Geobiology (2005), 3, 179–193

Living under an atomic force microscope

O R I G Iunder
Living
Blackwell NA L an
A Ratomic
T I CLtd
Publishing L Eforce microscope

An optimized approach for in vivo investigations on surface alterations towards biomineral

nucleation on cyanobacterial cells
M. OB ST AN D M. D I T T RI C H

Limnological Research Center, Swiss Federal Institute for Environmental Science and Technology (EAWAG),
Kastanienbaum, Switzerland

ABSTRACT

An approach for long-term in vivo investigations on cyanobacterial cell surface changes at high spatial resolution
by Atomic Force Microscopy (AFM) was developed in this study.
Until recently, changes of bacterial cell surfaces due to changes of the chemical environment could neither
be investigated in situ nor in vivo. However, in vivo investigations give insights into kinetics of cell response
to environmental changes and mineral nucleation at the cell’s surface. Continuously cultured cyanobacteria of
the representative freshwater strain Synechococcus leopoliensis (PCC 7942) were washed and artificially immo-
bilized on poly-l-lysine-coated glass slides. Both immobilization and environmental conditions were optimized
in order to facilitate long-term experiments (> 100 h) with living cells. AFM samples were investigated in situ
in two different solutions: Culture medium was used for cultivation experiments and nutrient-free NaHCO3/
CaCl2 solutions (supersaturated with respect to calcite) for long-term characterizations of the changes in cell
surface topography. Cell viability under these conditions was investigated by AFM, TEM and epifluorescence
microscopy, independently.
No indications for extended starvation were found within the relevant timescales. Analysing the influence of
Ca2+ on the surface of S. leopoliensis, we found significant changes compared to a Ca-free solution. Few hours
after CaCl2 was added to the circumfluent solution, small protuberances were observed on the cell surface.
These are promising results to environmental scientists for a wide range of applications, as cell response
to environmental changes can now be monitored online and in vivo at timescales, which are relevant for natural
processes. Most especially studies of biomineralization and mineral nucleation on bacterial cell surfaces will profit
from this new approach.

Received 01 November 2005; accepted 14 November 2005

Corresponding author: M. Obst. Tel.: +41-41-3492111; fax: +41-41-3492168; e-mail: martin.obst@eawag.ch.

Merz-Preiss, 2000). Studies on the biogenic calcite pre-

INTRODUCTION
Autochthonous calcite precipitation in carbonate lakes can
result in large carbonate deposits, especially at sedimentation
rates of several g m−2 d−1 as for example were observed for
parts of Lake Lucerne in central Switzerland (calculated from
data of Bloesch, 1974). These lake sediments, high in calcite
content, are now intensively studied as high-resolution continental
archives for environmental change, e.g. with respect to the
past climate (Lotter et al., 1997). Several photoautotrophic
picoplankton species (single-cellular algae or cyanobacterial,
cell size 0.2–2 µm) were observed to have a major impact
on or might even cause carbonate mineral precipitation in
both marine and lacustrine environments (Golubic, 1973;
cipitation have been performed from various perspectives,
and finally, several possible mechanisms have been discussed
(for review see Dittrich & Obst, 2004). Besides field observations,
several microscopy studies have been performed. Transmission
electron microscopy (TEM) investigations focused primarily
on the paracrystalline proteinaceous surface layers (called S-layers)
of cyanobacterial cells that are supposed to function as a template
for crystal nucleation (Schultze-Lam & Beveridge, 1994a,b; for
review see Smarda et al., 2002). Schultze-Lam et al. (1992)
suggest that as a first step, hydrated Ca2+-ions are bound to the
regularly spaced and negatively charged proteins of the S-layer.
These suggestions were based on structural investigations of
S-layers and bulk analysis of their chemical composition.

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Journal compilation © 2005 Blackwell Publishing Ltd 179
180 M. OBST AND M. DITTRICH
However, the alteration of picoplanktonic cell surfaces and
the process of mineral nucleation on those have never been
observed online or in situ; most probably because no appro-
priate technique has been developed so far. One basic require-
ment of the technique is the capability of in situ and in vivo
observation of cells at a sufficient lateral resolution. Further-
more, long-term stability is necessary in order to be able to
perform experiments on timescales that are relevant for
investigations on cell surface changes and mineral nucleation.
Scanning force microscopies (SFM) like atomic force microscopy
(AFM), in principle, meet the first requirement as discussed in
the following section.
Applications of AFM and scanning tunneling microscopy
(STM) in biological research are reviewed in Marti & Amrein
(1993). One decade later Jena & Hörber (2002) presented a
nice overview of more advanced techniques including in situ
AFM in fluids.
In situ AFM allows imaging and characterization of topo-
graphy, physical and chemical properties of cell surfaces at the
nm-scale (Müller et al., 1999). Since in situ AFM has been
developed and applied to biological samples, one of the most
important limitations of the previously mentioned electron
microscopy studies could be overcome: Experiments on
biological samples can be performed in native environments
and thus under physiological conditions (Hoerber & Miles,
2003).
Various AFM studies have been performed mostly on
biological interactions at the molecular scale and the structure
of biological membranes (Hoerber & Miles, 2003; Dufrene,
2004). In situ, short-term AFM observations of cells in their
natural aqueous environment were targeted on the structure
and functionality of cell membranes (Kamruzzahan et al.,
2004), on cell growth (Touhami et al., 2004) and division
(Matzke et al., 2001).
As previously mentioned, many environmental processes
like, e.g. cell response to environmental changes or biomineral
nucleation occur on timescales of several hours or days. Until
recently, such processes were barely investigated in situ and at
nm resolution as no applicable methods were established and
tested.
Therefore it was the intention of this study to develop and
optimize an AFM approach for in vivo investigations on pico-
planktonic cell surfaces and their response to changes of the
chemical environment, which is an important step towards
in vivo studies on the nucleation of CaCO3 on the cell surface.
With special regard to the second step, the long-term capabilities
(> 100 h) of the approach had to be addressed and discussed
in detail as this is a prerequisite for further biomineralization
experiments in the AFM.
Cyanobacteria of the freshwater strain Synechococcus
leopoliensis PCC 7942 were chosen for the development of this
method as Synechococcus ssp. are representative of many
freshwater systems and the leopoliensis strain is well investi-
gated from various perspectives like physiology (Wanner et al.,
1986; Arino et al., 1995; Goerl et al., 1998; Sauer et al.,
2001; Barker-Astrom et al., 2005), metabolism (Ritchie et al.,
1996; Kaplan & Reinhold, 1999; Allakhverdiev et al., 2000)
and biomineral precipitation (Schultze-Lam et al., 1992;
Thompson et al., 1997). Thus, choosing S. leopoliensis PCC
7942 enabled us to compare the results with those of previous
studies by our and other groups.
METHODS
The methods, culturing and experimental conditions were
chosen with respect to the following concerns.
The first is the relevance of the experiments in order to
simulate natural processes. Second, the viability of the immo-
bilized cells had to be ensured because a proper immobiliza-
tion is necessary in order to obtain high-quality images of the
cell’s surface. Finally, the experimental setup has to ensure
long-term stability, which is necessary in order to attain quality
results at the designated timescale.
Cell cultivation
Synechococcus leopoliensis cells were cultured continuously in a
2.5 L chemostat with Z/10 culture medium (5.9 mg L−1
Ca(NO3)2·4H2O, 46.7 mg L−1 NaNO3, 4.1 mg L−1 K2HPO4·
3H2O, 2.5 mg L−1 MgSO4·7H2O, 168 mg L−1 NaHCO3,
11.45 mg L−1 Na-EDTA, 3 mg L−1 FeSO4·7H2O, H3BO3
248 µg L−1, MnSO4·H2O 135 µg L−1 (NH4)6Mo7O24·4H2O
7.2 µg L−1, ZnSO4·7H2O 23.2 µg L−1, Co(NO3)2·6H2O
12 µg L−1, CuSO4·5H2O 10.4 µg L−1 without vitamin
solution) at a flow rate of 200 mL d−1. The culture medium
Z/10 was chosen in order to simulate low nutrient conditions
as observed in oligotrophic lakes. Therefore, the cell physiology
of the cyanobacterial should be adapted to low nutrient
conditions. The bioreactors were aerated with sterile filtered
air. The pH of the cell suspension was measured with long-
term stable combination electrodes (InPro 3030; Mettler-
Toledo GmBH, Greifensee, Switzerland). In order to optimize
growth conditions, CO2 was added to the aeration air when
the pH rose above the maximum value of 7.5. The cultures were
tempered to 27 °C. Fluorescent lights provided a permanent
light intensity of 7 µE m−2 s−1. Cells were harvested on the
same day as the experiment was started. The cell suspension was
vacuum filtered with 0.45 µm membrane filters (Schleicher &
Schuell OE67), resuspended in nanopure water and centrifuged
at 2500× g for 15 min. The supernatant was drawn out of the
centrifuge tube, and the cells were resuspended in fresh
nanopure deionized water. This washing step was repeated three
times in total until the cells were used for further experiments.
Sample preparation for AFM experiments
For sample preparation, aseptic techniques were followed. All
preparation steps until mounting the sample onto the AFM
© 2005 The Authors
Journal compilation © 2005 Blackwell Publishing Ltd

