latter serves as a reference.
The ratio rls = gls / gfix is shown in Fig. 68 for the twodimensional configurations space (Als, fls) for Pls =
8mW for an array of two beads (left and right) as representative. The result differs up to rls = 30% within the
3
configuration space of one trap and varies within the same margin between both traps, as well. The reason,
1
If, for example, the fluctuation is hindered by an external force, the autocorrelation time changes affecting the detector sensitivity g.
2
If Als is too small, the mean diffusion distance of the bead <b²(1/fls)> ≈ Als is of comparable size thus affecting the result. Similarly, if fls is
too small or Pls too high, the bead is dragged slightly during every scan and its position is influenced.
3
The power Pstatic = 400mW of the static traps was chosen to be Pstatic / Pls = 50 times larger than the power of the laser scan, thus
efficiently suppressing kicks of the scanning focus and large (x < 50nm) diffusive motion x.
97
4. Microtubules
especially for the difference of both traps, is not clear but has likely to be addressed to the diffraction angle
dependent properties of the AOD.
fls (Hz) fls (Hz)
200 400 600 800 1000 200 400 600 800 1000
Als (nm)
Als (nm)
1.0 1.0
400 400
0.9 0.9
0.8 500 0.8
500
0.7 0.7
600 600
Fig. 68. Determining the optimal frequency fls and amplitude Als of the
calibration line scan. Twodimensional configuration space (Als, fls) for trap 1
(left) and trap 2 (right). The color scale indicates the ratio rls between free
and immobilized beads. Trap separation y = 20µm. Red crosses mark values
usually used for calibration.
By comparing the results for both traps and based on further tests (not shown), the parameter set Als =
500nm and fls = 500Hz has been chosen for calibration. The detector sensitivity is directly proportional to the
trapping laser power (see chapter 2.4.3), so gls can be scaled if a laser power different to Pls = 8mW is used in
1
the actual experiment .
One general drawback of this method is, that it can be only applied to the lateral dimensions x and y since
no fast axial scanner is available. As pointed out in 6.11, all experimental configurations used in this work
result in twodimensional problems, so axial displacements are negligible.
160
2,MT = 331µs
ACx (nm²)
5 AC(0)=2.6% 5
4
1,MT = 364µs
4
140
100 3
3
9 1 = 273µs
120 8 2
2
ACx,1(0) = 185nm² 7 ACy,1(0) = 118nm² 2 = 293µs
ACx,2(0) = 174nm² ACy,2(0) = 196nm² 1 = 273µs
0 40 80 120 0 40 80 120
10 ACx,1,MT(0) = 182nm² 1 = 289µs 10 ACy,1,MT(0) = 115nm²
ACx,2,MT(0) = 164nm² 2 = 303µs ACy,2,MT(0) = 186nm²
7 7
0 200 400 600 800 1000 0 200 400 600 800 1000
t (µs) t (µs)
Fig. 69. Autocorrelation analysis to determine trap stiffness .
Autocorrelation ACx (left) and ACy (right) of calibrated positions bx,i, by,i and
bx,i,MT, by,i,MT of single beads and beads with microtubules attached,
respectively, for both traps (i = 1, 2). Trap separation y = 10µm.
Concerning trap calibration, i.e., finding the detector sensitivity , the Langevin method is still eligible if a
microtubule is attached to the bead. The autocorrelation value AC(t = 0) = kBT / sys of the calibrated position
signal b(t) of a static trap results in the sensitivity sys of the system trap (t ≥ 10pN/µm) + microtubule (MT <
1pN/µm, typically MT ≈ 0.15pN/µm). As pointed out in 6.13.2, sys = t + MT ≈ t, so the influence of the
microtubule on the trap stiffness using the yintercept of the autocorrelation is negligible. This is shown
representatively in Fig. 69 for a two trap arrangement with beads only (triangular markers) and with a
1
Most experiments were carried out at P = 64mW, so g = 64mW / 8mW · gls was used to obtain b = S / g.
98
4. Microtubules
microtubule attached (circular markers). Here, the same beads were used in order to not falsify the result by
1
different bead diameters , for example.
As indicated in Fig. 67, traps were displaced along y and thus, the microtubule was also aligned in that
direction. Consequently, the spring constant MT and drag coefficient MT have a stronger impact on the y
autocorrelation ACy. In fact, the autocorrelation times i and i,MT (i = 1, 2) with and without microtubule of
both traps differ by up to 50% in y direction and 30% in x direction. The autocorrelation time hence is not
suitable for calibration. Nevertheless, the yintercepts ACx,i(0), ACx,i,MT(0), ACy,i(0) and ACy,i,MT(0) for both
traps differ by less than 10% for both directions and are well suited for calibration (AC(0) = ACi(0)  ACi,MT(0)
< 10%·ACi(0)).
Alternatively, the results of a Langevin calibration of different but unperturbed beads can be used as a
reference. The position of the traps should be close to the positions of the traps used in the actual microtubule
experiment because of the angle dependent diffraction efficiency of the AOD.
A 50 B 150 800
600
100
25 400
by,1, by,2 (nm)
bx,1, bx,2 (nm)
50
200
yL (nm)
0 0 0
200
50
25 400
gx,1 = 612.8 kV/m 100 gy,1 = 625.0 kV/m
600
gx,2 = 544.3 kV/m gy,2 = 497.0 kV/m
50 150 800
0 5 10 15 20 0 5 10 15 20
t (ms) t (ms)
C 1.5 D 3 800
600
1.0 2
400
Fy,1, Fy,2 (pN)
Fx,1, Fx,2 (pN)
0.5 1
200
yL (nm)
0.0 0 0
200
0.5 1
400
1.0 x,1 = 28.6 pN/µm 2 y,1 = 21.9 pN/µm
600
x,2 = 30.3 pN/µm y,2 = 30.8 pN/µm
1.5 3 800
0 5 10 15 20 0 5 10 15 20
t (ms) t (ms)
Fig. 70. Calibrated signals of an arrangement of a trap oscillating along y (red,
Aa = 800nm, fa = 100Hz) and a static trap (blue). (A), (B) Displacement bx and
by in x and y direction of bead 1 (red) and bead 2 (blue) relative to trap
center yL (black). (C), (D) Optical forces Fx and Fy in x and y direction of
bead 1 (red) and bead 2 (blue) relative to the trap center yL (black).
The resulting position and force signals b(t) and F(b(t)), respectively, are shown in Fig. 70 for the exemplary
signals S(t) presented in the foregoing chapter (Fig. 66). Beads diffuse up to b ≈ ±30nm inside the trap due to
Brownian forces of up to F ≈ ±1pN. Trap 1 was oscillated sinusoidally at fa = 100Hz and Aa = 800nm along y.
The bead follows this motion but is hindered due to viscous forces up to F = v ≈ ±2.5pN, resulting in
displacements of up to by,1 ≈ ±150nm from the actual trap center.
1
The bead used in microtubule experiments have a specified diameter 2R = (1062 ± 35)nm. See appendix 6.6 for details.
99
4. Microtubules
The displacement of the actor bead is shown in Fig. 71 where the actor trap (trap 1) is actuated at fa =
200Hz and Aa = 600nm. The displacement by,1(t) of the bead relative to the trap at position yL,1(t) is highly out
of phase. This phase lag b,1 is caused by the viscous drag force F = v acting on the bead moving at velocity
v. The actual bead displacement relative to its initial position y0,1 is given by
a j (t ) rL, j (t ) b j (t ) (4.25)
1
In the particular situation shown here this is ay,1(t) = yL,1(t) – by,1(t). Due to sine addition theorems , the
actual phase lag a,1 between the trap and the true bead position is small but present. The maximum and
2
minimum amplitude of the bead position a(t) is determined for positive A+y,1 and negative Ay,1 edges of the
oscillating trap as indicated by the orange and green markers in Fig. 71B.
A B by,1
600
Trap 1 oscillating 200 yL,1
yL,1(t) = y0,1 + Aasin(2fat) ay,1
400
100 
z A
+
x 0
y,1
0
b,1 200
y 100
Aa 0 Aa a,1 400
200
600
0 2 4 6 8 10 12 14
t (ms)
Fig. 71. Movement of a bead trapped by an oscillating trap. (A) Schematic of
experiment. (B) Displacement by,1 of the bead relative to the current position
yL,1 of the trap center and true bead position ay,1 relative to its initial position
at y0,1 = 0.
To address the influence of hydrodynamic coupling on the bead signals, a two trap arrangement separated
by d(1,2) = y0,2 – y0,1 = 11µm is used as shown in Fig. 72A. Again, trap 1 is actuated at fa = 200Hz and Aa =
600nm. The coupling of the second bead held in a static trap is clearly visible in Fig. 72B. A sine fit results in an
amplitude Ay,2 ≈ 14nm and a phase lag of b,2 = 0.79 relative to the actuating trap.
x
0
y 10
Fig. 72. Coupling between beads in a oscillating and a static trap separated by
y = 11µm. (A) Schematic of experiment. Trap 1 (red) is oscillated while trap
2 (blue) is held static. (B) Displacement by,2 of the second bead relative to its
trap at y0,2.
To analyze the oscillation of both beads more systematically, the frequency 0.1 ≤ fa ≤ 1000Hz of the actor
bead (bead 1) is varied in nearly logarithmic steps (fa = 0.1Hz, 0.2Hz, 0.5Hz, 1Hz, …) as well as the amplitudes
Aa = 200nm, 400nm, 600nm, 800nm. The result is shown in Fig. 73. For frequencies fa ≤ 5Hz, the actor bead
(Fig. 73A) follows the movement of its trap, while for fa > 5Hz the viscous drag starts to become significant and
1
sin( + ) = sin()cos() + cos()sin( ), sin() – sin( ) = 2cos(½( + ))sin(½(  )) and cos() – cos() = 2sin(½( + ))sin(½( – ))
2
To account for Brownian fluctuations, the maximum (or minimum) of the signal for each positive (or negative) edge is determined and
the mean of a window with a width of 510% of the oscillation period is calculated.
100
4. Microtubules
the bead cannot follow the complete oscillation amplitude of the trap anymore. At around the same
frequency, an oscillation can also be seen in the signal of the sensor bead (bead 2) as shown in Fig. 73B.
However, due to hydrodynamic coupling at a large distance (d(1,2) = 11µm), the maximum amplitude Ay,2 ≈
0.1Ay,1 is small compared to that of the actor bead (also see chapter 4.1.2, especially Eq. (4.10)). The
amplitudes A+y and Ay of both beads for the positive and negative edge of the actor are symmetric indicating
that there is no difference if the beads move towards or away from each other.
A 200 B 15
150
10
100
5
50
by,2 (nm)
by,1 (nm)
0 0
50
5
Aa = 200nm Aa = 600nm Aa = 200nm Aa = 600nm
100
Aa = 400nm Aa = 800nm Aa = 400nm Aa = 800nm
+ 10 +
150 Positive actor edge A y,1 Positive actor edge A y,2
 
Negative actor edge A y,1 Negative actor edge A y,2
200 15
Regarding microtubule experiments, the distance change y = Aa  Ay,1 – Ay,2 between both beads is
relevant since it reflects the filament’s elastic component. This is shown in Fig. 74A together with the total
force F1 + F2 = 1b1 + 2b2 in Fig. 74B acting on both beads and their phase lag a = a,1 – a,2 in Fig. 74C.
The latter is relatively small (˂10%) even for high frequencies. The movement of the actor bead is conducted
1
by the molecules of the surrounding medium (water) at the speed of sound , thus the response of the distant
sensor follows nearly immediately. The total force at low frequencies is governed by Brownian motion and
thus the absolute value of its average amplitude is constant. At high frequencies, the optical force depends on
the maximum amplitude Aa of the actor trap because it balances the viscous force, which increases if the
displacement Aa per time 1 / fa increases.
A 800 B 7 C
Aa = 200nm Aa = 600nm
Aa = 200nm Aa = 600nm
6 Aa = 400nm Aa = 800nm
6 Aa = 400nm Aa = 800nm Positive actor edge
Positive actor edge Negative actor edge
600 Negative actor edge
5
4
F1 + F2 (pN)
A y, A y (nm)
4
a (%)
400

3 2
Aa = 200nm Aa = 600nm
+
Aa = 400nm Aa = 800nm 2
200 0
Fig. 74. Influence of the oscillation frequency fa and amplitude Aa on (A) the
relative distance between both beads, (B) the total optical force acting on
them and (C) their relative phase lag.
1
The acoustic velocity of water is v ≈ 1500m/s. Thus, the time a sound wave needs to propagate the separation distance d(1,2) = 11µm of
the beads is t = d(1,2) / v ≈ 7.3ns.
101
4. Microtubules
The rheological methods introduced in chapter 4.1.2 already account for the analysis of displacements,
forces and phases shown here. However, in order to be able to interpret signals and results with microtubules
attached to beads correctly, it is important to have a reference of the behavior of single beads in the same
experimental conditions.
