Particle Tracking

Review
Vol. 7, No. 4 / December 2015 / Advances in Optics and Photonics
713
Recent advances in holographic

3D particle tracking
PASQUALE MEMMOLO,1,2,* LISA MICCIO,1 MELANIA PATURZO,1
GIUSEPPE DI CAPRIO,3,4 GIUSEPPE COPPOLA,3
PAOLO A. NETTI,2 AND PIETRO FERRARO1
CNR-ISASI Institute of Applied Sciences & Intelligent Systems E. Caianiello, via Campi Flegrei 34,
80078 Pozzuoli (NA), Italy
2
Center for Advanced Biomaterials for HealthCare@CRIB, Istituto Italiano di tecnologia,
Largo Barsanti e Matteucci 53, 80125 Napoli, Italy
3
Institute for Microelectronics and Microsystems, Unit of NaplesNational Research Council,
Naples 80121, Italy
4
Currently at the Rowland Institute at Harvard, Harvard University, Cambridge, Massachusetts 02142, USA
*Corresponding author: p.memmolo@isasi.cnr.it
1
Received May 29, 2015; revised September 9, 2015; accepted September 15, 2015; published
November 9, 2015 (Doc. ID 242050)
Particle tracking is a fundamental technique for investigating a variety of biophysical

processes, from intracellular dynamics to the characterization of cell motility and migration. However, observing three-dimensional (3D) trajectories of particles is in general
a challenging task in classical microscopy owing to the limited imaging depth of field of
commercial optical microscopes, which represents a serious drawback for the analysis of
time-lapse microscopy image data. Therefore, numerous automated particle-tracking approaches have been developed by many research groups around the world. Recently,
digital holography (DH) in microscopy has rapidly gained credit as one of the elective
techniques for these applications, mainly due to the uniqueness of the DH to provide
a posteriori quantitative multiple refocusing capability and phase-contrast imaging.
Starting from this paradigm, a huge amount of 3D holographic tracking approaches have
been conceived and investigated for applications in various branches of science, including optofluids, microfluidics, biomedical microscopy, cell mechano-trasduction, and cell
migration. Since a wider community of readers could be interested in such a review, i.e.,
not only scientists working in the fields of optics and photonics but also users of particletracking tools, it should be very beneficial to provide a complete review of state-of-theart holographic 3D particle-tracking methods and their applications in bio-microfluidics. 2015 Optical Society of America
OCIS codes: (180.6900) Three-dimensional microscopy; (100.4999) Pattern recognition, target tracking; (350.4855) Optical tweezers or optical manipulation;
(090.1995) Digital holography
http://dx.doi.org/10.1364/AOP.7.000713
1. Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 715
2. Particle-Tracking Methods: An Overview . . . . . . . . . . . . . . . . . . . . . . . 716
2.1. Highlights from the State of the Art . . . . . . . . . . . . . . . . . . . . . . . 716
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2.2. Digital Holography and 3D Tracking . . . . . . . . . . . . . . . . . . . . . .

3. Principles, Methods, and Applications of Holographic Tracking. . . . . . . .
3.1. Axial Localization of Particles through Refocusing Criteria . . . . . . .
3.2. Methods for Transverse Particle Localization . . . . . . . . . . . . . . . . .
3.3. Special Case: Methods for Simultaneous 3D Coordinates Recovery . .
4. Holographic Tracking for Microfluidics Particle Dynamics . . . . . . . . . . .
4.1. Optical Tweezers for Particle Manipulation and Tracking . . . . . . . . .
4.1a. Back-Focal-Plane Displacement Detection . . . . . . . . . . . . . . .
4.1b. Holographic Tracking in Optical Tweezers . . . . . . . . . . . . . . .
4.2. Holographic Particle Image Velocimetry . . . . . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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Recent advances in holographic