scanner were performed in a clean bench (Gelair HF 36).
Round glass cover slips with a diameter of 13 mm were
precleaned with pure ethanol. After drying, the cover slips
were treated with 0.05% poly-l-lysine (Sigma Aldrich, no.
P1524) in nanopure deionized water for 48 h. The cover
slips were removed from the solution and air-dried overnight,
standing vertically on a paper towel. The cover slips were
mounted onto metal plates with adhesive carbon stickers and
stored in the dark at 4 °C until usage. For the cell cultivation
and cell division experiments, a glass cover slip was treated with
a drop of the cell suspension for 60 min. Then the cover slip was
vigorously rinsed with nanopure deionized water in order to
remove all cells that were not well immobilized by the poly-l-lysine.
The wet sample was mounted on the scanner of the AFM.
Gelatin-coated glass slips were prepared similar to method
described above. We followed the protocol of Doktycz et al.
(2003) using the Sigma Gelatin G6144 that gave the best
results in their study.
AFM experiments
All AFM experiments were performed in a Digital Instruments
MultiMode III AFM with a Multimode IIIa controller with
the Tapping Mode extender. Olympus Bio-Levers (type B
levers, 100 µm, 0.006 N m−1, sidewall angle 45°) mounted
into a tapping mode fluid cell were used for the experiments.
The volume of the fluid cell is approximately 100 –150 µL,
depending on the adjustment of the O-ring. During optimization
of the setup, Veeco NP tips (spring constants of 0.06 and
0.12 N m−1, sidewall angle 35°) and Ultrasharp CSC12 tips
(spring constants 0.03 and 0.05 N m−1 cone angle 30°)
were also tested. In comparison, the Olympus Biolevers
gave the best results because the least amount of cells was
removed during scanning. During the experiments, all scans
were performed at an angle of 90°.
The AFM fluid cell was cleaned before mounting the tip and
the Z-shaped silicon O-ring, using pure ethanol. Before and
after the experiment, the O-ring was cleaned with pure etha-
nol as well and stored in nanopure water when it was not in
use. All AFM tips were exposed to intense UV light for 20 min
in order to clean the tips from potential organic contamina-
tions and to avoid bacterial contamination. All tips were used
for one experiment only.
The microscope was operated in tapping mode. The reso-
nance frequencies of the cantilevers were between 7 and
9.5 kHz. Tip velocities smaller than 12 µm s−1 were used.
Prior to the installation of the fluid cell, the cantilever was
covered with nanopure deionized water. Z-shaped O-rings
were used to keep the cell watertight. The fluid cell of the AFM
was carefully mounted onto the sample in order to avoid
air bubbles within the cell. One of the outlets of the cell was
connected to a reservoir bottle and the other to a syringe
pump (Braun Perfusor IV, adapted to suction instead of
pressure) via previously autoclaved silicon tubes.
© 2005 The Authors
Journal compilation © 2005 Blackwell Publishing Ltd
Living under an atomic force microscope 181
Cultivation experiments in the AFM
BG11 (1.491 g L−1 NaNO3, 100.5 mg L−1 KNO3,
35.8 mg L−1 CaCl2·2H2O, 40.7 mg L−1 Na2(EDTA)·
2H2O, 74.7 mg L−1 MgSO4·7H2O, 70.6 mg L−1 Na2HPO4,
254.3 µg L−1 FeSO4·7H2O, 152.7 µg L−1 ZnCl2, 1.3 mg L−1
MnCl2, 55.6 µg L−1 CuSO·5H2O, 33.3 µg L−1 CoSO4·7H2O,
271 µg L−1 NaMoO4·2H2O, 1.99 mg L−1 H3BO3) or Z/10
culture medium (described previously) was drawn through
the fluid cell at flow rates of 0.6 or 3.0 mL h−1. The first
images were taken after an equilibration time of about 1 h
after mounting the fluid cell. The flow-through experiments
were run up to 6 days. In order to test the viability of the
artificially immobilized cells by an independent method, one
of the cell-treated cover slips was deposed in a sterile 50 mL
centrifuge tube with 15 mL of Z/10 culture medium and
incubated at 27 °C under permanent illumination.
Cell division and surface alteration experiments
The samples for the cell division experiment were prepared
in the same way as described above. During long-term
experiments, these samples were enclosed in the fluid cell of
a Digital Instruments Multimode AFM. Several cells were
monitored in tapping mode while a solution of CaCl2 / NaHCO3
(with no additional nutrient concentration) was pumped
through the fluid cell by a syringe pump. The concentration of
NaHCO3 was 1.5 mM for both experiments. The concentration
of CaCl2 was 5.9 mM in the experiment where we observed the
cell division and 11.8 mM in the CaCO3 nucleation experiment.
The saturation with respect to calcite was 9.2 times for the first
case and 13.7 times for the second case. The saturation states
were calculated using PHREEQC INTERACTIVE (version 2.8) using
the wateq4f database and assuming a CO2 concentration of
370 p.p.m. in the ambient air.
Investigation of EPS by AFM in air
For investigations of the production of EPS by S. leopoliensis
PCC 7942 samples were prepared for tapping mode AFM in
air. In order to excite the production of extracellular sheath
material, samples previously used for cell division experiments
were slowly exposed to drought conditions, causing stress
on the cells. The specimen was placed in a closed Petri dish
together with a drop of water and left for air-drying for 14 days.
The same microscope as described above was used for scanning.
The AFM was run in tapping mode in air with Veeco RTESP
silicon tips (125 µm, 40 N m−1) at a driving frequency of
318 kHz.
Sample investigation by epifluorescence microscopy
The initial samples for epifluorescence microscopy were
prepared and analysed immediately after treating the
182 M. OBST AND M. DITTRICH
poly-l-lysine covered glass cover slip with the cell suspension.
More samples were prepared after 1, 2 and 7 days of the flow-