In this chapter, single microtubules are subject to slowly varying pushing forces exerted by laterally
attached beads causing the filament to buckle as explained in chapter 4.1.1.
A
Trap 1
(t = t0) (t = t0 + Nt)
Trap 2
stepwise displacement static z z
x x
y y
y0,1 Ny y0,2 y0,1 Ny y0,2
B y(t)
t
y0,1
y N
yL ,1 (t ) y0,1 y t it
y0,2 yL ,2 (t ) y0,2 i 1
t
L’MT
C
0 pLMT 0
z
0 R R 0 x
y
1 by,1 yL by,2 2
Fig. 75. Schematic of experiment. (A) An initially (t = t0) straight filament (left)
is buckled (right) due to a displacement of trap 1 towards trap 2. (B) The trap
is moved in discrete steps of y every t seconds. (C) Geometrical
representation of the bend filament with rotated beads caused by a torque.
(D) Fluorescence image of a buckled filament. Beads are indicated by white
circles. Scale bar: 5µm.
The situation is depicted in Fig. 75A+B. While trap 2 is held static, the position of trap 1 is moved towards
trap two at a step size of y every t seconds (y = 50nm and t = 100ms typically). The principle is similar to
204
the approach shown by {Kikumoto 2006} who also used two optically trapped beads, generated by splitting
102
4. Microtubules
up the laser beam with beam splitters, as handles to exert forces on a microtubule. However, the authors used
DIC video microscopy to analyze the deflection of the filament at the critical escape force when one of the
beads is pushed out of its trap. The escape force was determined independently by applying an increasing flow
to a trapped bead without microtubule. Here, back focal plane interferometry is used to measure the beam’s
deflection and the currently applied force below the escape force at the same time, thus resulting in a more
general and flexible method.
Due to the lateral attachment of the microtubule to the beads, torque is generated which causes beads to
rotate as depicted in Fig. 75C. Thus, the beam’s projected length pLMT, and thereby its deflection, does not
agree with the displacements by,1 and by,2 of the beads from their initial positions. According to Fig. 75C, the
buckling or compression pLMT of the projected length relative to the total arc length LMT of the filament
between its attachment points is given by
pLMT LMT pLMT LMT yL by ,1 1 ( A ) by ,2 2 ( A ) (4.26)
For convenience, only absolute values of by,1 and by,2 are regarded. The distance of the attachment
projected onto the y axis to the center of the bead is 1,2 = Rsin(A) with A the angle to the normal of the
attachment point. Here, neither pLMT nor A can be measured directly. However, the relation A = 4pLMT /
LMT between both quantities is given by Eq. (4.3). Together with Eq. (4.26), this results in a quadratic equation
for the angle A
8R b
A2 A 4 1 0 (4.27)
LMT LMT
Here, b = yL + by,1 + by,2 is used as a shorthand. Since Eq. (4.26) is only a small angle approximation, 1,2
= Rsin(A) ≈ RA is used, too. Physically, only one of both possible solutions makes sense resulting in
4R LMT
2 MT
A 1 L b 1 (4.28)
LMT 4R
If this result is substituted in Eq. (4.26), all quantities can be determined experimentally and a plot of
F(pLMT) reveals the true buckling behavior of the filament as shown in Fig. 76A for a short (LMT = 5.1µm) and
a long (LMT = 15.9µm) example. In order to exclude hysteresis effects or structural damage, experiments on
the same filament are performed two to three times in both directions, i.e., when increasing the buckling
strength and when subsequently relaxing back to the initial position. Although results sometimes deviate
1
qualitatively from those shown in Fig. 76A, none of these effects are visible in general . According to Eq. (4.2),
a perfect beam does not buckle until the critical force Fcrit is reached. Here, this perfection is violated due to
fluctuations caused by Brownian motion on the one hand, and the lateral attachment of the filament on the
other hand. This results in a finite steep rise of the applied force F for small pLMT ˂ 200nm. To properly
2
determine Fcrit, f(x) = Fcrit (1  exp(x / x0)) + mx is used as fit function , motivated phenomenologically. Results
for Fcrit are shown for twelve filaments covering a range of the microtubule length of 3µm ≤ LMT ≤ 22µm. In
3
order to obtain the persistence length characterizing the bending stiffness of the filament, two different
model fit functions for Fcrit(LMT) are used
A A
fit1 ( x) fit2 ( x) (4.29)
x2 x 2 l '2
1
In some experiments small dips in the force shortening curve F(pLMT) are visible. These might be addressed to structural damage of the
filament, but more likely is a problem with the NeutravidinBiotin binding to the bead. However, such events are discarded or treated with
care.
2
The fit function is motivated phenomenologically. The exponential increase with characteristic constant x0 is due to the imperfection of
the buckling beam, i.e., laterally attached beads. A theoretical description of the principle phenomenon of imperfection is described in
{Gross227 2009}. The parameter m is linked to the spring constant MT of buckling filaments, i.e., if F > Fcrit.
3
The persistence length lp = EI / kBT is a microscopic quantity relating the bending modulus EI to the thermal energy, thus being a
measure of rigidity. Loosely speaking, it describes the minimal length of a filamentous structure that is needed to bend it to a circle
without damage.
103
4. Microtubules
fit1 is motivated by the classical buckling beam as introduced in chapter 4.1.1. Here, A = ²EI = ²kBTlp with
211
a constant persistence length is found. Pampaloni et al. {Pampaloni 2006} describe a persistence length
lp(LMT) depending on the actual length of the filament, thus serving as motivation for fit2. Here, A 2 k BTl p
where l p is the persistence length for filaments longer than a critical length l’. Qualitatively, both fits agree
1
well to the measured values. The relatively high ²/DOF values can be explained by the scattering of values,
especially at large LMT. This can be addressed to defects in the microtubule structure, impurity during
225 226
polymerization, bundling of filaments or different numbers of protofilaments {Zhang 2014; Zhang 2014}
which is hardly or simply not at all observable during experiment. However, exclusion of bad data points (at
LMT ≈ 14µm and LMT ≈ 18µm, marked in yellow) does not change fit results significantly.
A 1.2 B
Fcrit
1.0
6.0 A 2 k BTl p (7.50 0.17)10 23 N
m2
0.8
5.5 l p (1.83 0.04)mm
F (pN)
0.6
5.0 2
DOF 57
0.4 4.5
Buckling
0.2 Relaxing 4.0 l ' (3.26 0.39) µm
LMT = 15.9µm Fit
0.0 3.5 A 2 k BTl p (9.50 0.36)10 23 N
Fcrit (pN)
m2
0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 3.0 l (2.20 0.08) mm
p
pLMT (µm)
4 Fcrit 2.5 2
DOF 49
2.0
3
1.5
F (pN)
1.0 Fcrit
2
0.5 fit1
fit2
1 Buckling 0.0
Relaxing
LMT = 5.1µm Fit 5 10 15 20
0 LMT (µm)
0.0 0.2 0.4 0.6 0.8 1.0
pLMT (µm)
Fig. 76. Results for the buckling beam. (A) Force shortening curve F(pLMT)
for a long (top) and short microtubule (bottom) during buckling and
subsequent relaxation. Error bars result from the standard deviation of the
mean obtained by averaging sample points at each trap position. (B) Critical
buckling force Fcrit depending on the length of the filament LMT.
From fit1, lp = (1.83 ± 0.04)mm is obtained which fits well to the range of observed values listed in
193
{Hawkins 2010}, for example. The critical length l’ = (3.26 ± 0.39)µm gives significantly rise to a length
dependent persistence length, but is nearly one order of magnitude smaller than l’ = 21µm reported by
{Pampaloni 2006}. l p = (2.20 ± 0.08)mm again fits well to other reported values of the persistence length
211
and to the result obtained by fit1, but differs form the result l p = (6.3 ± 0.8)mm given by {Pampaloni 2006}.
211
The difference to the results of Pampaloni et al. can be addressed to a different polymerization and
207
stabilization of the filaments, as well as to the end suspension as argued in {Hawkins 2013}.
Stretching experiments are the direct opposite of buckling experiments. For an initially straight filament,
one trap is moved away from the other thus exerting pulling forces on the filament. The situation is depicted in
Fig. 77A. Similarly to buckling, beads are exposed to a torque caused by optical forces and the tensile strength
of the filament. Filaments are bent locally at the point of attachment to the bead as illustrated in Fig. 77C+D.
1
This is not the critical length of approximately 12 tubulin dimers when tubulin protofilaments start to assemble laterally as mentioned in
the introduction of chapter 1.
104
4. Microtubules
218
{van Mameren 2009} modeled this situation using the classical beam equation which relates the bending
moment to local curvature of a beam. The authors introduced a fit function which can be directly used to
extract the bending modulus EI and thus the persistence length lp from data obtained by optical tweezers
experiments:
2
EI 8 2
F ( ) 2 22 2 (4.30)
D 3 1
Here, = (b – LMT) / D is a dimensionless variable reflecting the current distance b = yL + by,1 + by,2
between the beads with yL = yL,1 – yL,2 and the bead diameter D.
A Trap 1 Trap 2 (t = t0) (t = t0 + Nt)
stepwise displacement static z z
x x
y y
Ny y0,1 y0,2 Ny y0,1 y0,2
B y(t)
t
y0,1
y
y0,2
C t
z
x
by by
y
R ½0 ½0 R
0 0
Bead
Biotin
Neutravidin
Tubulin
Fig. 77. Schematic of experiment. (A) An initially (t = t0) straight filament (left)
is stretched (right) due to a displacement of trap 1 away from trap 2. (B) The
trap is moved in discrete steps of y every t seconds. (C) Geometrical
representation of the bend filament with rotated beads caused by a torque.
(D) Fluorescence image of a stretched filament. Beads are indicated by white
circles. Scale bar: 5µm.
The displacement by of each bead from its trap center during such an experiment is shown in Fig. 78A.
Experiments were typically performed several times for the same filament in both directions, stretching (red
markers) and relaxing (blue markers) back, in order to test for structural damage. In some cases, sudden
position changes are observed without a particular notice in the fluorescence images. For the sake of safety,
these events are sorted out. Fig. 78B shows the dependence of the total force acting on the filament versus
105
4. Microtubules
the dimensionless stretching parameter for filaments with comparable length but different stabilization. An
immediate observation is the smooth rise for the Taxol only stabilized filament while the doubly stabilized
1
(Taxol + GMPCPP ) microtubule increases much faster. This is also reflected by the persistence length obtained
218
from the function fitmame according to {van Mameren 2009}, i.e., Eq. (4.30). The doubly stabilized filament (lp
≈ 5µm) is approximately five times stiffer than the Taxol only treated specimen (lp ≈ 1µm) at comparable
207
length (LMT = 18µm and LMT = 15µm, respectively). This has been reported previously {Hawkins 2013} and is
likely due to the integration of GMPCPP into the microtubule structure since it replaces GTP during
polymerization.
Filaments with a brought spectrum of lengths have been analyzed and the result is summarized in Fig. 78C.
The persistence length of single stabilized microtubules (Taxol only) seems to be constant. This is expected
2
because the experimental configuration probes mainly the local mechanical properties . Nevertheless, a fit
211
function fitpam according to {Pampaloni 2006} (also see fit2, last chapter) is used to address this question. As
result, l p = (0.63 ± 0.04)µm at a critical length l’ = (5.3 ± 1.0)µm. The latter value is nearly exactly the same
size as the shortest measured filament length LMT = 4.6µm. The persistence length l p for filaments longer
than that is approximately three times smaller than the values obtained by buckling experiments shown in the
211
last chapter and one order of magnitude smaller than the value reported by {Pampaloni 2006}. Thus, length
dependence can be excluded.
Doubly stabilized microtubules again seem to be much stiffer; however, only four values demand for a
better statistics to draw definite conclusions.
A 150
by,2 (nm)
100
50
C
0 4 Taxol
Taxol + GMPCPP
by,1 (nm)
50 fitpam
100 Stretching 3
Relaxing
150
lp (mm)
15 ²/DOF = 2.4
0
10
0 5 10 15 20 25 30
5 LMT (µm)
lp = (0.91 ± 0.15)mm
²/DOF = 2.6
0
1
See footnote 3 on page 85.
2
Based on the description of tubulin dimer and interprotofilament bonds in {Pampaloni192 2008} and illustrated by the inset of Fig. 77C,
bonds located close to the point of attachment to the bead are stretched or compressed much more than remaining bonds.
106
4. Microtubules
As depicted in Fig. 79, the same experimental configuration as in the foregoing chapters is used here, i.e., a
single microtubule filament held by laterally attached, optically trapped beads. In contrast to the slowly
varying, unidirectional trap movement, the trap is oscillated sinusoidally at frequency fa and amplitude Aa.
1
Both parameters are typically swept over a brought twodimensional parameter space (fa, Aa) which makes
2
the use of active microrheology techniques possible. This approach allows testing the frequencydependent
transmission of a mechanical stimulus by a single filament of the otherwise rather intricate microtubule
cytoskeleton. Since this is the simplest configuration possible, elementary physical laws and conclusions made
here may serve in a bottomup approach as basis for more complex networks consisting of multiple filaments
as shown in the next chapter.