3D particle tracking
PASQUALE MEMMOLO, LISA MICCIO, MELANIA PATURZO,
GIUSEPPE DI CAPRIO, GIUSEPPE COPPOLA,
PAOLO A. NETTI, AND PIETRO FERRARO
1. INTRODUCTION
Today, the astonishing development of bio-microfluidics technology strongly demands substantial improvements in multifunctional tools for characterization, monitoring, and manipulation in microfluidic environments. In recent years, the number of
proposed and implemented techniques for diagnostic purposes has greatly grown,
such as novel microscopic imaging techniques ([14] and their citations), lab-ona-chip applications [5], optical manipulation and trapping [68], point-of-care analysis [9], and particle tracking [10]. In particular, for the last case, the emerging and
growing number of contributions on this issue has prompted Chenouard and coauthors
to propose an objective comparison of particle-tracking methods based on an open
competition, organized in 2012, with the aim to establish performances of approaches
proposed by research groups worldwide, on the same representative training data (see
http://www.bioimageanalysis.org/track/ and Ref. [10]). Recently, digital holography
(DH) in a microscopic configuration [1116] has rapidly gained credit as one of the
most elective techniques for biological sample analysis. DH is a full-field and labelfree imaging technique able to provide quantitative phase-contrast information. In recent years, many improvements have been achieved, either in optical arrangements or
in numerical image processing algorithms, in order to make DH a high-throughput
imaging instrument able to furnish a diagnostic tool for applications in various
branches of science, including optofluidics [17], microfluidics [18,19], biomedical
microscopy [20,21], cancer cell detection [22], and three-dimensional (3D) cell
morphometry [2325]. Furthermore, nonlinear optical techniques for coherent optical
contrast between tracer particles and the background scatterings have been developed,
namely, harmonic holography [2628].
Among other features, DH in microscopy allows the accurate retrieval of the spatial
coordinates of multiple targets performing 3D particle tracking in the entire imaged
field of view (FOV). The main motivation is related to the ability of DH to address
a posteriori numerical quantitative multiple refocusing phase-contrast imaging.
Starting from this property, classical holographic tracking methods are typically composed of two main steps: 1) numerical refocusing, for retrieving the position of the
targets along the optical axis, and 2) evaluation of the transverse position of the
refocused object through quantitative phase map segmentation methods. Several contributions in recent years have been proposed in the literature for investigating increasingly accurate refocusing strategies [2948] as well as suitable two-dimensional (2D)
localization approaches for quantitative phase-contrast images [4954]. DH in microscopy is mainly accomplished through two configurations: in-line or off-axis. Smart
solutions for tracking have been found for both arrangements. This papers intent is a
deep overview of the tracking methods in off-axis geometry. Nevertheless it is worthwhile to note the importance of in-line holography for the detection of 3D trajectories
and velocimetry of multiple particles in a microfluidic environment [5557]. In-line
holography has also been demonstrated to be useful for imaging [58], besides being a
successful tool for out-of-laboratory measurement campaigns [59].
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However, referring to participants of competition reported in [10], holography-based