through experiment (Z/10 culture medium) within the fluid
cell of the atomic force microscope.
Water was carefully removed from the glass slides using a
syringe needle. The samples were dried in an air stream. After
about 1 min, the slides were covered with a drop of immersion
oil (Zeiss 518N) and covered by a second glass cover slip. The
samples were analysed immediately after preparation by a Zeiss
Axioscope 2 epifluorescence microscope. The microscope was
equipped with a HBO 100 W/2 mercury arc light source and
a SenSys CCD camera (KAF 1401E, 1317 × 1035 pixels).
CCD images were taken with a Plan-Neofluar 100×. Overview
images were obtained with a Plan-Neofluar 20× in order to
characterize the homogeneity of the cell distribution. For
imaging the cell distribution and testing the viability of the
immobilized cells, the HQ filter set Cy3 (Chroma Technology
Corp.) was used. All slides were also checked for contamina-
tion (e.g. by heterotrophic bacteria) using the Zeiss filter sets
02 and 09.
TEM imaging and sample preparation
Cell suspensions for TEM experiments were prepared as
described in the section on cell cultivation. These cell suspensions
were analysed comparing three different preparation methods:
First, cell culture samples were prepared immediately after the
washing step. Next, the cells were incubated for 24 h in
1.5 mM NaHCO3 solution. Finally, the cells were incubated in
1.5 mM NaHCO3/11.6 mM CaCl2 solution for 18 and 45 h.
The final solution was 13.4 times saturated with respect to
calcite when assuming a CO2 level of 370 p.p.m. in the
ambient air. The saturation was calculated with PHREEQC
INTERACTIVE (version 2.8), using the wateq4f database. The
suspensions were centrifuged at 2000× g for 10 min, the
supernatant solution was decanted. The concentrated cell
suspension was then sucked into cellulose capillary tubes,
high-pressure frozen and freeze substituted and embedded in
Epon as described by Hohenberg et al. (1994). Ultrathin
sections were prepared on a Reichert Jung Ultracut microtome
and put onto collodium/carbon-coated 300 mesh copper
grids. The sections were stained with uranyl acetate and lead
citrate according to the method cited above. Images were
taken on a Zeiss 912 Ω enegery filter TEM.
RESULTS
Cell immobilization
In order to perform in situ AFM experiments effectively, a
reproducible and reliable method for cell immobilization had
to be developed. We tested two different approaches for their
suitability for long-term experiments: immobilization on
gelatin-coated mica slides and immobilization on poly-l-lysine
coated mica and glass surfaces. In our study, the gelatin
approach did not result in a suitable long-term immobilization.
Synechococcus leopoliensis cells could not be attached to the
surface firmly enough in order to be able to perform one or
even several scans of the same area without removing the
majority of cells in this area. Furthermore, the gelatin-coated
surface often caused line artefacts, probably caused by the tip
sticking to the gelatin surface. Thus, we were not able to
obtain high quality images working with gelatin-coated slips
in situ, and no images from that method are presented in this
study. For this reason, the immobilization by poly-l-lysine was
found to be superior to the former described method. When
screening several concentrations between 0.01% and 0.05%
and different exposure times, best results were obtained when
the glass cover slips were placed into a 0.05% solution of
poly-l-lysine for 48 h.
After treating the glass cover slips with a drop of the cell
suspension, the homogeneity of the cell distribution was inves-
tigated by epifluorescence microscopy. When the poly-l-lysine
coated glass cover slip was treated with freshly prepared
suspension of S. leopoliensis, this technique resulted in a more
homogeneous distribution of cells, whereas a day old sus-
pension resulted mainly in agglomerates of cells. Thus, for each
of the following experiments, the suspension was prepared
immediately before observation, in order to obtain homoge-
neously distributed cells on the cover slips.
Cell cultivation in the fluid cell of an AFM
In order to test the vitality of the immobilized cyanobacteria,
cultivation experiments were performed in the fluid cell of the
AFM. Results of these experiments are shown in Fig. 1. In the
AFM experiments (first and second columns), the same area is
shown at each time step, whereas the epifluorescence series
(third column) shows different samples because the sampling
is disruptive. Relatively large areas of 60 × 60 µm were
scanned several times within 6 days of cyanobacterial growth.
Because of the high tip velocity, weakly attached cells were
removed from the observed areas (compare AFM experiment
2 after 3 and after 20 h). In the AFM experiment 1 of Fig. 1,
the growth of cell clusters from a previously much lower
number of cells can be clearly observed (indicated by the
arrows). Clusters of cells could not be scanned accurately,
probably because of the production of EPS as discussed later
on. After 20 h, the first cell clusters were formed, and after
3 days, the topography of the cell-covered glass slide became
too high for the z-range of the scanner and the whole image
occurred blurry. In the epifluorescence microscope the formation
of a biofilm containing several cell layers was observed. These
results totally coincide with the AFM results and explain the
difficulties of AFM imaging after several days of cell growth.
All cover slips used for the cultivation were checked for
contamination by other bacteria using different filter sets. No
cells were observed which exhibited different fluorescence
© 2005 The Authors
Journal compilation © 2005 Blackwell Publishing Ltd
 Living under an atomic force microscope 183