The chapter is divided into two parts: first, an oscillation in longitudinal direct of the filament is regarded
and the principle analysis of oscillation based experiments is explained. The second part then treats an
oscillation lateral to the filament axis. While both types of experiment have different physiological relevant
counterparts (e.g., a radial indentation of the cell and tangential shear), they also serve as basis for oscillation
experiments of complex networks, where, depending on the topology of the network, a superposition of
longitudinal and lateral oscillation is inevitable (see the triangular network configuration in chapter 4.3.2).
Longitudinal oscillation
B t = t0 z D t = t0 + Ta/2 z
x x
1 3
y y
Aa y0,1 Aa y0,2 time Aa y0,1 Aa y0,2
C t = t0 + Ta/4 z E t = t0 + 3Ta/4 z
x x
2 4
y y
Aa y0,1 Aa y0,2 Aa y0,1 Aa y0,2
1
Typically, fa is swept in nearly logarithmic steps between 0.1 – 100Hz, i.e., 0.1Hz, 0.2Hz, 0.5Hz, 1.0Hz, … Such a frequency sweep is
typically performed for amplitudes Aa = 200nm, 400nm, 600nm or 200nm, 500nm, 800nm to study the influence of different applied forces
and velocities of the actor.
2
Introduced in chapter 4.1.2.
107
4. Microtubules
When oscillated at a low frequency (fa < 1Hz), the complete experiment resembles a successive
combination of filament stretching and buckling as shown and analyzed in the last two chapters and illustrated
in Fig. 79BE. Besides the complete time dependent displacements by,1(t) and by,2(t) of the beads from the
current positions yL,1(t) = y0,1 + Aasin(2fat) and yL,2(t) = y0,2 = const. of their traps, the maximum amplitudes
A+y,1, Ay,1, A+y,2 and Ay,2 of by,1(t) and by,2(t) during the first (‘+’, for a displacement in positive direction) and
second (‘‘, for a displacement in negative direction) half period of the actor trap oscillation are regarded.
Here, ‘+’ and ‘‘ can be used as synonym for buckling and stretching. However, in more complex networks, one
filament can buckle while another one is stretched and a universal notion as used here is needed.
Fig. 80 shows bead displacements b(t) for actuation frequencies fa = 0.1Hz (Fig. 80A) and fa = 100Hz (Fig.
80B) for the actor (left) and sensor (right). Due to the symmetry of the experiment and same coordinate
systems of both traps, displacements are inverted to each other. The current state of the filament is indicated
by small insets and numbered according to Fig. 79. If the actor trap (trap 1) is moved in positive direction (first
half period in Fig. 80), the tensile strength of the filament pulls on the sensor bead (bead 2), thus it is displaced
in positive direction. The same tensile strength hinders bead one from completely following its trap and thus, it
is displaced in negative direction regarding the current trap center (see Fig. 79C). The spring constant MT =
²lpkBT / 2L³ ≤ 0.1pN/µm of the buckling microtubule is rather low compared to the force constants T ≈
25pN/µm typically used for the traps. This causes the microtubule to buckle during the second half periods in
Fig. 80A (fa = 0.1Hz) and the beads are only slightly displaced (b ≈ 10nm) from the trap centers. In particular,
this means that the actor bead follows the complete amplitude Aa of the sinusoidal movement of its trap
during each second half period. The situation becomes different at high frequencies as shown in Fig. 80B. Here,
the position signals b(t) are symmetric for both half periods. While the stretching amplitudes A+(100Hz) ≈
A+(0.1Hz) are approximately identical for low and high frequency, the microtubule seems to stiffen
tremendously since the buckling amplitudes A(100Hz) ≈ 10A(0.1Hz) increase by nearly an order of
magnitude.
Actor Sensor
A
2
1 4 b,2=0
b,1=/2 4
3 1 1 3
2
?
B 4
2
b,2>0
b,1˂/2 ?
4
2 3 1 1 3
Fig. 80. Bead displacements by,1(t) of the actor (left) and by,2(t) of the sensor
(right) during oscillation at (A) fa = 0.1Hz and (B) fa = 100Hz. Aa = 500nm in
both situations. Dashed lines indicate the position of the actor trap.
To address the frequency dependence of the amplitudes A+(fa) and A(fa), a custommade, automated
1
amplitude finding algorithm is applied to the signals by(t) of the complete parameter space (fa, Aa). The result
1
A sine fit would be the easiest implementation. However, automated fitting is prone to errors, especially for a large amount of data
points N to fit (e.g., for fa = 0.1Hz and sR = 25kHz, N = 250,000). Therefore, the maximum (or minimum) of every half period has been
108
4. Microtubules
is shown exemplarily for a filament with LMT = 5.3µm in Fig. 81A+B. The amplitudes A+ during stretching
display only a slight dependence on high frequencies and are approximately proportional to the actor
amplitude Aa. The latter is intuitive since larger oscillation amplitudes are equal to higher optical forces acting
on the beads whose displacements are hindered by the filament. In contrast, the amplitudes A during buckling
display a plateau for low frequencies and rise by an order of magnitude for frequencies fa > 1Hz. The rise of
the amplitude A of the displacement of the bead from the trap is equivalent to a decrease of the distance
= yL  Ay,1.  Ay,2 between both beads (see appendix 6.13.1). Thus, the filament buckles less which implies an
increased apparent stiffness MT ~ EI. Comparing the situation to Fig. 73B, the response of the sensor (bead 2)
cannot be explained purely by hydrodynamic interaction of both beads.
A 2 Stretching (A )
+ B 2 Buckling (A )
 C
100 100
(nm)
A y,2 (nm)
6 6
5 5
y,2
4 4
+

A
3 3
2 2
with MT
A y,1 /
/ y,1

+
10 10
A
without MT
6 6
5 5
4 Aa = 200nm 4 Aa = 200nm
3 Aa = 500nm 3 Aa = 500nm
Aa = 800nm Aa = 800nm
2 2
Fig. 81. Oscillation amplitudes. (A) Mean maximum amplitudes A+y,1 and A+y,2
of the actor and sensor displacement during the first half periods for the
stretched filament. (B) Mean maximum amplitudes Ay,1 and Ay,2 of the actor
and sensor during the second half periods for the buckled filament. (C)
Analytical solution and Brownian dynamics simulation of the overdamped,
driven coupled oscillator.
Analytically, the experimental situation can be modeled by a set of two coupled differential equations of
the total force:
1 T by ,1 yL ,1 B
d
dt
d
dt
1
2
by ,1 MT by ,2 by ,1 Fcrit MT LMT (by ,2 by ,1 ) 0
(4.31)
2
dt dt
1
2
T by ,2 yL ,2 B by ,2 MT by ,2 by ,1 Fcrit MT LMT (by ,2 by ,1 ) 0
d d
Fopt
F B F MT FMT
B = 6R is the viscous drag coefficient of a bead resulting in the viscous drag force FB. MT is a general
1
drag coefficient for the microtubule (see appendix 6.13.5) resulting in a drag force FMT if the filament’s ends
2
move relative to each other (e.g., during buckling). Fopt is the optical trapping force and FMT the buckling force
according to Eq. 4.1.1. As shown in appendix 6.13.2, the system can be rewritten in two decoupled differential
equations by introducing relative coordinates by,1 + by,2 and by,1 – by,2. The oscillation of trap 1 yL,1(t) = y0,1 +
Aasin(2fat) yields a driven, overdamped motion of both beads that can be expressed by
detected by a builtin method (wavestats) of the analysis software Wavemetrics Igor Pro. To reduce effects of the particle’s fluctuation,
points within a time window of T = 1 / 20fa around this maximum were averaged (indicated by the error bar of the xaxis in Fig. 80). This
yields a reliable method of finding the amplitudes A+ and A.
1
The rod shaped filament with length LMT and diameter DMT can be regarded as a cylinder with parallel and perpendicular viscous drag
coefficients MT,par = 2LMT / (ln(LMT/DMT) – 0.2) and MT,per = 4LMT / (ln(LMT/DMT) + 0.84). As discussed in appendix 6.10 and 6.13.5, due
to the shape of the buckled filament, the true viscous drag is not just a simply the sum of both parameters.
2
To account for filament stretching and buckling, different MT have to be used and both cases have to be treated separately.
109
4. Microtubules
by ,1 (t ) Ay ,1 sin 2 f a t b ,1 C y ,1
(4.32)
by ,2 (t ) Ay ,2 sin 2 f a t b ,2 C y ,2
Here, the amplitudes Ay,1(fa) and Ay,2(fa) as well as the phase lags b,1(fa) and b,2(fa) depend on the
frequency fa of the actor trap. Cy,1 and Cy,2 are constants determined by the boundary conditions of the
experiment, i.e., Aa, y0,1, y0,2, Fcrit, MT, T, but not on the frequency fa. The dependence of Ay,1(fa) and Ay,2(fa)
on MT and MT is analyzed systematically in appendix 6.13.2. Fig. 81C shows the course of both amplitudes Ay,1
and Ay,2 for the realistic set of parameters MT = 5MT,par ≈ MT,par + MT,per and MT = 0.1pN/µm of a
1
microtubule with length LMT = 10µm. Here, the analytical solution according to Eq. (4.32) is compared to the
result from a Brownian dynamics simulation of Eq. (4.31), additionally. Besides the fact that simulation and
analytical description overlap perfectly, the amplitudes of both, actor and sensor, exhibit the same plateau for
low frequencies and rise at a critical frequency fcrit ≈ 1Hz as observed in experiments. Even the absolute value
of A(fa) is approximately identical to the experimental values. The slightly faster rise of the sensor Ay,2
compared to the actor Ay,1 is not present in the experiments. However, the introduction of a friction for the
2
filament made in Eq. (4.31) is rather simple and might be too general . Remarkably, both amplitudes Ay,1 ≈ 0
and Ay,2 ≈ 0 are negligible if only MT ≠ 0 is considered and MT = 0 is set to zero. This implies that the viscous
drag of the filament is crucial for conducting a mechanical stimulus and that the apparent stiffening of the
filament for high frequencies is caused, at least to a large amount, by an increasing drag.
So far, all results still include the optical traps and beads, i.e., the trap stiffnesses and viscous drags. In
order to draw conclusions on the conduction of stimuli in vivo, these effects have to be subtracted. One way is
to use microrheology techniques as they were introduced in chapter 4.1.2. The apparent stiffening of the
filament should be visible in an increase of the elastic component G’(1,2) of the coupling between distant sites
‘1’ and ‘2’ (equal to the positions of both beads).
3 3 3
y0 = (1.7 ± 0.2)mPa 2
2 2
A = (0.14 ± 0.01)mPa/Hz y0 = (2.2 ± 0.8)mPa
(Pa)
(Pa)
(Pa)
(1,2)
(1,2)
y0 = (1.4 ± 0.3)mPa =
²/DOF = 0.22
= = =
G'
G'
A = (0.10 ± 0.03)mPa/Hz
G'
6 6
pow = 1.11 ± 0.47
6 ftrans
5 5 5
4 4 ²/DOF = 0.05 4
3 3 3
2 2 2
10µM Taxol 100µM Taxol
long filaments long filaments GMPCPP
ftrans short filaments ftrans short filaments long filaments
0.001 0.001 0.001
As shown in Fig. 82, G’(1,2) indeed rises by approximately an order of magnitude similar to the amplitudes

A (see Fig. 81B). Here, differently stabilized filaments have been analyzed since this has an effect on the
207
mechanical properties of microtubules ({Hawkins 2013} and chapter 4.2.1) and thus on conducting a
mechanical stimulus. Filaments can be grouped according to their length as shown for results of individual
filaments in appendix 6.13.4. The results shown in Fig. 82A+B are weighted averages of three to six single
filaments denoted as short (4.6µm ≤ LMT ≤ 6.0µm) and long (15µm ≤ LMT ≤ 25µm). For doubly stabilized
microtubules shown in Fig. 82C, only data for long filaments (10µm ≤ LMT ≤ 16.0µm) is available.
1
MT = ²lpkBT / 2L³ ≈ 0.1pN/µm follows from the theory of the buckling beam introduced in chapter 4.1.1. MT = 5MT,par is motivated
during the estimation of forces acting on both beads during an oscillation experiment given in appendix 6.13.5.
2
This is discussed in appendix 6.13.2 and 6.13.5. Further improvements are: accounting for the full slope of force profile as shown in Fig. 9
and integration of the hydrodynamic interaction as shown and discussed in chapter 4.1.2.