methods were not considered, since the training data were on images coming from infocus fluorescence microscopy. Obviously, some of the techniques considered in [10]
could also be applied to holographic images, but only for the estimation of the particle
positions in the transverse plane, since the numerical refocusing step is uniquely and
intrinsically associated exclusively with the DH imaging technology. These considerations further confirm the importance of a review about holographic particletracking techniques, because they refer to a methodology different from the one
presented in [10].
On the other hand, holographic particle tracking is often used in different branches of
bio-microfluidics, ranging from the study of the migration and motility of biological
samples [49], and the investigation of optically trapped particle motion by using optical tweezers (OT) [68], to calculating the force exerted by the single-beam optical
trap [6062] and characterization of the fluid motion by using the holographic particle
image velocimetry (HPIV) paradigm [50,63,64].
Extensive literature on these research issues is available, and recently, the holographybased tracking approach has represented a suitable choice, becoming a very viable
alternative to conventional techniques. Considering the huge amount of holographic
tracking methods and related research fields, our aim here is to present a careful and
extended review of the most important techniques of 3D tracking by DH, with
emphasis on their applications in optics and photonics. This review is aimed at investigators that are approaching 3D tracking in DH, but it also presents a complete
overview for those directly involved in the topic. However, if one considers other
journals in chemistry and materials sciences and biotechnology, potentially a much
wider community of readers should be interested in such a review, i.e., also scientists
working in fields different from optics that use particle-tracking tools. For this reason,
a brief overview of the most important noninterferometric approaches of 3D particle
tracking is also discussed in Section 2. In Section 3, we will focus on the holographic
tracking framework, describing in detail the most significant methods proposed in this
field and their practical applications in bio-microfluidics. Finally, Section 4 will be
dedicated to the holographic particle-tracking methods developed specially for
optically trapped particles and for fluid motion analysis.
2. PARTICLE-TRACKING METHODS: AN OVERVIEW
2.1. Highlights from the State of the Art
Over the last three decades, particle tracking has appeared to be the key technology for
quantitative analysis of intracellular dynamic processes. Typically, detecting and following individual particles in a time series of images is referred to as single-particle
tracking, while, when the number of particles becomes very large, one can introduce
the concept of multiple-particle tracking. Thus, two main steps may be identified to
implement a particle-tracking analysis: 1) particle detection, where elements that
stand out from the background are labeled by implementing suitable image segmentation approaches, and 2) particle linking, in which detected particles are connected
from frame to frame using another set of criteria to form tracks. For each of these
steps, many methods have been developed, often originating from other areas of data
analysis ([10] and its references). In addition, many software tools have been
developed for particle-tracking applications [10,65].
On the other hand, a fundamental aspect regards the possibility to establish whether
the best particle-tracking method exists. For this purpose, several comparison studies
have been published in recent years [6670]. However, as is well described in [10], the
cited studies were limited to either one aspect of the task or one specific application,
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thus neglecting the general effectiveness of the compared methods. As pointed out in
Section 1, an independent comparison of 14 particle-tracking methods, proposed by as
many research teams, has been reported in [10]. In particular, the competing
teams applied their particle-tracking method on the same time series of fluorescent
images, and a variety of performance parameters were used to establish the best
particle-tracking method. Remarkably, there was no overall winner. On the other
hand, the competition demonstrated the interest of many research groups in the development of even more accurate particle-tracking techniques. In order to make readers
aware of both the techniques and the teams participating in this competition, we report
the list of participants/methods (see Table 1) and their achieved performances
(see Fig. 1).
However, as said above, holography-based methods were not considered, mainly due
to the choice of using time-series fluorescence images even if, in the last two decades,
DH has gained huge credit as the natural imaging technique to investigate dynamic
processes related to particle tracking in 3D. In fact, after a simple keyword search in
the main databases of scientific documents, we reveal that holography-based approaches represent about 30% of all documents related to methods of particle tracking
in the last 30 years (see Fig. 2).
The first paper that addressed this topic dates back to 1989, when a coherent optical
correlation technique for real-time simultaneous tracking of several different objects
making independent movements was described [71]. Notice that, through 2000, few contributions were found, but the technological development of computers and solid-state
sensors in the late 1990s, which made it possible to record holographic information on a
digital sensor [72], has significantly contributed to the development of numerical
methods in all the hot topics concerning holography, including particle tracking.
These considerations further confirm the meaning of the current review regarding the
holographic particle-tracking framework, because it represents an ever-hotter topic in
many branches of scientific research.
2.2. Digital Holography and 3D Tracking
DH in microscopy [1116] is a powerful tool for the imaging of micro-objects