Fig. 1 AFM cell cultivation experiments (series AFM1 and AFM2) and epifluorescence images (series epi). Each image shows a size of 60 × 60 µm2. Series AFM1
images show the same area after starting the flow-through cultivation experiments. The arrows highlight two areas where the formation of agglomerates of freshly
divided cells (smaller size) can be observed. These cells are remarkably smaller in diameter than the ones visible at the beginning. Series AFM2 images show results
of another experiment. The formation of agglomerates can be observed until finally the vertical range of the scanner is not sufficient for imaging the area. Because
of the high tip velocity weakly attached cells were removed from the observed areas (compare AFM2 at 3 and 20 h). Series epi shows epifluorescence images of
different areas of equally treated samples. The formation of agglomerates is clearly visible until a biofilm of several cell-layers developed (138 h).

© 2005 The Authors

Journal compilation © 2005 Blackwell Publishing Ltd
184 M. OBST AND M. DITTRICH
Fig. 2 AFM images showing 3D projections of Synechococcus leopoliensis
cells at the beginning of a flow-through cultivation experiment. The image of
the enlarged section (20 × 20 µm2) was taken 2 h after the flow-through of Z/
10 culture medium was started. The overview image (60 × 60 µm2) was taken
1 h later. Cells at different states of cell division were observed in the enlarged
image: Arrow 1 is indicating a cell division resulting in almost spherical cells. The
arrows 2–5 are indicating increasing progress in cell division and separation.
behaviour from that of S. leopoliensis. Thus, no indication for
contamination by, e.g. heterotrophic bacteria was found
during all long-term cultivation experiments.
A typical 3D projection of immobilized S. leopoliensis cells
during the first hours of a flow-through experiment with
culture medium is presented in Fig. 2. From the overview
scan, it is obvious that the cells are firmly attached to the
glass substrate: Almost no cells were removed during the scan
with higher magnification, which was taken first. Cells at increas-
ing progress states of cell division and separation are highlighted
by the arrows 2–5.
Long-term stability of scanning conditions
In order to be able to perform meaningful experiments on cell
surface alteration, in this series of experiments, on the one
hand, we had to pay special attention to the observation of
cells with respect to their vitality. On the other hand, it was
important to establish stable scanning conditions for long
periods of time. Using a solution of NaHCO3/CaCl2 without
additional nutrients facilitated these long-term experiments
as no perturbing amounts of EPS were produced by the cells
in this solution. This enabled us to find stable settings for the
feedback loop while working with the same tip for several days
(> 100 h).
Cell viability
The viability of the cells did not seem to be affected by our
technique. As shown in Fig. 2, cells at all stages of their life
cycle were immobilized on our samples. During one experiment
of 4 days, we were able to observe the continuation of a cell’s
life cycle. We observed the sequence of two cell divisions of an
initially rod-shaped cell into four daughter cells (Fig. 3A–D).
Fig. 3 AFM images showing a 3D projection (first column) and the phase lag
(second column) of a dividing Synechococcus leopoliensis cell at different time
intervals. Thirteen hours after the experiment was started (A), only the division
in the lateral axis is visible. Thirty hours after the beginning (B), a line structure
appears, but only in the phase-image. Twenty-four hours later (C), a furrow can
be observed in the 3D projection as well. One hundred and one hours after
starting the experiments (D) the cell division is followed by growth of the
resulting 4 cells.
The cell division in lateral direction was already observed when
the cell was scanned the first time, 13 h after the flow-through
experiment was started. Thirty hours after the beginning of
the experiment, we observed a line structure in the phase lag,
dividing the cell in the longitudinal axis, although at this stage,
the topographical image did not show any indication of
division. The phase lag in tapping mode is influenced mainly by
the stiffness of the sample and by adhesion forces between
sample and tip (Raghavan et al., 2000; James et al., 2001).
The reduction of the phase lag in this region therefore might
indicate an increase of stiffness in this region, most likely due
to the formation of the new cell membrane between the
evolving cells.
After a time lag of about 24 h, this structure also appeared
as a constriction in the topography of the cell. This division
was then followed by little longitudinal growth of the resulting
cells. We infer that imaging the phase lag facilitates determin-
ing the onset of cell division before it becomes visible in the
topography. Figure 4 shows a 3D projection of the result of
the cell division after 101 h. The whole sequence of a cell divi-
sion was observed once. However divided, but not yet separ-
ated cells were observed more often during our experiments,
© 2005 The Authors
Journal compilation © 2005 Blackwell Publishing Ltd