110
4. Microtubules
Measurements for different amplitudes Aa of the same filament are treated as individual measurements and
averaged, too, since no quantitative difference of the results for different Aa is found. Error bars are statistical
errors, i.e., standard deviations of the (weighted) mean. In order to analyze the frequencydependent rise of
1
G’(1,2), power law fits according to fitpow(x) = y0 + Axpow have been performed to obtain the principle slope of
(1,2)
G’ . Interestingly, the exponent pow implies a linear dependence (pow ≈ 1) for all Taxol treated filaments
and a quadratic dependence (pow ≈ 2) for GMPCPP stabilized microtubules. Similarly, the transition frequency
ftrans ≈ 3Hz, when G’(1,2) starts to rise, is approximately identical for all Taxol filaments but is one order of
magnitude larger (ftrans ≈ 25Hz) for GMPCPP filaments, indicating a link between the mechanical rigidity EI ~
G’ of the filament and ftrans. After the exponent has been determined by the power law fit, corresponding line
(fitline = y0 + Ax) or parabolic (fitpar = y0 + Ax²) fits (with fixed exponent) were applied to determine the slope A
and plateau value y0 = G’(1,2)( f < ftrans).
Here, stabilization seems to have no effect on the plateau value y0 ≈ 2mPa for long filaments, while the
slope is different only for Taxol and GMPCPP treated filaments due to different power laws. For short
filaments, only the lower Taxol concentrations exhibit the previously observed rise of the curve, while it
scatters around a constant value of G’ ≈ 0.02Pa for a 100µM concentration (fa = 50Hz and fa = 100Hz were
excluded from the fit shown in Fig. 82B). Comparing long and short filaments for 10µM Taxol, y0,short ≈ 2y0,long as
well as Ashort ≈ 2Along differ by a factor of two indicating a lengthdependent behavior. As explained in chapter
4.4, both observations are likely due to a larger value Fcrit(L) for short filaments of the lengthdependent
critical force and an overall increased stiffness of filaments treated 100µM Taxol compared to 10µM.
A 5 B 5 C 5
4 4 4
3 3 3
2 2 2
y0 = (24.2 ± 0.4)mPa
A = (1.16 ± 0.14)mPa/Hz
0.1 0.1 0.1 pow = 1.15 ± 0.34
²/DOF = 0.68
(Pa)
(Pa)
(Pa)
6 6 6
5 5 5
(1)
(1)
(1)
= = =
4 4 4
G'
G'
G'
3 3 3
2 2 2
The local elastic components G’(1) shown in Fig. 83A+B do not display a clear frequency response for Taxol
stabilization, rather they scatter within approximately one order of magnitude. Doubly stabilized filaments
(Fig. 83C) again behave differently by exhibiting a clear linear rise (pow ≈ 1) for ftrans ≈ 6Hz which is below the
corresponding value of the global components G’(1,2).
An interesting behavior display the viscous components G’’(1) and G’’(1,2) shown in Fig. 84. While the local
components G’’(1) are larger than the viscous part of water and no difference between different length and
stabilizations is observed, the global components G’’(1,2) are below the corresponding slope of water and
''(1,2)
display a length dependence. The general fact than GMT GH''(1,2)
2O
seems to be absolutely counterintuitive. An
explanation is given in chapter 4.4.
1
Materials are typically grouped according to their power law behavior. Typical examples are glassy, rubber or fluidlike materials with
similar properties as glass, rubber or water.
111
4. Microtubules
A B
= =
Fig. 84. Local (A) and global (B) viscous components G’’(1) and G’’(1,2).
Lateral oscillation
During lateral oscillation, the filament is stretched the whole time and is therefore under tensile forces. The
principle behavior can be expected to be the same as for a longitudinal oscillation, but the quantitative
1
measures very likely differ. For this type of experiments, only data of long filaments (10µm ≤ LMT ≤ 16µm)
stabilized with 100µM Taxol and GMPCPP is available. Data was analyzed the same way as explained above,
i.e., results for different amplitudes Aa of single filaments are averaged and these results are the summarized
by a weighted mean of different filaments. The result is shown in Fig. 85.
A 1 B 5
C
4
3
y0 = (0.58 ± 0.05)mPa y0 = (27 ± 7)mPa
A = (85 ± 2)µPa/Hz 2 A = (16 ± 6)mPa/Hz
0.1 B = (16 ± 1)µPa/Hz pow = 0.37 ± 0.07
pow = 1.53 ± 0.02 ²/DOF = 3.04
²/DOF = 40 0.1 ˫
(Pa)
(Pa)
7
6
0.01 5
(1,2)
(1)
˫ ˫ 4
G'
G'
3 ˫
2
0.001
0.01
ftrans GMPCPP 7 GMPCPP
long filaments 6 long filaments
0.0001 5
For both elastic components, global (Fig. 85A) and local (Fig. 85B), there is a clear frequency dependence
but less pronounced than for the longitudinal oscillation. The exponent pow ≈ 1.5 of the rise of the global
elastic component G’(1,2) is lower than the quadratic behavior of the longitudinal oscillation of this type of
filaments. Since this rise is between a linear and a quadratic dependence, a third order polynomial fitpoly = y0 +
Ax + Bx² fit was applied. The relative amplitudes of the linear and the quadratic term A ≈ 85µPa/Hz ≈ 5B and
B ≈ 16µPas/Hz indicate that both dependences are of importance. The transition frequency ftrans ≈ 3Hz is one
order of magnitude lower than for longitudinal oscillation. The local elastic component G’(1) displays only a
1
Three measurements in total.
112
4. Microtubules
slight frequency response with an exponent pow ≈ 0.37. The local and global viscous components G’’(1) and
G’’(1,2) are similar to the case of longitudinal oscillation, i.e., GMT
''(1,2)
GH''(1,2)
2O
''(1)
and GMT GH''(1)2 O .
Final remarks
For an isotropic material, the Young’s modulus E and the elastic component G’ are related by Poisson’s
ratio P. As mentioned in the introduction of chapter 1, microtubules are mechanically anisotropic, thus a
211
simple conclusion form G’ on E cannot be drawn. {Pampaloni 2006} found a ratio E / G’ ≈ 106. Here, for lp ≈
2mm as determined in chapter 4.2.1 and 4.2.2, EI = 10 Nm² = 10pNµm2 follows. With I = 1.6·1032m4 (see
25
footnote 2 on page 87), E ≈ 6·106Pa and thereby E / G ≈ 6·108 is derived, highlighting the mechanical
anisotropy of these filaments.
In longitudinal oscillation experiments, the filaments are stretched and buckled during oscillation which
results in a superposition of local filament bending and global buckling in the results of microrheology
analyses. This effect is further argued in the discussion (see chapter 4.5).
The schematic of the experiment is shown in Fig. 86. Three traps are used to establish a network of two
microtubules with length LMT ≈ 10µm in series (100µM Taxol stabilization). The configuration is aligned parallel
to the ydirection. The chapter is again divided in two parts: an oscillation in longitudinal (y) and lateral (x)
direction. Here, only the results of a twoparticle active microrheology (2pAMR) analysis are shown. This
method only allows to investigate connections to the actor, thus only the complex viscoelastic components
G(1,2) and G(1,3), but not G(2,3), can be analyzed.
z
x
MT 12 MT 23
Fig. 86. Schematic of experiment. (Top) Three traps are arranged in a line.
Trap 1 is used as actor to apply a sinusoidal force on the network consisting
of two filaments in series. Bottom: corresponding fluorescence image during
actual experiment. Scale bar 5µm.
113
4. Microtubules
Longitudinal oscillation
The global elastic components G’(1,2) and G’(2,3) shown in Fig. 87A display the same power law behavior
with exponent pow ≈ 1 but different transition frequencies ftrans (1,2)
≈ 2Hz and ftrans
(1,3)
≈ 6Hz. The former is similar
to ftrans ≈ 3Hz of a single filament as shown in Fig. 82B. The much larger plateau value y0 ≡ G’(1,3)(f < ftrans) of
the connection 13 compared to y0 ≡ G’(1,2)(f < ftrans) of the first connection together with the larger transition
(1,3)
frequency ftrans ftrans
(1,2)
is likely an effect of a doubled critical force for two filaments compared to a single (see
chapter 4.4). Another effect is the second bead itself, which connects both filaments. By acting as a hinge
which is free to rotate and move, this gives the system another degree of freedom.
(1) (1) 1
The local elastic components G ''1,2 and G ''1,3 scatter less than their single filament counterparts (Fig. 83B)
(1)
and seem to be constant within a factor of two. G ''1,2 shows a slight frequency dependence. A power law fit
fitpow revealed an exponent pow ≈ 0.26 ± 0.14 but this has been omitted from the graph due to a lack of
significance.
The local and global viscous components display the same behavior than for single filaments except for the
global component G’’(1,3) of the connection 13. This can again be addressed to bead two, which dissipates
more energy than a piece of a microtubule filament of equivalent size.
A B 5 C
4
4
y0 = (29 ± 3)mPa
2
A = (1.42 ± 0.06)mPa/Hz
pow = 0.83 ± 0.12 3
=
0.1
8
²/DOF = 10.8
6
2
4
(Pa)
G' (Pa)
(i,j)
2
(i)
= =
G'
=
0.01
8
6 0.1
(1,3)
4 ftrans 9
8
y0 = (5.3 ± 0.2)mPa
A = (0.64 ± 0.4)mPa/Hz 7
2
pow = 1.13 ± 0.15 i = 1, j = 2 6 i = 1, j = 2
²/DOF = 1.89 (1,2)
0.001
ftrans i = 1, j = 3
5
i = 1, j = 3
Lateral oscillation
The lateral components also display the same qualitative dependence as single filaments. Due to different
stabilizations (single filaments: 100µM Taxol + GMPCPP; here: 100µM Taxol), results cannot be compared
quantitatively. Single GMPCPP filaments displayed a larger transition frequency (ftrans ≈ 6Hz, ftrans ≈ 3Hz) and
smaller exponent (pow ≈ 1.0, pow ≈ 1.5) in the longitudinal global elastic component compared to the lateral
direction. The principle situation here is the same regarding the exponents pow ≈ 1.0 and pow ≈ 2.0. Since the
transition frequencies are clearly different for the connection 12 and 13 in the longitudinal direction, a
definite conclusion is hardly possible, but transition frequencies seem to be slightly larger in lateral direction
contrary to single filaments.
1
The local component is always probed at the position of the actor. However, as given in chapter 4.1.2 and expressed by Eqs. (4.6) as well
as (4.10), the local component G(i) is determined by the inter particle response function A(i,j) as well, and thus two different G(i) can be
(1) (1)
estimated. However, a significant difference between G1,2 and G1,3 makes no physical sense.
114
4. Microtubules
The local elastic components G’(1) display the same qualitative and quantitative behavior for both
connections with an exponent pow ≈ 0.75 twice as large as for single GMPCPP filaments. The transition
frequencies ftrans ≈ 3Hz are identical, too.
So far, the local and global viscous components were always symmetrically above and below the
corresponding component of pure water in all experiments. Here, the local component G’’(1) overlaps with
the viscous component of water and the global components G’’(i,j) differ by approximately an order of
magnitude between both connections.
A 1
10 B 0.1 C
9
y0 = (1.56 ± 0.10)mPa
8
A = (6.4 ± 0.6)µPa/Hz
2
pow = 2.26 ± 0.17 7
²/DOF = 4.17 y0 = (39 ± 1)mPa
10 ˫
6 A = (0.51 ± 0.04)mPa/Hz
pow = 0.78 ± 0.26
²/DOF = 0.56
(Pa)
G' (Pa)
5
3
10
(i,j)
(i)
˫ ˫
G'
4
˫
Final remark
Unfortunately, only a single measurement for this configuration is available. The significance of the findings
shown here is therefore arguable, but the slopes are clear and distinct. Nevertheless, the measurement has to
be repeated to check for reproducibility.
Here, a welldefined configuration of three traps in a distance D xL2 yL2 = 15µm is used to set up an
equilateral triangular network of microtubules as illustrated in Fig. 89. In this configuration, a pure longitudinal
and lateral oscillation, as it was performed in the foregoing experiments, is not possible, since the three
filaments are not aligned in parallel. However, oscillation is performed again along the x and yaxis resulting in
oscillations in directions radial and tangential (shown in Fig. 89) to the connection between the center of mass
of the network and the actor bead. Due to this symmetry and the fact that filaments buckle in one direction
but are tense in the other, an oscillation in x and ydirection cannot be used as orthogonal coordinate system
to conclude on material parameters parallel and perpendicular to each filament.
The analysis shown here bases on two different measurements of identical networks with different
filaments. Additionally, two different laser powers, resulting in different trap stiffnesses, were used for one of
the networks, resulting in three measurements in total.
115
4. Microtubules
Tangential oscillation
Position scatter plots in the plane of oscillation of an oscillation in xdirection at fa = 100Hz and Aa = 600nm
are shown in Fig. 89 for all three beads. All axes are true to scale and spacing of different graphs is identical.
Positions are colorcoded over the current phase a of the actor trap’s oscillation. The direction (2 = 32° and
3 = 38°) of oscillation of the sensor beads is roughly in direction ( = 30°) of the microtubule spanning to the
actor. All beads move on a slight ellipsoidal trajectory caused by buckling of the filament during one half period
of the oscillation and stretching in the second half period.