contained in a 3D volume, by single-shot image acquisition.
A basic DH setup consists of an illumination source, an interferometer, and a digitizing camera (CCD or CMOS camera). Most often a laser is used for illumination with a
certain coherence to produce interference, but even a LED can be sufficient for
holographic microscopy [73]. Also, DH using a 10.6 m CO2 infrared laser [74], deep
UV (193 nm) [75], and a 32 nm soft x ray [76] has been demonstrated. Typically, two
types of interferometers are employed for imaging of micro-objects, the Michelson
interferometer for reflective objects and the MachZehnder interferometer for transparent objects. For the purpose of this review, only the MachZehnder interferometer
is considered for time-lapse recording of our holographic 3D particle motility investigation. In Fig. 3(a), sketches of its optical arrangement are depicted. The captured
hologram pattern is digitized by the CCD as a 2D array of integers with 8-bit or higher
grayscale resolution.
An example of the DH process is shown in Figs. 3(b)3(e) using a resolution target
with FOV 200 150 m. In Fig. 3(b) the recorded hologram is reported, while the
interference fringe detail is shown in the inset. Figure 3(c) shows the amplitude
Fourier spectrum, where the three diffraction orders are clearly visible. Typically,
one of the twin terms is selected with a numerical bandpass filter (yellow circle)
to extract the object wavefield. Then, the filtered hologram is numerically
W. J. Godinez, K. Rohr
Y. Kalaidzidis
L. Liang, J. Duncan,
H. Shen, Y. Xu
K. E. G. Magnusson,
J. Jaldn, H. M. Blau
P. Paul-Gilloteaux
Either thresholding + centroid or

maxima + Gaussian fitting
Lorentzian function fitting to structures above
noise level
Gaussian mixture model fitting
Adaptive Otsu thresholding
Morphological opening-based clump splitting
Watershed-based clump splitting
The information is extracted from Table 1 in Ref. [10].
J. Willemse, K. Celler,
G. P. van Wezel
H.-W. Dan, Y.-S. Tsai
Nearest neighbor + Kalman

filtering
Nearest neighbor
Sequential multiframe
assignment
Linear assignment problem
Viterbi algorithm on state-space

representation
Nearest neighbor + global
optimization
Gaussian template matching
Multiple hypothesis tracking
Multitemporal association
tracking
Kalman filtering + probabilistic
data association
Dynamic programming
Combinatorial optimization
Principle
Remarks
Interacting multiple models using motion models as

specified.
Track assignment by the weighted sum of multiple
features.
Interacting multiple models with forward and
backward linking.
Brownian motion is assumed in all cases.
MM
MM, GC
MF, MT,
MM, GC
MT, GC
Two-step approach (frame-to-frame and segment

linking).
Allows merging and splitting of particles and uses a
linear motion model.
Essentially a 2D method keeping track of maximum
intensity in z.
Global linking cost minimization.
MF, MT, GC Global optimization of associations using simulated

annealing.
Only local and per-trajectory particle linking.
MF, MT
MF, MM
MF, GC
MF, MM
MF, MT, GC Post-tracking refinement of detections.
MF, MT, GC Greedy hill-climbing optimization with topological

constraints.
MF, MT,
Motion models are user specified (near-constant
MM
position and/or velocity).
MF, MT,
Motion models are user specified (near-constant
MM, GC
position and/or velocity).
Approaches
Linking
2D
3D
2D
3D
2D
3D
2D
2D
3D
2D
3D
2D
2D
2D
3D
2D
3D
2D
2D
3D
2D
3D
2D
3D
Dim.
[56,57]
[54,55]
[52,53]
[35,50,51]
[4749]
[45,46]
[43,44]
[42]
[41]
[29,4]
[38,39]
[3537]
[33,34]
[32]
Refs.
14
13
12
11
10
Maxima after thresholding two-scale wavelet

products
Adaptive local-maxima selection
Iterative intensity-weighted centroid calculation
Remarks
Watershed-based clump splitting and parabola