Fig. 4 AFM 3D projection of the divided cells after 101 h. The fourfold division,
which is caused by two subsequent divisions perpendicular to each other, is
clearly visible.
for example, during the cultivation experiment presented in
Fig. 2. An observation that is similar to the starting point of
the previously described sequence – a division resulting in two
almost spherical cells of S. leopoliensis – is highlighted by arrow
1 in Fig. 2. Another example of almost spherical daughter cells
is indicated in the phase image of Fig. 7, also indicated by an
arrow.
When we analysed ultrathin sections of freeze substituted
S. leopoliensis cell suspensions from both culture broths
(Fig. 5B) and precipitation experiments in NaHCO3/CaCl2
solutions and ‘blanks’ in NaHCO3 solution (Fig. 5A) by
TEM, we also observed several stages of cell divisions. Most of
them resulted in rod-shaped daughter cells and some in almost
spherical cells. All divisions of elongated cells observed by
TEM were perpendicular to the longitudinal axis.
Surface alteration of Synechococcus leopoliensis
When CaCl2 was added to the surrounding NaHCO3 solution
during our CaCO3 nucleation experiment, the surface structure
and properties of the cyanobacteria changed. The results of
these experiments are presented in Figs 3 and 6. In the 3D
projection on the left side of Fig. 6(A), the cell surface appears
rather smooth 2 h after CaCl2 was added. Only some little
protuberances can be observed. The phase image on the right
also presents a rather homogeneous surface. The line structures
parallel to the cell’s shape are artefacts caused by a non-ideal
shape of the tip sidewalls. Six hours after the addition
(Fig. 6B), these protuberances are more pronounced in both
the topographical image and the phase image. Twenty-one
hours after CaCl2 was added to the solution (Fig. 6C), the
surface topography appears more textured than at the beginning
of the experiment. Also the phase lag is not homogeneous over
cell surface anymore.
In principle, the same observations but for different time
intervals are presented in Fig. 3. In the first image (Fig. 3A),
which was taken 13 h after the addition of CaCl2 to the solu-
tion, the surface of the cells already looks rough. Comparing
the two lower cells at the time steps of 54 h (Fig. 3C) and
© 2005 The Authors
Journal compilation © 2005 Blackwell Publishing Ltd
Living under an atomic force microscope 185
101 h (Fig. 3D), the enlargement of some protuberances is
conspicuous. At this time nuclei of CaCO3 crystals on could
not be observed on the surface of the analysed cell.
Formation of extracellular polymeric substances (EPS)
The formation of a biofilm after several days of the cell
cultivation indicates that the Synechococcus cells are likely to
have started producing extracellular polymeric substances.
As EPS or sheath production is known to be strongly
influenced by the culture conditions (Merz & Zankl, 1993)
and, in particular, enhanced by drought (Potts, 1994), this
suggestion could be proven by exposure of drought stress to
the immobilized cells. In Fig. 7(A), a 3D projection of the
sample is shown. A bulky structure is visible and some
cell-sized objects seem to be embedded in this blurry matrix,
but no single cells are visible. In contrast to the height data,
the cells are clearly visible in the phase image of this sample
(Fig. 7B). The suggestion of cells embedded in a matrix is
confirmed by this result. In air, the EPS becomes rigid, enough
to form a surface that can be scanned by AFM. However, it was
very difficult to scan samples covered by EPS in an aqueous
environment. Figure 8 shows a topographical image of the
same area during a flow-through experiment at different time
intervals. After 2 h of pumping culture medium through the
fluid cell, a good image quality was achieved (Fig. 8A), whereas
after 4 h (Fig. 8B), the first artefacts were observed. However,
17 (Fig. 8C) and 18 h (Fig. 8D) after beginning to culture
cyanobacteria within the fluid cell of the microscope, no
parameter set for the feedback loop could be found in order
to run the microscope under stable conditions. The images
contained many line artefacts, probably caused by the tip
occasionally sticking to a bulk of EPS.
DISCUSSION
Influence of culturing and experimental conditions
on cyanobacteria
With respect to both the design of the long-term in vivo
experiments in the AFM and the relevance of the experiments
for simulating natural conditions, the culturing conditions
had to be considered carefully. BG11 is a widely used medium
for the cultivation of cyanobacteria. This medium, which was
originally optimized for the cultivation of coccoid cyanobacteria
(Stanier et al., 1971), is rich in both nitrate and phosphate.
It is not representing conditions that are observed in oligotrophic
freshwater systems, which are often phosphate-limited. For
some sensitive phycoerythrin-rich red pigmented Synechococcus
species, BG11 was even shown to slow down growth compared
to lower concentrated media (Ernst et al., 2005). Thus, culturing
and experimental conditions are influencing physiology,
ultrastructure and metabolism of the cells (reviewed by
Allen, 1984; de Marsac & Houmard, 1993); therefore surface
186 M. OBST AND M. DITTRICH

Fig. 5 TEM images showing the ultrastructure of variously treated Synechococcus leopoliensis PCC7942. Cell viability was not affected after 24 h of treatment in
1.5 mM NaHCO3 solution without addition of other nutrients (A), as cell divisions were observed in this solution. However, starting separations of the thylakoids
were observed (indicated by S), these are the first indications of adaptation to these conditions. An almost finished separation of two cyanobacterial cells in culture
medium Z/10 is presented in the figure (B). After 45 h in NaHCO3/CaCl2 solution (C), which is used for the experiments on surface alteration and CaCO3 nucleation,
the thylakoids of the cells are intact. However, cell metabolism adapted to the nutrient-poor conditions by accumulating glycogen granules (indicated by G).

properties of the cells as well (Borrok et al., 2004; Haas, 2004).
When the experimental conditions were not representative of
those in nature, the relevance of the experiments with respect
to simulating and understanding natural processes would be
strongly affected. One example with respect to our experiments
is the production of EPS by the cells. Both composition and
production were shown to be strongly dependent on: (i) the
physiological state and growth state of the cells, (ii) the
composition of the nutrient media, and (iii) the ionic and
physical conditions of the media (reviewed by Decho, 1990).
Another more ecological aspect is the importance of pico-
plankton within the phytoplankton community. Picoplankton
can dominate the primary production especially in oligo-
trophic lakes (Stockner et al., 2000). Nutrient concentrations

© 2005 The Authors

Journal compilation © 2005 Blackwell Publishing Ltd
 Living under an atomic force microscope 187

Fig. 6 AFM images showing a 3D projection (first

column) and the phase lag (second column) of the
surface alteration of a Synechococcus leopoliensis
cell at several time intervals after the cell was
exposed to Ca2+: 2 h after the beginning of the
experiment (A), the surface appears rather smooth
in both, topography and phase. After 6 h (B) small
protuberances are more noticeable and after 21 h
(C) the cell surface seems to be rather rough in
topography. The protuberances are still clearly
visible in the phase image.

Fig. 7 Three-dimensional projection (A) and phase

image (B) of Synechococcus cells embedded in
huge amounts of EPS. While it is not possible to
identify the cell structures within the bulk mass of
sheath material in the topographical image, they
can easily be identified in the phase image. These
images were taken in tapping mode in air. The
arrow is indicating a previous cell division resulting
in almost spherical daughter cells.

and temperature were shown to control the contribution
of picoplankton to phytoplankton biomass and production
(Agawin et al., 2000a,b). Therefore, the environmental
relevance of the processes we investigate in the laboratory
is strongly linked to the experimental conditions. In order to
represent these conditions and to culture a sufficient amount
of cells for our experiment in reasonable time, we decided to
culture S. leopoliensis in Z/10 medium. The major nutrients:
phosphate, nitrate and sulphate are 25, 33 and 30 times less
concentrated than in BG11-based media that were used for
many experiments, e.g. studies on sulphur starvation (Arino
et al., 1995) or nitrogen starvation (Sauer et al., 2001; Barker-
Astrom et al., 2005) or both (Wanner et al., 1986). Thus, the
metabolism and therefore the growth rate of our cultured
cyanobacteria were adapted to these conditions. This is indi-
cated by a specific growth rate µ that is reduced by more
than one order of magnitude in continuous Z/10 cultures
compared to BG11 batch cultures. We measured equilibrium
growth rates µ of 0.1 d−1 in our chemostats (light intensity
7 µE m−2 s−1), compared to measured maximum µ-values
between 1 d−1 and 2 d−1 (average 1.8 d−1, light intensity 12 µE
m−2 s−1) in BG11 medium in batch experiments. However, the
nutrient concentrations were sufficient in order to maintain
photosynthetic active and viable cells. The cultures showed the
typical blue-green colour. No indications of starvation-induced
chlorosis could be found as described in Sauer et al. (2001)
and Goerl et al. (1998). In their studies, the authors describe
the acclimation and the loss of photosynthetic pigments as an
adaptation to long-term survival. Concerning growth, our
results also coincide with the findings of Timmermans et al.
(2005). They demonstrated that small phytoplankton species
can grow under much lower nutrient conditions and concluded
that picoplankton will hardly ever stop growth totally by nutri-
ent limitation under natural conditions. Collier & Grossman