600
Trap 3 static
100 3 ≈ 38
xL,3(t) = x0,3 50
bx,3 (nm)
400 0
50
100
0 Trap 1 oscillating
50
xL,1(t) = x0,1 + Aasin(2fat) bx,2 (nm)
50
100
Bead 3
200
100 50 0 50 100
by,2 (nm)
Color bar
1.0
Bead 1
400 0.8
a (2)
0.6
0.4
0.2
Bead 2
0.0
600
40 0 40
by,1 (nm)
Surprisingly on the first glance, no clear frequency dependence is visible in the microrheology results
shown in Fig. 90. The elastic components are constant with approximately an order of magnitude difference
between the local and global part G’(i) and G’(i,j). A difference of one to two orders of magnitude between
these two quantities has also been observed in single filament measurements. Data for the connection
between bead one and two scatters less than for connection one to three which is likely due to a slight
116
4. Microtubules
asymmetry of the system originating from the construction process of the network rather than being
systematically. This (slight) asymmetry is also visible in the oscillation signals b shown in Fig. 89.
The viscous components G’’(i) and G’’(i,j) display the same qualitative frequency dependence as for single
filaments. Quantitatively, both components differ by approximately one and a half to two orders of magnitude
which is the same in single filament experiments. However, both components are shifted relative to the
corresponding component of water, similar to the linear network.
A 2 B 2 C
0.1 1 ˫
8 8
6 6
4 4
(Pa)
G' (Pa)
2 2
(i,j)
˫
(i)
˫
G'
˫
0.01 0.1
8 8
6 6
4 4
i = 1, j = 2 i = 1, j = 2
i = 1, j = 3 i = 1, j = 3
2 2
Radial oscillation
Individual frames captured at the extremes of one period during an oscillation in ydirection (fa = 0.1Hz and
Aa = 600nm) of a combined movie of fluorescence and brightfield illumination are shown in Fig. 91. The
exposure time of the camera was Texp = 100ms, thus snapshots of the nearly static situation of the network are
made.
Relaxed
Tense
+ + + +
x
y
For t = 0 and t = T/2 = 1/2fa, the network is in its initial position, thus all filaments are relaxed. After one
quarter of the oscillation (t = T/4), the actor bead is displaced maximal and the filaments attached to it (MT12
117
4. Microtubules
and MT13) are tense. A careful comparison to the initial state reveals that the third filament (MT23), spanned
between bead two and three, buckles slightly. This is caused by a net shortening of the distance b between
bead two and three in ydirection by ≈ 200nm. The exact opposite is the case at t = 3/4T, i.e., when the actor is
displaced maximal in direction towards the other beads. The first two filaments are buckled, causing beads
two and three to displace away from each other, thus stretching the third filament.
An interesting question, also regarding single filaments, is if filaments still buckle at high frequencies, e.g.,
fa = 100Hz. This is shown in Fig. 92. Here, the oscillation frequency was fa = 50Hz and the exposure time of the
camera Texp = 100ms. Thus, five complete oscillations are averaged in a single camera frame. The oscillation of
the actor bead is clearly visible while both microtubules connected to the actor definitively also buckle during
oscillation. However, the buckling amplitude cannot be estimated because it is smeared during the five
oscillations and filaments likely also buckle out of the plane of focus as indicated in Fig. 91. As argued in
chapter 4.2.3, an increasing viscous drag at high frequencies is likely a major effect of the response of single
filaments or networks analyzed here. A quantitative measure of the filament buckling amplitude would be an
experimental proof.
Tense
The microrheological results for a radial oscillation shown in Fig. 93 are qualitatively the same as for the
tangential direction. Both elastic components G’(i) and G’(i,j), do not display any distinct frequency response
and differ by approximately one order of magnitude, similar to single filaments. Except of a slightly different
gap in the distance between local and global viscous components G’’(i) and G’’(i,j), the result here is identical
to the tangential case, as well.
A 2 B 2 C
0.1 1 =
8 8
6 6
4 4
(Pa)
G' (Pa)
2 2
(i,j)
(i)
= =
G'
=
0.01 0.1
8 8
6 6
4 4
i = 1, j = 2 i = 1, j = 2
i = 1, j = 3 i = 1, j = 3
2 2
The reason for the qualitatively completely different behavior compared to single filaments or the linear
network can be motivated: in single filaments experiments, one period of the longitudinal oscillation (y) is a
superposition of stretching and buckling of the filament. Here, the same is true for the filaments attached to
the actor (MT12 and MT13), but the filament spanning between both sensors (MT23) is always deformed
118
4. Microtubules
the opposite way during each half period as explained above and illustrated in Fig. 91. In lateral oscillation (x)
experiments of a single filament, the microtubule is stretched during both half periods. Here, one filament
attached to the actor always buckles while the other one is stretched at the same time. This results in a
superposition of buckling and stretching; not only during a complete oscillation period (as it is the case during
longitudinal oscillation of a single filament), but during each half period, as well. Thus, the principle
deformations of the triangular network are completely different to a linear configuration.
6) The global viscous components G’’(i,j)(f) depends on the length of the filament LMT with G’’(i,j)(LMT =
5µm) < G’’(i,j)(LMT = 15µm).
All of these points, except of the last, can be explained by introducing a frequency ( = 2f / 0) dependent
filament stiffness ( ) = 0 pow’ in the else purely viscous true response function = i /
Here, 0 = 1Hz is a scaling constant ([] = m / N) and 0 = H2O + MT is introduced as friction of the system
microtubule (MT) immersed in water (H2O). As pointed out in chapter 4.1.2, the latter is different for a bead
(B) with radius R locally or the hydrodynamic coupling (HC) between beads separated by the distance L
globally, i.e., H2O = B = 6R and H2O = HC = 4L. MT = 2L / (ln(L/D) – 0.2) (≈ 0.35L for LMT = 10µm)
is the viscous drag of a cylinder with diameter D = 25nm and length L = LMT in longitudinal direction of the
filament axis. The local and global viscoelastic components G(i) = 1 / 6R(i) = 1 / (i)(i) and G(i) = 1 / 4L(i,j) =
1 / (i,j)(i,j) can be obtained from the true response function according to Eq. (4.10), resulting in
Here, = (i) and = (i,j) for the local and global component is used as shorthand. The experimentally
observed power law dependences (observation 1) and 4)) follow directly from a comparison of the nominators
to denominators as given by the right hand side of Eqs. (4.34) and (4.35). This is not surprising regarding the
dependence on of the real and imaginary part as it was introduced in the true response function in Eq. (4.33)
, but illustrates that the frequency dependence of the elastic and the viscous component of G can be tuned
119
4. Microtubules
independently. However, a possible length dependence of the stiffness 0(L) and drag 0(L) influences both
parts, real and imaginary. The first is expressed according to {Pampaloni 2006} and chapter 4.1.1: 0 = ∞ /
211
(1 + l’ / L ) and = lp kBT / 2L with the persistence length lp of filaments with length L » l’ much larger
2 2 ∞ 2 ∞ 3 ∞
than the critical length l’ (see appendix 6.13.6 for corresponding plots). Both values can be varied to study
their impact on the viscoelastic components as shown in Fig. 94. A lower persistence length lp∞ (solid
compared to dashed lines) results in a lower spring constant 0 and thus in a higher amplitude of the elastic
component G’ because of its ultimate dependence on 1/0. In order to explain the plateau value and transition
frequency ftrans observed in experiments, a constant term motivated by the critical buckling force Fcrit is
introduced additionally:
According to chapter 4.1.1, Fcrit = 20L = 2lp∞kBT / L2, thus shorter filaments have a higher critical force
1
resulting in a larger plateau value G’(f = 0) (≡ y0 for the power law fits in chapter 4.2.3). The results shown in
Fig. 94A correspond perfectly to the measurements (see Fig. 82) for long filaments (red, LMT = 15µm) by
displaying the same plateau value and transition frequency. Besides, there is no significant difference for
2
differing persistence lengths or transition frequencies . However, both quantitates have a clearly perceivable
effect on short filaments (LMT = 5µm). Since the local elastic component only reflects the mechanical
properties of a short piece of the filament (L ≈ R), its plateau value is high and ftrans > 100Hz, which is not
resolved in the experiments shown in chapter 4.2.3. Taken together, the critical force Fcrit and 0(L) explain
observations 2) and 3).
Neglecting the stiffness 0 in Eq. (4.33) for a short while allows to estimate the impact of the drag from the
microtubule on the amplitude of the viscous components, i.e., G’(i) = G’(i,j) = 0, G’’(i) = i0 / (i) and G’’(i,j) =
i0 / (i,j)
B MT 0.35 L
G ''(i) ( ) i 0 i 1 0 i 2.20 2.2G ''H O
6 R 6R 2
MT 0.35
G ''(i, j) ( ) i HC 0 i 1 0 i1.10 1.1G ''H O
4 L 4 2
affects the global component G’’(i,j) only little (≈ 10%) but has a strong impact on the local component
MT
(i)
G’’ (≈ 120%). This is a first indication for an explanation of observation 5), i.e., that the local viscous
component G ''(i ) ( f ) G ''(Hi ) O ( f ) is larger than the viscous part of water.
2
As shown in Fig. 94B, the viscous component G’’ clearly depends on the length of the filament but only
little on its mechanical properties. Especially, the global viscous component G ''(i , j ) ( f ) G ''(Hi ,2jO) ( f ) is smaller
than the viscous component of water due to its dependence on 0(L) and 0(L) (see Eq. (4.35)). Thereby,
observation 5) can be explained, too.
Observation 6), i.e., G’’(i,j)(LMT = 5µm) < G’’(i,j)(LMT = 15µm) cannot be explained by the considerations
made here. However, similar to the analytical description of bead signals in chapter 4.2.3 (Eq. (4.31)), only a
drag in parallel direction of the filament is considered so far. The perpendicular component likely plays an
important role especially during buckling, but a correct treatment of the complete viscous drag of the filament
depending on its current buckling amplitude is complicated as outlined in appendix 6.10. Secondly, filaments
are stretched and buckled during oscillation experiments. This effect has also not been addressed so far.
1
This is also confirmed in the static buckling experiments shown in chapter 4.2.1.
2
The pairs of values chosen in Fig. 94 are motivated by the findings in chapter 4.2.1 and the work presented in {Pampaloni211 2006}.
120
4. Microtubules
A B
Fcrit
Fcrit
Fcrit
Generation of an array of dynamical optical traps: to create an array of static traps, the laser focus jumps
between individual focal positions in a circulating manner, resting at each position for on = 20µs = 1 / fAOD,
which is the reciprocal of the AOD deflection frequency fAOD = 50kHz. Dynamical traps, i.e., each moving on a
defined trajectory yL,j(t) (j = 1, …, N), are created by splitting the individual trajectories yL,j in pieces with
length on and combining them sequentially to account for the jumping of the focus between different traps. In
this way, traps moving on arbitrary spatial trajectories (e.g., linetraps or circular traps) with arbitrary time
constants (e.g., frequency fa of a line trap) can be combined with multiple static traps at flexibly tunable
positions.
Position tracking of an array of beads: back focal plane interferometry has been used to track the bead’s
positions. To account for the jumping of the laser focus between different traps, the position signals Siraw(t) (i
= x, y, z) obtained from the quadrant photo detectors are split in pieces of length on and sorted corresponding
to individual traps. To account for the finite propagation velocity of the ultrasonic wave through the crystal of
121
4. Microtubules
the AOD, data points corresponding to the first 7µs of each piece are cut off. By averaging the signal Siraw(t)
over the remaining length of each piece, the position signals Si,j(t) with temporarily equidistant sampling
points for each trap are obtained.
Calibration: similarly to the calibration of Spiroplasma, a combination of fast laser scans and Langevin
calibration is used here. In particular, the fluctuation of each bead is hindered by a strong point trap while it is
scanned by a fast (fa = 500Hz, Aa = 500nm) and weak sawtoothlike trap. In this way, the detector sensitivity g
is determined. The yintercept of the autocorrelation of fluctuation data of the calibrated position of each
bead is then used to determine the trap stiffnesses of each trap.
Motivation for this approach: this is advantageous because of several reasons. First, due to the angle
dependent diffraction efficiency of the AOD, the laser intensity depends on the position of each trap. This
necessitates a force and position calibration for each change of trap positions. Secondly, a broad distribution
of filament lengths LMT often necessitates repositioning of at least one trap during the assembling process of
the beadfilament construct. This necessitates calibration after the bead construct is assembled. Third,
filaments attached to beads impair their fluctuation and thus the autocorrelation (AC) time ac and the corner
frequency c = 1 / ac of a power spectral density (PSD). Thus, a calibration approach is needed which does not
depend on these two parameters. As demonstrated here (chapter 4.1.5), all three prerequisites are met by the
abovementioned calibration procedure.