fitting
Either maxima with pixel precision (2D) or
thresholding + Gaussian fitting (3D)
P. Roudot, C. Kervrann, Histogram-based thresholding and Gaussian
F. Waharte
fitting
I. Smal, E. Meijering
Gaussian fitting (round particles) or centroid
calculation (elongated particles)
J.-Y. Tinevez, S. L. Shorte Parabolic fitting to localized maxima
N. Chenouard,
F. de Chaumont,
J.-C. Olivo-Marin
M. Winter, A. R. Cohen
I. F. Sbalzarini, Y. Gong,
J. Cardinale
C. Carthel, S. Coraluppi
Authors
Method
Detection
Table 1. List of Participants and their Methods of the Comparison Study Reported in [10]a
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reconstructed, at a certain distance from the hologram plane, computing the discrete
diffraction Fresnel propagation integral, which results in a 2D array of complex numbers. The corresponding amplitude and phase images are shown in Figs. 3(d) and 3(e),
respectively. In particular, the phase image, with color scale ranges 2 from blue to
red, represents a phase profile of the optical field reflected from the object surface or
transmitted through a thickness of a transparent object. Notice that the object surface
in Fig. 3(e) is slightly tilted with respect to the reference wavefront, and such tilt and
other aberrations can be readily compensated by numerical techniques [77,78]. Most
importantly, the phase image is a quantitative representation of the object profile with
high resolution [79]. Figure 4 displays a few more examples of quantitative phase
microscopy images by DH. Figure 4(a) is one group of three bars on a resolution
target. Figures 4(b) and 4(c) are fixed SKOV-3 ovarian cancer cells, where one
can discern several intracellular components such as the nuclear membrane and chromosomes. Figure 4(d) shows several red blood cells, and in Fig. 4(e) one can notice a
Figure 1
Top three best-performing methods for each performance measure and combination of
biological scenario, particle density, and SNR. The cells are color coded according to
method number (Table 1). Reprinted by permission from Macmillan Publishers Ltd.:
Chenouard et al., Nat. Methods 11, 281289 (2014) [10]. Copyright 2014.
Figure 2
Particle-tracking methods enveloped from 1981 to 2014. The blue bars count the
holographic methods.
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fold of the cheek epithelial cell, as well as its nucleus and mitochondria. Finally,
Fig. 4(f) shows an image of a small quartz crystal in common sand.
All phase images in Fig. 4 are reconstructed knowing the in-focus distance of each
imaged object. However, this information could not be available to the user, or more
than one object, with different reconstruction distances, can be observed in the imaged
FOV. To point out this aspect, in Fig. 5 we report an example of intensity reconstructions, calculated at different distances, of a recorded digital hologram of a MG63
osteosarcoma cell line, cultivated in reconstituted bovine collagen gel (Sigma
Aldrich, St. Louis, Missouri, USA) [54].
Figure 5(a) shows 10 cells in the FOV that appear out of focus, while in Figs. 5(b) and
5(c) the blue arrows indicate two cells very close to their in-focus plane. Recovery of
the correct reconstruction distance, i.e., the correct position of particles along the optical axis, is a very challenging task, and several automatic methods have been employed [2948]. In the next section, we review some of the most well-known methods
of automatic focusing.
The example reported in Fig. 5 clearly demonstrates the benefit of the holographic
imaging method with respect to classical microscope imaging due to the possibility to
recover 3D positions of the imaged particles by using only one recorded image.
3. PRINCIPLES, METHODS, AND APPLICATIONS OF HOLOGRAPHIC
TRACKING
In this section, we describe the fundamental steps to obtain an efficient holographic

particle-tracking strategy. As highlighted in Subsection 2.1, the first step to be
performed is particle detection. A large number of existing methods can be applied
Figure 3
(a) MachZehnder interferometer for digital holographic microscopy of transmissive