© 2005 The Authors

Journal compilation © 2005 Blackwell Publishing Ltd
188 M. OBST AND M. DITTRICH
(1992) measured continued cell division of S. leopoliensis
PCC 7942 in their nutrient deprivation experiments, but
depending if P, N or S was depleted, the time steps between
cell divisions increased to certain extents. Thus, the expression
‘starvation’, which is used in most of the studies cited above,
can easily be misinterpreted to some extent. The expression
‘adaptation’ describes the process more accurately as supported
by the studies of Sauer et al. (2001) and Goerl et al. (1998).
Another aspect, the influence of nutrient depletion on cell
physiology, morphology and ultrastructure of cyanobacteria,
was proven carefully in our study. Ultrastructural changes
affecting the cell wall were not observed in any of our experi-
ments. Hardie et al. (1983) describe in detail several steps of
intracellular changes due to iron starvation. The first step is the
separation of the thylakoids and was observed between 16 and
60 h of iron starvation. Degradation or deterioration was not
observed in any of their cells at this stage. However, after >60 h
of starvation, they observed deterioration of the thylakoid
system and in some of the cells a separation of the cytoplasmic
membrane and the peptidoglycan layer. This separation could
change the morphology of the cells and was never observed
during the first step. Iron starvation did not affect carboxysomes
or the extracellular glyocalyx in their experiments.
The reversible disintegration of the thylakoids membranes
was also observed after 9 days of sulphur starvation experiments
on Gloeothece sp. PCC6909 by Arino et al. (1995).
Fig. 8 The production of sheath material during
an in situ flow-through AFM experiment is shown
for several time intervals. Two hours after the
cultivation experiment was started (A) the image
is rather clear, many cells of small diameter were
observed in the middle of this image. Six hours after
starting (B) the cell agglomerate can still be easily
identified. Twenty-one hours after the start (C) the
image was already blurry as the tip started sticking
to the sample. Twenty-two hours after starting
cultivation (D), it is not possible any more to
identify single cells within the blurry mass of sticky
sheath material.
In contrast to their results, these ultrastructural changes
were observed to much less extent during our experiments.
In Fig. 5(A) (taken after 24 h in NaHCO3 solution), the thy-
lakoids are less separated than in Fig. 2(A) of Hardie et al.
(1983), which is described as an early stage of separation. For
our experiments, Fig. 5(A) is representative as we observed
this stage for the majority of the cells we analysed by TEM
(>15).
Furthermore, we found that Ca2+ most probably plays an
important role in the stability of the thylakoid system as the
separation was quite reduced when S. leopoliensis cells were
suspended in NaHCO3/CaCl2 solution even after extended
periods of time (45 h in Fig. 5C) compared to the Ca-free
solution of NaHCO3. In the Ca-containing solution, the vast
majority of the cells did not indicate separations of the thyla-
koids (>15 cells analysed). Thus, morphological changes of
the cell and, therefore, of the cell surface due to starvation are
very unlikely to happen in the timescale and under the condi-
tions of our in vivo AFM experiments. Only the very first steps
of this process were observed by TEM in an even more nutri-
ent depleted solution than the one used for the nucleation
experiments.
A noticeable ultrastructural change that occurred in the
NaHCO3 /CaCl2 sample compared to the cell from the cul-
ture medium is an accumulation of glycogen granules that
makes the cell appear more electron translucent. Our findings
© 2005 The Authors
Journal compilation © 2005 Blackwell Publishing Ltd