Technical limits: timemultiplexed optical trapping and tracking is mainly limited by the beam steering
device. In the present configuration, the optical grating inside the AOD, caused by the acoustic wave, needs
acc ≈ 7µs to be fully established. This limits the maximal AOD deflection frequency to fAOD = 1 / acc ≈ 140kHz
and ultimately the maximum sampling rate sR = fAOD / N for the position tracking of N beads. The maximum
number of parallel trapped beads mainly depends on the trap stiffnesses of each trap, and thus on the
maximum intensity of the laser. A P = 10W laser as used in the present work, should in principle allow to trap
and track 100 beads or more.
Comparison to other techniques: the generation of N static, timemultiplexed traps has been
101
demonstrated by {Ruh 2011} earlier. Here, this approach has been extended to dynamical traps, i.e., each
moving on a defined trajectory yL,i(t) (i = 1, …, N) over time as described above.
Holographic optical tweezers: the only other technique which is able to flexibly create and alter an
arbitrary array of optical traps is holographic optical traps (HOT), created by spatial light modulators (SLM).
SLMs are typically limited by the maximum frame rate f ˂ 100Hz the liquid crystal array can be refreshed or
1
updated. In the present case, this is problematic regarding the sinusoidal oscillation of the actor bead for
frequencies fa > 50Hz. The maximum frequency used in the experiments shown here was fa = 200Hz.
Video tracking: the position of beads could also be tracked by videobased tracking methods. This has the
advantage that the sampling rate does not depend on the number of traps as it is the case in back focal plane
tracking. However, video tracking algorithms have two major disadvantages. First, although threedimensional
particle tracking is possible in principle, it relies on complicated algorithms or techniques especially in axial
direction. Secondly, high sampling rates for spatially large bead arrays necessitate cameras with high frame
rates for large fields of view. Using an array of 100 beads, for example, timemultiplexed tweezers in the
present technical configuration would still be able to achieve a sampling rate sR = 1.4kHz which is hardly
possible with any camera. However, regarding the experiments presented in this thesis, only lateral
dimensions of bead displacements were tracked and the maximum oscillation frequency for microtubule
experiments was fa = 200Hz. Thus, although at the limit, videotracking would have been an alternative
method, too.
Conclusion: timemultiplexed optical tweezers combined with back focal plane interferometry are superior
to holographic optical tweezers combined with videotracking, concerning their application to three
dimensional problems, tracking precision and temporal resolution.
1
According to the Nyquist theorem, a signal with frequency f has to be sampled at least with rate 2f.
122
4. Microtubules
Microrheology techniques have been used to analyze the frequencydependent response of single
filaments and networks thereof. The method is wellestablished and tested by various publications
229 230 241
{Atakhorrami 2006; Mizuno 2008; Buchanan 2005}. However, like all rheology techniques, it is usually
used to research bulk material properties, e.g., solutions of proteins. It is the first time that this technique is
applied to single filaments or small, defined structures. A further adaption of the method might be necessary,
also to better reflect the network topology.
Description of the method: the particle displacements bs(t) of a sensor bead as response to a sinusoidally
applied oscillating optical force Fa(t) on an actor bead is analyzed in Fourier space. The resulting apparent
response function A( ) bs (t ) / Fa (t ) reflects the viscoelastic properties of the material under investigation,
the shape and size of the probe (bead) and the properties of the optical traps. The latter two can be subtracted
according to Eqs. (4.6)  (4.10) resulting in the complex function G() = G’( ) + iG’’( ) reflecting the elastic
(G’) and viscous (G’’) properties of the material only.
Test of the method: in order to test this technique, Brownian dynamics simulations of active one and two
particle microrheology (1pAMR and 2pAMR) as well as passive single particle microrheology (1pPMR) of
beads in water have been compared to experimental results, which agreed well. For 2pAMR, the
hydrodynamic coupling between beads even at a separation distance of d > 10µm was clearly visible in the
interferometric position signals. This effect is predicted by theory because coupling decays monotonically (1/d )
with the separation distance d. However, since water is ideally viscous, isotropic and homogenous, GH2O( ) =
i is purely imaginary and independent of direction or separation distance. This has been confirmed by
experiments, serving as ideal test for the analysis of microtubule experiments because the experimental
situation is similar.
Alternative methods: twoparticle passive microrheology (2pPMR) could be used instead of 2pAMR, as
well. In 2pPMR, the thermal fluctuations of two beads are compared by calculating crosscorrelation power
spectrums. Since correlations and power spectrums are analyzed over the complete frequency range, this
allows analyzing the frequencydependent response over a much larger bandwidth. Although thermal forces
might be not strong enough to get significant and distinct results, 2pPMR has the potential to replace or
complement 2pAMR and should be tested. Instead of performing microrheology analysis in Fourier space, the
mean square displacement (MSD) is often used for an analysis in time domain. However, to the best
knowledge of the author, only oneparticle methods have been reported so far, which is not sufficient to probe
frequencydependent mechanical properties of extended microtubules.
In order to obtain the mechanical rigidity of microtubules which is important to interpret results of
rheology experiments, filaments have been investigated under slowly varying compressive and tensile forces.
Filament buckling: a straight beam or filament buckles under a compressive mechanical load. Here, a
description from classical mechanics based on the Euler beam approach is used to determine the rigidity to
227 228
bending of microtubules {Gross 2009; Gross 2007}. A perfect beam bears its load and stays straight until
the applied force F > Fcrit is larger than a critical force Fcrit = 2EI / L2. If buckled, the filament’s deflection w(x)
can be described by a sine function according to Eq. (4.1). For a nonperfect beam, e.g., due to a nonaxially
applied load, the beam already starts to buckle if F < Fcrit, but the deflection amplitude is small. In the
experiments shown here, beads are attached laterally to the filament and are free to rotate in the optical trap.
Thus, the situation resembles a nonprefect beam. In order to get the true projected length pL of the
compressed filament, this rotation of beads has to be taken into account as shown in chapter 4.2.1.
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4. Microtubules
Experimental results: the compressive load on the filament is increased slowly but steadily by the trapped
beads. The resulting force compression curves F(pL) showed to increase rapidly for small pL < 200nm until a
threshold is reached. To properly identify this threshold, which is identified with Fcrit, F(pL) was fit by an
exponential function f(x) = Fcrit (1  exp(x / x0)) + mx. The last term accounts for the slow rise of the force for
large compression pL > 200nm, corresponding to a high compliance of the already buckled filament. The
resulting critical force Fcrit(LMT) showed a clear dependence on the filament length LMT as predicted by theory.
In order to determine the persistence length lp = EI / kBT as a measure for the material’s lateral elasticity, two
different models for lp were fit to the results of Fcrit. The first model assumes lp = const. while the second
211
addresses the question of a lengthdependent persistence length as it was demonstrated by {Pampaloni
2006}. Both fits revealed lp ≈ 2mm with a similar significance for filaments stabilized with 100µM Taxol. Thus,
a lengthdependent persistence length can neither be excluded nor can it be clearly confirmed.
207
Discussion of results: as mentioned by {Hawkins 2013}, the observation of a lengthdependence might
by an effect of suspension of the filament’s ends. These observations are typically made during analysis of
shape fluctuations of the filament caused by thermal forces. Besides the absence of a suspension of at least
one end, filaments are bent laterally and thus not restricted to F > Fcrit. This is a major principle difference to
the approach shown and used here.
Comparison of technique: a similar method using two optical traps and the same principle theory of
204
filament buckling has been demonstrated by {Kikumoto 2006}. However, the authors used video microscopy
to measure escape forces when beads are pushed out of their traps due to the resistance of microtubule
filaments. The approach shown here allows measuring the applied force during the whole experiment and
beads are not pushed out of their traps. Therefore, experiments on the same filament can be repeated several
times, also with different parameters for the trap stiffness for example. Thus, the present approach is more
flexible. (For a comparison of results see further down)
1
Filament stretching: microtubules have a high resistance to tensile forces and stretching is negligible. Due
to the lateral attachment of beads, filaments are bent locally at the point of attachment during stretching
experiments. This local bending has a much higher resistance to applied forces than the global bending of
buckling filaments as described above.
Experimental results: force extension curves F() with the dimensionless variable reflecting the current
distance b between beads were fit according to a special fit function given by Eq (4.30). Fits including and
excluding a length dependence of lp again did not reveal a clear length dependence. However, taxol stabilized
microtubules exhibited a persistence length lp ≈ 0.5mm, while doubly stabilized GMPCPP filaments with lp ≈
2.5mm are approximately five times stiffer.
Discussion of results: the experimental configuration probes the mechanical properties of filaments only
locally on the scale of the bead radius (LMT ≈ R ≈ 500nm). This is also illustrated by the inset of Fig. 77C which
192
is motivated by the description of axial and lateral intertubulin connections by {Pampaloni 2008}. Similar to
the description of stretching and compression of fibers above and below the neutral fiber in a laterally bent
beam, tubulin bonds are stretched and compressed heavily, but only locally.
Comparison of technique: stretching of microtubule filaments has only been performed by {van
218
Mameren 2009}. The authors used the same technique as shown here, i.e., two optical traps combined with
back focal plane interferometry. In contrast to timemultiplexed traps created from the same laser source, the
authors used two independent lasers for trapping which is less flexible but has some advantages, too, such as
reduced noise. The fit function according to Eq (4.30) was introduced by van Mameren et al.
1
{van Mameren218 2009} estimated a resistance to stretching of ≈10pN/nm for 10µm long filaments based on the stretching stiffness
≈50pN/nm of 1µm long actin filaments. Their argument was that the stiffness directly scales with the length and has to be doubled for
microtubules since they have double the crosssectional area compared to actin. This attempt seems to be too simple. Actin filaments
consist of two protofilaments while microtubules are typically assembled by thirteen protofilaments. So the number of bonds per cross
section is much higher. Secondly, individual bonds are likely different for actin and tubulin, thus also their resistance to stretching.
However, these considerations imply that microtubules have a stretching stiffness in the order of >10pN/nm. In the experiments shown
here, the maximal applied load was 50pN, so the filament has surely stretched by less than 5nm which cannot be separated from filament
bending.
124
4. Microtubules
Comparison of results: The results for buckling and stretching of filaments obtained here agree well to the
193
brought range of 0.5mm ≤ lp ≤ 10mm reported so far by various publications (see {Hawkins 2010}). Different
207
persistence lengths for filaments stabilized by Taxol or GMPCPP has been reported previously {Hawkins
2013} and is likely due to the integration of GMPCPP into the microtubule structure since it replaces GTP
during polymerization, in contrast to Taxol. The theoretical details are still under discussion.
Neutravidinbiotin linker elasticity: the effective elongation of streptavidin is on the Angstrom scale even
242
at applied forces of 100pN and more {Wong 1999}. The rupture force Fr of avidin strongly depends on the
243 244
temperature {Lo 2002}, the rate of applied force {de Odrowąż Piramowicz 2006} and ranges between
100pN ≤ Fr ≤ 400pN. Here, maximum forces 50pN < Fr were used which is clearly below the rupture force.
Assuming similar properties for the related neutravidin protein, the elasticity of the beadfilamentattachment
245
can be neglected. Since microtubules have a negative surface charge {Heald 2002}, neutravidin is used here
for biotin binding because it has a neutral isoelectric point.
The oscillation frequency fa in these experiments was typically swept over three orders of magnitude, i.e.,
0.1Hz ≤ fa ≤ 100Hz in nearly logarithmic steps. Additionally, experiments were typically carried out for three
different oscillation amplitudes Aa = 200nm, 400nm and 600nm.
General experimental observations: for pushing forces (second halves of the oscillation periods), already
the displacement of beads from their trap centers showed a clear dependence on fa. At low frequencies fa <
1Hz, beads were displaced only little (≈10nm) from the trap centers meaning that filaments buckle strongly. At
high frequencies fa > 10Hz, beads were displaced strongly (≈100nm) from trap centers, indicating that
filaments buckle less; an apparent frequencydependent stiffening. The maximum bead displacements were
approximately proportional to the oscillation amplitude Aa ~ Fa as a function of the applied force.
For pushing forces (first halves of the oscillation periods), the relative bead displacements did not show a
distinct dependence on fa and scaled approximately linearly with the oscillation amplitude Aa ~ Fa similar to
buckling forces.
Theoretical explanation and simulation: by analytically solving (see chapter 6.13.2) the coupled differential
equations of the driven, overdamped oscillation of actor and sensor, the viscous drag MT of the filament has
shown to play an important role in the apparent frequencydependent stiffening of single filaments during
buckling. Without MT, the displacement of the sensor from its trap displays no frequency dependence and its
amplitude is negligible. By varying MT and MT, a realistic pair of parameters (MT ≈ 5MT,par ≈ MT,per and MT ≈
0.15pN/µm) has been identified that nicely reproduces the experimental observations. The theoretical
description of the buckling beam has been used to derive the total viscous drag acting on the filament during
oscillation (see chapter 6.10 and 6.13.5). Here, the drag MT,per acting in perpendicular direction of the filament
has shown to be dominant. Additionally, bead displacements for different oscillation frequencies Aa have been
simulated by a Brownian dynamics simulation (see chapter 6.13.3). Results displayed a remarkable
correspondence to the experimentally obtained trajectories and to the analytical solution of the equations of
motion.