specimen. BS, beamsplitters; L, lenses. (b)(e) Digital holographic microscopy process (resolution target) (FOV 200 m 150 m, 1024 768 pixels): (b) hologram,
with detail shown in inset; (c) angular spectrum, with the yellow circled area passfiltered for reconstruction; (d) amplitude image; and (e) phase image. Reprinted with
permission from [11].
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in DH to identify single particles, and several research groups are working on this. For
example, Javidis group demonstrated several techniques of pattern recognition in DH
[8082], and our research group proposed an identification method in DH to detect
spermatozoa heads [25]. Since many of these approaches are inherited from the field
of image segmentation and pattern recognition, thus requiring a very detailed description, in this review we choose to consider the particle detection step already done,
focusing our description on the step of locating particles.
One of the main fields of application of holographic particle tracking is the study of
the migration and motility of biological samples. Motility is a main characteristic of
certain types of cells, and it is essential to understand the motility of cells by
Figure 4
Examples of quantitative phase microscopy by digital holography: (a) resolution

target (25 m 25 m, 452 452 pixels); (b) SKOV-3 ovarian cancer cells
(60 m 60 m, 404 404 pixels); (c) SKOV-3 ovarian cancer cell
(60 m 60 m, 404 404 pixels); (d) red blood cells (50 m 50 m, 404
404 pixels); (e) cheek epithelial cell (60 m 60 m, 404 404 pixels); and
(f) quartz crystal of sand (60 m 60 m, 404 404 pixels). Reprinted with permission from [11].
Figure 5
Intensity reconstructions of MG63 osteosarcoma cell line varying the reconstruction

distance. In (b) the distance equal to zero indicates the middle plane of the sample
volume. Blue arrows indicate cells very close to their focus plane.
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measuring their 3D motion. DH in microscopy is an eligible method for this purpose,

offering a rapid and efficient way to monitor the 3D motile behavior and the dynamic
processes of various types of cells. The effectiveness of this method is quantified in
terms of the maximum number and speed of the moving cells that can be tracked, as
well as the direction of the motion (lateral and axial). The earliest studies, involving
DH, show fibroblast migration, proposed by Kims group [83], and cancer cell
migration in in vivo-like circumstances, investigated by Duboiss group [84].
Other important contributions were conducted for the investigation of 3D motion
of human RBCs [85], free-swimming micro-organisms [8691], submicrometer objects [92], fast-moving bubbles in airwater mixture flows [93], predatory dinoflagellates [94], crawling cells in matrix gels [54], seminal cells [95,96], and algae and
bacteria [55]. A very complete review of these applications and others is realized by
Kims group in [97]. Here, we focus on the holographic tracking methods that allow us
to observe these phenomena. Since in the holography-based tracking the target positioning is typically addressed by decoupling the calculation of the optical axis position
and the transverse coordinates, we divide in the first two subsections the description of
the most used methods for both steps. Finally, the last subsection is dedicated to some
approaches capable of tracking, simultaneously and in a single step, all the three
coordinates of micro-objects.
3.1. Axial Localization of Particles through Refocusing Criteria
The digital holograms can be processed numerically with a reconstruction algorithm