coincide with the results of Wanner et al. (1986). In Fig. 2 of
their study, they present very similar cells of the S. leopoliensis
PCC 6301 strain, which were observed in stagnating and in
nitrate-starved cultures after 9 h of starvation. Size and shape
of their cells did not change significantly even after extended
periods of starvation (70 h). These results coincide with our
findings.
To sum up, the results of this study emphasize the impor-
tance of the culturing and experimental conditions. As this was
done extensively in the case of our study, we could show the
relevance of our first results with respect to simulating natural
processes in order to clarify the mechanisms. The reason why
we cannot find strong indications for starvation even after 45 h
in nutrient-free NaHCO3/CaCl2 solution, is most probably cell
metabolisms, which are already adapted to low nutrient
conditions (and slowed down compared to BG11 cultures)
because of the cultivation conditions in Z/10 medium.
Cell immobilization
The limiting factor of cell imaging is a proper immobilization
method that attaches the cells firmly to the substrate but avoids
denaturizing the sample (Dufrene, 2004). Some microscopists
tried to work around this problem when they performed
experiments in situ and then dried the samples for the AFM
imaging. Camesano et al. (2000) for example covalently bound
bacteria to the support. They could perform experiments with
the immobilized cells in an aqueous environment and air-dry
the samples for imaging. Liu et al. (2004) also performed
AFM experiments in air and observed structural differences
in the cell membrane between La3+ treated Escherichia coli
cells and nontreated ones. Gently air-dried polysaccharide
macromolecules are suggested to keep their hydration water
(Santschi et al., 1998) but it is not clear if biopolymers maintain
their conformation (Wilkinson et al., 1999). Bolshakova et al.
(2001) reported two kinds of artefacts induced by drying
biological samples when they compared images of bacteria
scanned in air and in situ, respectively. First, the height and
width of the cells decreased when dried. Second, a surface
pattern appeared in air, which could not be observed when
imaging in water. Therefore, cell imaging in water seems to be
superior with respect to an easier interpretation of the obtained
data.
However, scanning cells in an aqueous environment is
still a challenge (Dufrene, 2003) because of the delicate im-
mobilization. The optimization of the immobilization is
necessary as an improvement of cell adhesion was indicated to
promote cell resistance to the disruptive effect of the scanning
cantilever (You et al., 2000) and, therefore, reduces artefacts.
In principle, five different immobilization methods have been
established. (1) Mechanical trapping of cells in the filter pores,
e.g. an isopore polycarbonate membrane (Touhami et al.,
2003, 2004). (2) Physical adsorption of cells onto chemically
treated surfaces, e.g. poly-l-lysine (Bolshakova et al., 2001;
© 2005 The Authors
Journal compilation © 2005 Blackwell Publishing Ltd
Living under an atomic force microscope 189
Doktycz et al., 2003) or polyethylenimine-coated supports
(Crawford et al., 2001). (3) Embedding cells into a soft
layer of agarose (Gad & Ikai, 1995) or gelatin (Doktycz et al.,
2003). (4) Direct growing of the cells onto a suitable sub-
strate (Kuznetsov et al., 1997; Gebeshuber et al., 2003). (5)
Drying of cells on a surface of an aluminium oxide anodisc
filter for several hours and the following rehydration of the
cells (Yao et al., 2002).
All of these methods have advantages and disadvantages
that will be discussed in the following section with special
regards to their potential of performing long-term in vivo
experiments. Vadillo-Rodriguez et al. (2004) compared the
first two methods and concluded mechanically trapping
bacterial cells in filter pores to be more reliable. This method
has at least three possible drawbacks. First, it is not applicable
to rod-shaped micro-organisms. Second, EPS might accumulate
on top of the anchored cells, and third, the rigid entrapment
of cells in holes of limited diameter might affect the viability
of the cells (Gad & Ikai, 1995). Thus this method might be a
good option for short-term in vivo investigations of un-
disturbed cell surfaces. Doktycz et al. (2003) compared the
immobilization of different bacterial strains by adsorption
onto different poly-l-lysine coated surfaces to different
gelatine-coated ones. In their study, gelatin turned out to be
superior to poly-l-lysine as it attached the cells more firmly.
Both methods are capable of immobilizing rod-shaped cells.
The cell surface at the interface between cell and substrate
might be artificially altered by linker molecules like poly-
l-lysine. However, as the thickness of the coating of the
substrate is very little (on the molecular level in case of
poly-l-lysine), the AFM-accessible parts of the cell are likely
to be unaffected.
The fourth method is restricted to organisms or cells, which
tend to grow on solid substrates, but it is not applicable for
example on single-cellular organisms living in a water body.
The last of the five techniques was described to retain a suf-
ficient number of Gram-negative bacterial cells attached to
the membrane surface in order to perform experiments in an
aqueous milieu for hours. The authors claim that this method
was developed particularly considering the minimization of
surface alterations, which might be caused by binding agents.
However, drying of the cells might cause changes of the cell
metabolism and enzyme activity (e.g. Potts et al., 1984) and
surface alterations due to membrane modifications in particu-
lar. A review of observed cell responses to desiccation can be
found in Potts (1994). An alteration of the surface, however,
would be critical with respect to the surface-related nucleation
processes that we wanted to observe in our experiments.
Beside these alterations, another cell response is limiting this
approach: In his review, Potts (1994) reports that many
cyanobacterial strains secrete conspicuous amounts of EPS
when they are exposed to water stress. As discussed later in
this chapter, EPS is one of the major limiting factors when
performing long-term experiments in the AFM.
190 M. OBST AND M. DITTRICH
With respect to our requirements, we proved that the
second method using poly-l-lysine was superior to the third
method using gelatin for our experiments. Poly-l-lysine-coated
glass cover slips were shown to be suitable for immobilizing
single-cellular strains of cyanobacteria for in situ AFM investi-
gations in tapping mode. In contrast to the findings of
Doktycz et al. (2003), who investigated the immobilization
of Staphylococcus aureus (Gram-positive) and E. coli (Gram-
negative), in our study with S. leopoliensis (PCC7942), poly-
l-lysine was found to be superior to gelatin. Using gelatin, we
could immobilize cells only for short periods, but after one or
a few scans, the majority of the cells was shifted away from
the tip. Another disadvantage of the gelatine method was the
frequent occurrence of line artefacts in the background. These
artefacts were most probably caused by the tip sticking in the
soft gelatin layer that covered the glass substrate. Thus, it was
rather difficult to find the right settings in order to establish
a stable feedback loop. With gelatin, high-quality images
of S. leopoliensis could not be recorded. In contrast to this
method, the poly-l-lysine-coated cover slips were shown to be
capable of immobilizing S. leopoliensis cells properly for peri-
ods longer than 100 h. During this period of time, the cell
shown in Fig. 3 could be scanned more than 20 times in total!
The preparation mode and age of the used cell suspension
have an influence on the homogeneity of the cell distribution.
The preparation can be optimized using epifluorescence
microscopy as an effective tool for analysing the distribution of
cells on the cover slips. Immobilized Synechococcus cells rapidly
exhibited cell division and growth without a pronounced
lag phase after starting the experiments in growth medium.
This result provides additional evidence that cyanobacteria
cultivated in homogeneous suspension are viable under
‘biofilm conditions’ (Becker et al., 2004). Thus, we conclude
that immobilization of cells on flat surfaces by poly-l-lysine is
suitable for long-term experiments. Especially for in situ inves-
tigations of the cell surface under changing environmental
conditions, this method is superior to physical entrapment and
embedding in agarose or gelatin. However, this method is
most probably restricted to cells with negative surface charges
and most likely has to be optimized for each individual strain.
First results of long-term surface alteration experiments
After culturing conditions and cell immobilization were
optimized, we could successfully perform initial long-term in
vivo experiments on the changes of cyanobacterial surfaces due
to the treatment with supersaturated solution with respect to
calcite. The relevance of these results for natural processes was
ensured as discussed in detail in the preceding section.
Ca2+ ions were observed to alter the surface of the cells
significantly within the timescale of hours in a NaHCO3 /CaCl2
solution. These alterations do not only change the surface
microtopography of S. leopoliensis but also the physical prop-
erties of the cell surface as the changes could be observed in
the phase lag as well. Nuclei of initial CaCO3 crystals could not
be identified on the analysed cell. However, from SEM analysis
of precipitates of bulk experiments (data not shown), we know
that only a fraction of S. leopoliensis cell nucleates calcite on its
surface. Thus, we conclude that a series of experiments has to
be performed until the sequence of crystal nucleation can be
observed.
The observed changes in the fine structure of the cyanobac-
terial cell surface, however, could be shown not to be caused
by starvation, but by Ca2+ ions of the supersaturated solution.
This is supported by the following results: The cell surface
observed by AFM is rather smooth in Z/10 culture medium
(Fig. 2), pure NaHCO3 solution (data not shown here) and
at the beginning of the nucleation experiments after 2 h in
NaHCO3/CaCl2 solution (Fig. 6A). The most advanced steps
of the starvation process in our experiments, however, were
observed by TEM after treatment in pure NaHCO3 solution.
These changes were described as early stages of the separation
of the thylakoids in the work of Hardie et al. (1983). This
stage is described as the initial step of an iron starvation
process and still far away from affecting the structure of the
cell wall.
However, the physicochemical principles of the observed
surface changes during our nucleation experiments could not
be clarified in this first study. More advanced techniques like
chemical force microscopy could help in order to characterize
the changes more in detail.
Cell division
Another but rather unexpected result was the observed second
cell divisions perpendicular to the first divided cells (Fig. 3),
as Synechococcus usually divides perpendicular to the length axis
as shown in the TEM image of Fig 5(A,B). The starting points
of the unusual division are two almost spherical cells that seem
to be already divided, but not yet separated. The unusual divisions,
perpendicular to the first one, to the author’s knowledge have
never been observed before for S. leopoliensis. This observation
was only made once during our long-term nucleation experiments.
However, it is rather unlikely to observe ongoing cell division
online, simply based on statistics. In Z/10 medium, the
specific growth rate µ of our culture was 0.1 d−1 at 27 °C and
an illumination of 7 µE m−2 s−1. With regard to the study of
Collier & Grossman (1992), we suppose the growth rate to
be smaller in NaHCO3/CaCl2 solution. Nutrient supply is
limited at one side of the cell because of immobilization and,
furthermore, the temperature was only 20 °C. The illumination
in the fluid cell cannot be measured with conventional
instrumentation. Additionally, the lateral resolution needed
to be sufficient in order to be able to recognize cell divisions,
i.e. the scan area has to be small, the resolution settings high
(512 × 512 pixels) and the scan speed slow. Combining these
premises, it is evident that the probability of observing ongoing
cell divisions is rather small. Completed cell divisions of
© 2005 The Authors
Journal compilation © 2005 Blackwell Publishing Ltd