Microrheology results: here, the local and global elastic components G’(1) and G’(1,2) as well as the local
and global viscous components G’’(1) and G’’(1,2) of the viscoelastic material response function G = G’ + iG’’
at the position of the actor (1) and between actor and sensor (1,2) were analyzed in directions of the filament
axis (longitudinal) and lateral to it. Concerning the mechanical transport of information, mainly the global
components are of importance since they reflect properties between distant sites.
Results of single filaments displayed no significant difference for different oscillation amplitudes Aa and
were averaged. Furthermore, results for long (LMT ≈ 15µm) and short (LMT ≈ 5µm) filaments differ
quantitatively between both groups but are approximately the same within one group. Thus, results for
filaments of similar size were averaged, as well.
125
4. Microtubules
Longitudinal oscillation: filament stabilized with 10µM Taxol, 100µM Taxol and 100µM Taxol + GMPCPP
were investigated.
The global elastic components G’(1,2) displayed the same principle frequency dependence for all three
stabilizations: a plateau value for low frequencies fa < 1Hz and a power law behavior for high frequencies fa >
10Hz. The transition frequencies ftrans ≈ 3Hz and exponents pow ≈ 1 for high frequencies are approximately
the same for all Taxol stabilized filaments but much larger ftrans ≈ 20Hz and pow ≈ 2 for GMPCPP filaments. This
difference can be addressed to different mechanical properties of the differently stabilized filaments. The
plateau value G’(1,2)(fa < ftrans) ≈ 2mPa is approximately the same for all long filaments regardless of
stabilization. Remarkably, the plateau value for short filaments is up to an order of magnitude higher and
depends on the type of stabilization, indicating that mechanical properties of long and short filaments are
different and that the dependence on filament stabilization is more pronounced for short filaments. As
discussed later, the plateau can be explained by the constant critical force Fcrit of buckling microtubules and
the frequencydependent rise for f > ftrans with a frequencydependent spring constant of the filament.
The local elastic components G’(1) ≈ 50mPa for Taxol stabilized filaments display no distinct frequency
dependence and scatter within one order of magnitude. As shown later, this can be explained again by the
constant critical buckling force Fcrit. For GMPCPP stabilized filaments, a clear frequency dependence is visible
with an exponent pow ≈ 1 and transition frequency ftrans ≈ 6Hz smaller than for the global component.
The local and global viscous components G’’(1) and G’’(1,2) display the same linear frequency dependence
as water, but, interestingly, the local component G’’(1) > G’’H2O is larger than the corresponding component of
water while G’’(1,2) < G’’H2O is smaller. As shown later, this can be explained by the length dependence of the
viscous drag MT(LMT) of the rod shaped filament.
Lateral oscillation: lateral oscillation experiments are available only for long GMPCPP stabilized filaments.
Qualitatively, results are similar to results of the longitudinal oscillation for this type of filaments. However, the
transition frequency ftrans ≈ 3Hz and exponent pow ≈ 1.5 of the global elastic component G’(1,2) deviate. A
possible explanation is the principle difference of the experimental situation: during a longitudinal oscillation,
filaments are buckled and stretched while filaments are only stretched during lateral oscillation.
The local elastic component G’(1) displays a slight frequency behavior with exponent pow ≈ 0.4 and ftrans ≈
2Hz. Again, both parameters are smaller than for longitudinal oscillation. Both viscous components
qualitatively and quantitatively agree to the corresponding components of the longitudinal case.
Theoretical model explaining microrheology results: in order to explain the above summarized
experimental observations, the true response function of the system microtubule with two laterally attached
beads is modeled and the resulting implications for the material response function G are studied.
Frequency dependence of elastic components for f > ftrans: introducing a general frequency dependence of
the microtubule spring constant MT( ) ~ pow in the response function directly results in a frequency
dependence of G’ as shown in chapter 4.4. On the first glance, this seems to be a circular argument. However,
modeling the latticelike connection of bonds between tubulin dimers of single protofilaments and between
192
different protofilaments by springs as shown in Fig. 77C and motivated by {Pampaloni 2008}, this might
explain such a frequencydependent spring constant of the complete filament (lattice). A similar description is
246
used by {Tuszyński 2005} to theoretically investigate static mechanical properties of microtubules. To
investigate frequency dependence, such a model has to include an intrinsic friction of the shearing within and
inbetween protofilaments as well. This could also explain the different exponents observed for differently
stabilized filaments.
Plateau of global elastic components for f < ftrans: at low frequencies, the main component of the total force
during buckling is the critical force Fcrit = 2MTLMT. At high frequencies, the apparent stiffening dominates.
Thus, there is a constant, frequency independent elastic offset caused by the critical force. This can be
identified with a pressure G’(f < ftrans) = Fcrit / L2 = 2MT / L explaining the plateau value of the elastic
component G’. As a consequence, there should be no plateau visible for rheology experiments on prebuckled
filaments, i.e., where the distance between traps y0,2 – y0,1 + Aa < LMT is smaller than the length of
microtubule LMT which is an excellent further test.
126
4. Microtubules
Length dependence of elastic components: the length dependence of the global elastic components is
apparent for low frequencies f < ftrans. As explained above, this regime is dominated by the critical force Fcrit =
2MTL = 2lp(LMT)kBT / L2MT which depends explicitly on the filament length LMT and also implicitly by the
possible length dependence of the persistence length lp(LMT), thus explaining the experimental observations.
No frequency response of local elastic component: this also explains why no frequency response of the
local elastic components is observed, which reflects the properties of the system on the scale L ≈ R ≈ 500nm
of the bead radius, resulting in a large plateau value G’(f < ftrans) and thus in a transition frequency ftrans > fa
larger than the maximum oscillation frequency fa resolved here.
Transition frequency: if the left and right hand side of Eq. (4.36) are equal, a transition from the plateau to
1
the power law regime occurs. Solving this equation ultimately results in the transition frequency ftrans.
Slope of viscous components relative to water: the viscous drag of the filament MT(LMT) as well as its
spring constant MT(LMT) depend on the length of the filament. Both quantities determine the amplitude of
the power law slope of the viscous components G’’. As shown in chapter 4.4, this ultimately results in the
observation that the local viscous component G’’(1) > G’’(1)H2O is larger than that of water while the global
component G’’(1,2) < G’’(1,2)H2O is smaller.
Lateral oscillation: here, the critical force plays no role since filaments are only stretched. However,
filaments are also displaced laterally which is strongly influenced by the viscous drag of the filament. Thus, a
frequency dependence is not surprising. The plateau value can be explained by the local bending which causes
a constant offset similar to Fcrit.
Remark: according to Eq. (4.36), plateau values are governed by constant forces, i.e., the critical force Fcrit
during buckling and the nearly frequency independent stretching force probing the filament only locally. The
spring constant MT determines the amplitude of the frequencydependent regime of elastic components.
Conclusions: the experimental results can be explained by the critical buckling force Fcrit(L), a frequency
depend filament spring constant MT( ), a length dependence of the persistence length lp(L) and the viscous
drag MT(L) of the rodshaped filament. Theoretical considerations off the measurement signals and modeling
of the microrheology results clearly reveal that both, the mechanical properties of filaments and the viscous
drag acting on it, are essential for its frequency response. The power law dependence pow seems to be
determined by filament stabilization. A further connection to the molecular structure which differs between
differently stabilized filaments has to be addressed in the future. The transition frequency ftrans(Fcrit, lp) clearly
depends on the mechanical properties of the filament, ultimately also determined by filament stabilization.
The relative quantitative relevance of the mechanical and viscous properties has to be ruled out further by
more precise theoretical models and simulations thereof. However, the complex interplay of lp(L, lp∞, l’) and
MT(L), in principle, allows the cell to adapt a possible pathway for a mechanical mechanotransduction at high
frequencies (f > 1Hz) very flexibly.
The finding of a transition between two different behaviors of single filaments at a frequency of f ≈ 1Hz has
two interesting, physiological relevant implications. One the one hand, a lot of force generating movements
64
such as the beating of the heart or walking happen on a timescale of one second. Vogel et al. {Vogel 2006}
summarize several experiments and observations that emphasize the importance of this timescale. On the
other hand, a chemical conversion of a mechanical stimulus at the cell periphery and its transport to the cell
center is clearly limited to fa < 1Hz. Due to their high compliance, microtubules can function as a cushion for
these frequencies to protect the cell during migration, for example, by acting as a damping element, while it
has enough time to respond to the stimulus. To efficiently adapt the cell to more rapid occurring events, e.g., a
2
sudden rise of the heart rate , as well, another pathway is necessary. This is again offered by microtubules
which undergo a transition from nonconducting to conducting at around fa ≈ 1Hz. Thus, they might serve a
double role: passive protection for slowly applied forces and active signal transduction for fast applied loads.
1
Due to the power law dependence pow, a general solution for arbitrary exponents pow cannot be given.
2
The heart rate of humans typically ranges between 1Hz and 3Hz while small mice such as shrews can reach heart rates up to 18Hz
{Vornanen247 1992}.
127
4. Microtubules
Despite the fact that such a double role would be an efficient strategy, this interpretation bases on
observations made for single filaments. In vivo, microtubules are crosslinked and surrounded by plenty of
248
other components which cause localized or partial filament buckling {Brangwynne 2006}, for example. This
influence has to be further addressed.
Linear chain of two filaments: this configuration allows probing the ability to transport a mechanical
stimulus by a serial connection of filaments, similar to a serial connection of ohmic resistors. In particular, it
allows investigating the question if the observations and principle findings made for one filament can be
transported to a connection of filaments and if the connection has an influence on the response of a single
filament. Here, only one experiment for 100µM Taxol stabilized filaments is available.
Microrheology results: the qualitative and quantitative observations are similar to those of single
filaments.
In longitudinal direction, exponents pow ≈ 1 of the global elastic components of both filaments are the
same. The transition frequency f(1,2)trans ≈ 2Hz and plateau values G’(1,2) ≈ 5mPa for the first filament
(connection (1,2)) is the same as for a single filaments while f(1,3)trans ≈ 6Hz and plateau values G’(1,3) ≈ 29mPa
is slightly higher for the second filament. Interestingly, the local elastic components G’(1) obtained from the
different connections differ by a factor of two, indicating a principle difference of the connection (1,2) and
(1,3). A significant difference of the viscous components to that of a single filament cannot be observed.
In lateral direction, the quantitative observations are the opposite. The transition frequencies f(1,2)trans ≈
8Hz > f(1,3)trans ≈ 5Hz is larger for the first filament. Similarly, the plateau values G’(1,2) ≈ 1.5mPa > G’(1,3) ≈
0.25Pa. However, exponents pow ≈ 2 are the same. Here, the global elastic components display a frequency
response similar to lateral oscillation of single filaments. The plateau value G’(1) ≈ 37mPa is the same for both
connections.
Discussion: the qualitative observations can be explained by the same theoretical considerations made for
single filaments. However, the fact that the plateau value G’(1,3) > G’(1,2) of the global elastic component in
longitudinal direction is larger for the connection with the larger separation distance is in contradiction to the
model. This can be explained by the intermediate bead (2) which acts as a hinge between both filaments. This
hinge effect is very likely also responsible for the remaining observations, especially the difference of the local
elastic components.
Because of a different stabilization of filaments, the observations for a lateral oscillation cannot be directly
compared to the observations of single filaments made here, but the explanations of the general behavior still
hold.
Conclusions: the principle observations can be transported from single filaments to linear networks
thereof. However, quantitative conclusions of plateau values and frequency response are different which can
be addressed to the hinge effect of the intermediate bead. This effect is not included in the theoretical
considerations made for single filaments. Further, there is also a retroactive effect of the composite on single
filaments. This is likely also caused by the hinge.
Triangular network cell: the demonstration of a triangular network is the first realization of a complex,
defined elementary network cell. Due to the topology of the network, an oscillation purely along the
longitudinal and lateral direction of all filaments is not possible anymore. Instead, the actor trap was moved in
directions tangentially and radially to the direction to the center of mass of the network.
Experimental observations: twodimensional scatter plots of bead positions during an oscillation in
tangential direction revealed a slight ellipsoidal oscillation of all beads. This is caused by a superposition of
filament buckling and stretching during both half periods of the actuation period. The observation for a lateral
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4. Microtubules
oscillation is different, i.e., nonellipsoidal position scatterplots, due to the symmetric condition that both
filaments attached to the actor are either buckled or stretched at the same time.
Microrheology results: in contrast to all other experiments, the elastic components of the triangular
network do not show any frequency response in none of the oscillation directions. The mean amplitudes
0.01Pa < G’ < 2Pa are rather high compared to the plateau values G’ ≈ 0.002Pa of single filaments, for
example. However the viscous components still show a similar behavior as in all experiments, i.e., a larger gap
of approximately 1.5 orders of magnitude between the local and global component. In contrast to single
filaments, both components are shifted to larger amplitudes, i.e., G’’ > G’’H2O.