rod-shaped cells were observed frequently as shown for
example in Fig. 2. Although, divisions resulting in spherical
cells were only observed a few times during our experiments,
they were observed for both immobilized cells by AFM and
suspended cells in the free water by TEM.
However, reasons other than cell divisions can barely explain
our observations. Contamination by other strains is not likely
as aseptic techniques were followed as far as possible, a pure
culture was used for these experiments and contamination
was never observed when analysing the samples under the epi-
fluorescence microscope. The simple loss of cell pressure and
collapse of the cell for reasons of starvation were, on the one
hand, shown to be unlikely and, on the other hand, this proc-
ess would hardly lead to the observed fourfold structure in
topography (Fig. 4). It has been shown by Begg & Donachie
(1998) that round sister cells of an usually rod-shaped E. coli
strain divide at alternating planes at 90° to each other, when the
physical separation of the sister cells is prevented by immobi-
lization on agar. Therefore, we strongly believe that the observed
changes of our S. leopoliensis cell represent two subsequent
divisions of a single cell along perpendicular division planes.
Possible artefacts
When soft and fragile samples like cells are scanned by AFM,
possible artefacts induced by multiple scans of the same area
have to be addressed. Especially when we scanned large areas
for overview images at high tip velocities (e.g. 60 × 60 µm2 in
Fig. 1), some cells were removed from the surface simply due
to lateral forces applied by the scanning tip. Scanning smaller
areas allowed a significant reduction of the tip velocity and
still a reasonable acquisition time. Thus, scanning small areas
was found to be more convenient when working in aqueous
environments. Working at a scan-angle of 90° also improved
the image quality, because adherence artefacts only lead to
torsion of the cantilever, but influences the feedback-loop of
the AFM to a lesser extent. During the optimization of our
setup we also observed that using tapping mode facilitates the
long-term immobilization of our S. leopoliensis cells. Whereas
in contact mode the cells were frequently removed by the tip,
this problem was greatly reduced when working in tapping
mode. In comparison to contact mode, tapping mode applies
less shear stress to the sample. The shear stress in contact
mode was shown to cause artefacts as disruptive effects of the
cantilever in the study of You et al. (2000). Finally, artefacts
due to the tip geometry have to be considered when relatively
large objects in height-like cells are scanned. The upper surface
of the cells can be interpreted without problems, but the
sidewalls are affected by the tip geometry (Velegol et al., 2003).
Long-term capabilities of the in vivo AFM approach
Concerning the long-term capabilities of our approach, several
important topics have to be addressed. Cell viability and
© 2005 The Authors
Journal compilation © 2005 Blackwell Publishing Ltd
Living under an atomic force microscope 191
immobilization have already been discussed with respect to
long-term experiments in the preceding sections. Another
important topic is the stability of the scanning conditions. The
microbial production of EPS turned out to be the limiting key
factor, deciding if high-quality images of immobilized cells
could be obtained or not. In a nutrient-depleted medium like
the NaHCO3/CaCl2 solution used for our experiments, it was
possible to adjust feedback parameters of the AFM accurately
in order to have a stable feedback loop even after several days.
In culture medium, this was not possible with our setup,
basically because of the production of loosely bound EPS by
the cyanobacteria. Whereas in situ AFM is highly capable of
investigating thin mucilage layers that are strongly attached to
cells (e.g. Higgins et al., 2003), it is hardly possible to
elucidate the microtopography of thick, soft bulk exopolymers
produced by cyanobacteria (e.g. Fig. 8). The tips stuck to the
EPS frequently so that the induced line artefacts prohibit
reasonable interpretation of the obtained images. We emphasize
that experimental conditions have to be optimized not only
with respect to the cell viability and relevance for natural
processes, but as well as for the quality of the scanning
technique. In the case of our study, these ‘optima’ luckily fit
together. However, in the case of other geo-related in vivo
AFM studies, the optimization might require compromises.
CONCLUSION
For the first time, it is possible to investigate processes at the
interface between biology and geology on a relevant timescale,
in vivo and nearly online. The possibility of performing long-
term experiments on bacterial surfaces under nearly environmental
conditions offers great opportunities for investigations on
surface-related biogeochemical processes like biomineral
nucleation or metal adsorption. We tried to cover both potentials
and possible drawbacks of this approach in our study and we
hope to contribute some reassurance for future requirements
of long-term in vivo AFM studies.
ACKNOWLEDGEMENTS
This study was ETH funded (TH 0-20967-02). We are grate-
ful to M. Boller (Eawag) for loaning the AFM. We would like
to thank B. Wehrli, B. Sinnet (Eawag), Dr S. Akari and A. Korte
(NanoCraft) for useful and critical discussion of the results.
Finally we thank the reviewers for improving the quality of this
manuscript.
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