Discussion: due to the superposition of filament buckling and stretching of all three filaments the whole
time during oscillation, the constant force offset is rather high, especially compared to single filaments. This
causes high amplitudes of the constant plateau value of the local and global elastic components.
Consequently, the transition frequency determined by the balance between the constant and the frequency
dependent part of G’ according to Eq. (4.36) is much higher than measured here and only the constant plateau
value is resolved.
Conclusions: on the one hand, the elastic components of the triangular network cell did not show any
frequencydependent response. On the other hand, the constant plateau value is much higher than for single
filaments indicating a higher rigidity of this type of structure. This is not surprising since triangular supports are
very stiff scaffolds often used by engineers and architects. However, due to this high plateau value, such a
structure transports mechanical stimuli over the complete frequency range investigated here as well as single
filaments at high frequencies.
Remark: all rheology experiments were carried out at the same laser power P ≈ 20mW per trap (in the
focal plane) resulting in approximately the same force constant T ≈ 25pN/µm of each trap. Additionally, the
trap stiffness was doubled for the triangular network to account for the additional microtubules. However,
results did not show a quantitative difference.
Comparison to other techniques: the construction of artificial cytoskeletal structures has been
demonstrated by different techniques. However, these approaches only exploited a stochastic attachment or
215
growth of individual filaments to a substrate, i.e., micropillar arrays or trapped beads {Vignaud 2012;
220 221
Gardel 2004; Xu 1998}. The targeted construction of a biopolymer network with a defined topology is
here demonstrated for the first time. Microrheology experiments are typically carried out for bulk solutions of
220 221
proteins or cytoskeletal filaments {Gardel 2004; Xu 1998}. Rheological properties of whole cells as well as
222 223 224
of local cytoskeletal properties in vivo have also been investigated {Guo 2014; Hoffman 2006; Chen
2015}. However, these measurements do not allow drawing conclusions on the ability of longrange transport
of mechanical stimuli by individual components of the cytoskeleton. The experiments shown here is the first
experimental realization of studying the frequencydependent response of single biopolymers or small
networks thereof with a defined topology.
Conclusions and implications: the fact that structures with a different network topology behave
completely different by displaying a frequency response or not, raises two interesting questions concerning
the biological relevance: which network topologies can be used to transport a mechanical stimulus, in
principle, and which topologies are found in a typical cell? While the first question has to be addressed by
further experiments following the bottomup approach shown here, the latter has to be answered by careful
analysis of high resolution images of the cytoskeleton in vivo for different types of cells. However,
microtubules primarily are aligned radially in the cell emanating from the centrosome near the cell nucleus.
Comparing results of single filaments, a linear combination and the triangular network indicates that it is
neither an advantage nor a disadvantage to connect filaments in series concerning the mechanical transport of
information or to damp an applied force. In contrast, a triangular configuration increases the ability to
transport a mechanical impulse and thus is not eligible for protection from an applied load.
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4. Microtubules
General discussion
As shown here, the transport of information of a mechanical stimulus by microtubules at low frequencies is
governed by the critical buckling force. In vivo, microtubules surrounded by plenty of other structures tend to
buckle localized, sometimes exhibiting a periodic structure of several local buckles. Since the critical force Fcrit
~ 1 / L2 depends on the inverse of the length L of the buckle, the spatially restricting microtubule surrounding
248
tremendously reinforces the single filament. {Brangwynne 2006} demonstrated the repetitive (f ≈ 0.5Hz)
periodic buckling of single filaments in vivo caused by the beating of heart cells. Interestingly, neighboring
microtubules aligning in parallel stayed straight. This is a clear indication for a transport of mechanical stimuli
by single filaments. In the same publication, the authors also reported the localized buckling of (multiple)
filaments during a radially applied external load. The buckling of filaments is equal to a force transported from
one end to the other. Thus, the cell core can instantaneously sense the applied load and react by
reorganization of the cytoskeleton, for example. However, this also indicates the possibility of a feedback
mechanism. If the cell retracts its outer periphery for whatever reasons, a force pointing radially towards the
cell center results. The magnitude of this force can be transported to the nucleus by microtubules in order to
terminate or regulate further retraction in real time.
Final remarks
It has been demonstrated that timeshared optical tweezers can be used flexibly as a tool to investigate
mechanotransduction by single cytoskeletal filaments and small networks thereof. Tools to analyze data have
been presented, analytical models have been introduced and first conclusions have been ruled out. Different
network topologies displayed a different response to the rate of applied force. Thus, the approach shown here
yields a fruitful basis for further investigations in this field. The only obstacle that gets in the way of a
subsequent, straightforward construction and analysis of larger networks, also in combination with
crosslinking to actin or other components, is the biochemistry itself. A clean solution of perfectly labeled and
biotinylated filaments is the most important prerequisite for the success of such experiments. The second
most important requirement is the stability of filaments, i.e., suppressing fluorescence bleaching and filament
breaking due to the formation of reactive oxygen species. These can be scavenged by chemical agents such as
the GODCAT system which was inevitable in all experiments shown here. However, the GODCAT system
1
impairs the likelihood of binding beads to filaments, thus its application is just a necessary evil. A solution to
this dilemma, based on a reflected light darkfield microscopy approach, is presented in appendix 6.8. By
avoiding fluorescence illumination, no radicals are produced anymore and thus the scavenger can be omitted.
The lateral attachment of beads to the filament is not ideal and necessitates a complicated fit function in
stretching experiments. This effect has to be addressed in rheology experiments, as well. However, a direct
250
attachment to the filament’s ends as demonstrated by {Trushko 2013} would be a more sophisticated
approach.
Microtubules are a common target during cancer treatment and have shown to form bundles in this case.
The mechanical properties of such a bundle are different to that of single filaments and could be well
investigated with the methods shown here. A theoretical study on bundle formation of taxanestabilized
251
microtubules is presented by {Kim 2014}.
Electrooptical deflectors (EOD) are superior to acoustooptical deflectors (AOD) by exhibiting constant
103
diffraction efficiency over the complete range of diffraction angles {Valentine 2008}. Consequently, the laser
intensity does not change for different trap positions and an individual trap calibration would be no longer
1
As shown by {Swoboda249 2012}, the pH value decreases tremendously over time if a scavenger based on glucose oxidase is used. This
likely impairs the binding properties of neutravidin to biotin. However, the authors also demonstrated that a similar scavenger based on
pyranose oxidase is pH stable and would be an alternative, as well.
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4. Microtubules
necessary (accepting slight errors <5% due to a variation <3% of the bead size). Thus, an EOD would be a good
replacement for the AOD used here.
Outlook
Similar to the combination of individual filaments to a serial connection of multiple filaments shown in this
thesis, the effect of a combination of identical but also different network cells has to be addressed in the
future. Necessary, interesting and potentially fruitful next steps and experiments are as follows:
Implementation of a different illumination to avoid any chemical disturbance by oxygen scavengers.
A further analytical description of the experimental situation for single filaments and small networks
to rule out the relative importance of the viscous drag, resulting in an apparent stiffening of the
filament, and a true frequency dependence of the filament’s spring constant.
Consequent Brownian dynamics simulations thereof (an analytical solution of the coupled differential
equations of more than two anchor points (beads) is very likely not possible).
Rheological analysis of the simulations to test the influence of different models for (L) and its
dependence on the critical length l’ and overall persistence length lp∞.
Rheology experiments of single filaments with different prestretching and prebuckling to address
the effect of a superposition of stretching and buckling inherent in the experiments shown so far.
Rheology experiments of different elementary network cells, e.g., triangular and rectangular.
Rheology experiments of compositions of identical and mixed elementary network cells.
Development and test of passive microrheology. Here, no active oscillation is needed which
decreases the complexity and vulnerability of experiments.
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5. Summary and final conclusions
1
For realistic networks consisting of up to N = 20 anchor points. Theoretically, up to at least N = 100 anchor points should be viable until
single beads are able to escape their traps during the absence of the laser. However, the construction of networks with such a high
complexity evokes additional problems, such as the assembling process itself.
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5. Summary and final conclusions
for further experiments and theoretical models explaining mechanical mechanotransduction. Current
obstacles were identified and solutions were presented.
Rheological analyses of single filaments clearly revealed a mechanical stiffening at high frequencies with a
constant plateau value for small frequencies. The transition frequency ftrans ≈ 1Hz between both regimes is in a
physiologically interesting range, since the long distance chemical transport of information by diffusion or
molecular motors is clearly limited to frequencies f < ftrans. However, due to the plateau value governed by the
critical buckling force of filaments, a mechanical transport by microtubule filaments is also possible for f < ftrans.
The stiff microtubule transports a force directly from one end to the opposing end. In this way, a force can be
transported through the cell and sensed in its interior in real time. This would be an efficient and sensitive
feedback mechanism for the control of the retraction or expansion of the cell periphery, for example.
Furthermore, due to the high compliance of buckled microtubules, the cell interior, in particular the nucleus, is
protected from forces with high magnitudes that could be dangerous or even damage organelles.
A comparison of results for single filaments to a linear chain and a triangular network of filaments revealed
huge differences between different network topologies. This clearly has to be addressed further by
investigating elementary network meshes with other topologies and complex networks thereof. Here, the
triangular network did not display any frequency response which raises the question which elementary
network meshes and complex combinations thereof are well eligible to transport information, over which
frequency range and which meshes function as mechanical insulator by damping applied forces. The influence
of other cellular components such as actin filaments or molecular motors has to be ruled out by introducing
further spatial restrictions in the experimental configuration and cross linkers between different filaments. A
careful comparison of results thereof to the topology of the microtubule network and elementary network
meshes found in vivo may shed light on the origin of the structure and function of the eukaryotic cytoskeleton
in general.
255
Cells are also able to sense and transmit (extracellular) forces over long distances {Wang 2014}. Loosely
speaking, they are able to talk to each other. In this context, mimicking asterlike microtubule networks (which
is their primary alignment in the cell) might give interesting insights on the transport of mechanical stimuli
through multiple cells of a large cluster or in tissue, for example.
134
6. Appendix
6. Appendix
This chapter is intended to give some additional information on certain topics or detailed calculations
mentioned during the course of this thesis, which would distract from the relevant things if mentioned directly
in the corresponding chapters. There also are some notes and information that might be of particular interest
for successors of one of the projects or setups presented here.
Here, A(b) = 2P(t)sPDaPAEiEi(b) is the amplitude of the interference term and Ioff(t) ≈2P(t)sPDaPAEi the
incident intensity in the back focal plane. P(t) was a time dependent intensity modulation used, for example,
to create a harmonic potential in scan direction during Spiroplasma experiments. sPD and aPA are electronic
constants of the QPD.
6.1.1. Lateral
Here, the signal Sx in xdirection is shown. According to Eq. (2.28) and Fig. 12, this is the difference of the
left and right two quadrants. Using rBFP = k0NAD, a straightforward integration results in:
135
6. Appendix
rBFP
0 rBFP
S x A(t ) dk y
r dk x sin k b
x x k b
y y a 0 z z
k b dk x sin k x bx k y by a0 k z bz
rBFP BFP 0
r
A(t ) BFP
bx rBFP
dk y 2 cos k y by a0 k z bz cos rBFP bx k y by a0 k z bz cos rBFP bx k y by a0 k z bz
A(t )
bx by
2sin rBFP by a0 k z bz 2sin rBFP by a0 k z bz (A.2)
2 sin rBFP by cos a0 k z bz cos rBFP by sin a0 k z bz
cos a k b 4sin r b
2 A(t )
0 z z BFP y
bb x y
2 sin rBFP bx cos rBFP by cos rBFP bx sin rBFP by
2 sin r b cos rBFP by cos rBFP
BFP x b sin r b
x BFP y
16 A(t ) 2 rBFP bx
sin sin rBFP by cos a0 k z bz
bx by 2 (A.3)
A closer look on the final result of Eq. (A.3) and the relation sin(x) / x = sinc(x) results in
r b r b
2
16rBFP A(b)
Sx sinc BFP x sin BFP x sinc rBFP by cos a0 k z bz
2 2 2
bx2
(A.4)
2x r b r b
~e sinc BFP x sin BFP x
2 2
Due to the symmetry of the lateral directions, Sy follows accordingly. Resulting signals Sx(bx, by, bz = 0) and
Sy(bx, by, bz = 0) in the xy plane of a scan of the bead through the complete focal volume is shown in Fig. 95B.
Line scans Sx(bx, by = 0, bz = 0) and Sy(bx = 0, by, bz = 0) as shown in Fig. 95A show the complete slope of the
detection signal resembling a bipolar, sinusoidal function with a central, linear region (thick black line) typically
exploited in optical tweezers experiments. Line scans can be compared experimental data in Fig. 16. Signal
linearity and orthogonally for small displacements is also shown in Fig. 95C by equidistant isolines of Sx
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