to calculate the complex object wave [98]. Once the whole field is known, it is
possible to reconstruct the optical wavefront at different distances from the plane
of acquisition, as described by
Ox; y; z d F 1 fF fOx; y; z 0g expidv2 2 g:
(1)
DH provides a numerical investigation of the third dimension by performing plane-byplane refocusing. In Eq. (1), is the laser wavelength, F and F 1 denote the Fourier
transform and the inverse Fourier transform operators, respectively, and x and y represent the spatial and and the spectral coordinates. The variable z indicates the
plane in which the optical wavefront is reconstructed. Indeed, if digital holographic
reconstructions can refocus a sample slice-by-slice as the focusing stage of a classical
imaging system, the refocusing degree of an object has to be determined by an
external criterion. Typically, the refocusing stage starts defining a range of
reconstruction distances, and the hologram is numerically reconstructed at each distance in such an interval. Then, each amplitude reconstruction is processed by calculating a suitable image sharpness metric that is related to the refocusing criteria.
In this subsection we will describe some of the several refocusing criteria developed.
We start with a method proposed by Dubois [32,33]: it is based on the invariances of
both energy and amplitude. Those invariance properties allow building two focus
criteria, respectively, for pure amplitude and pure phase objects, based on the score
of the integrated amplitude modulus. It has been demonstrated in [32] that the criterion
is minimized for the amplitude object while it is maximized for the phase object. It is
assumed that a pure phase object is illuminated in transmission by a plane wave that is
propagating along the z axis and that the physical thickness of the object is small in
comparison with the depth of focus of the imaging system. Moreover, it is assumed
that the object is located at a distance d with respect to the focus plane of the imaging
system. As we consider a pure phase object, the emerging intensity is constant when
the focus image of the object is reached. Indeed the intensity image of the object
disappears from the focus image as is well known in optical microscopy. A further
result is that the modulus of the amplitude is constant in the focus plane. When the
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image of the object is defocused, the refraction created by the objects phase
modulates the amplitude and the resulting intensity. As a consequence the defocus
is at the origin of fluctuations of the amplitude modulus and the intensity. Let
I d x; y jOx; y; z dj; it can be demonstrated [32] that the refocusing criterion
is connected to the parameter
X
Md
I d x; y:
(2)
x;y
It is shown that this measure is minimized for pure amplitude objects, while it is maximized for pure phase ones. At this time, the selection of the pure phase or amplitude
criteria has to be decided by the user. As in microscopy, the objects can often be
considered as belonging to one of these two cases; there are few ambiguities. For
example, most of the living cells in liquid can be considered as pure phase objects,
while opaque particles are pure amplitude objects. However, we have to mention that
transparent particles can behave as amplitude objects when they are strongly scattering
the light. A similar behavior could also be observed for liquid droplets in air. In those
cases, the pure amplitude criterion gives better results. An upgraded criterion for refocusing based on the same principle has recently been proposed by the same author
[34]. The refocus criterion based on the integral of the amplitude modulus has been
generalized to be used in the same way for amplitude, phase, and mixed nature samples. To achieve this goal, the complex amplitudes are first adequately processed to
flatten the phase background and to remove the permanent defects in the FOV. Then,
high-pass filtering is applied to the complex amplitude. This process enhances the
high spatial frequencies, which are originated from the fast amplitude variations created by the objects. The result is that the new criterion provides minima when phase,
amplitude, or mixed nature objects are refocused. The comparison between the two
methods is shown in Fig. 6. A comprehensive review of four methods for the determination of the optimal focus position of digital holographic images of pure phase
objects is proposed in [29]. First, the fine-structure content in the image is analyzed by
the quantification of high spatial frequencies in the images and is taken into account in
a weighted spectral analysis (SPEC). Furthermore, a contrast-based evaluation of the
histogram of the gray value distribution is carried out to quantify the image sharpness
with the deviation of each gray value relative to the mean gray value (VAR).
Cumulated edge detection is used as a method for the quantification of the image
sharpness.
Therefore, the total sum of the gradient (GRA) or the Laplacian (LAP) of the image is
calculated for the quantification. In the following equations we reviewed four metrics:
X
logf1 F F fI d gv; g;
(3)
1 X
I x; y hI d i2 ;
N x N y x;y d
(4)
N
y 1 q
x 1 N
X
X
I d x; y I d x 1; y2 I d x; y I d x; y 12 ;
x1 y1
(5)
SPEC
v;
VAR
GRA
LAP
N
y 1
x 1 N
X
X
I d x 1; y I d x 1; y I d x; y 1 I d x; y 1 4I d x; y2 :
x1 y1
(6)
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In Eqs. (3)(6), hi is the average value operator, and F F denotes the bandpass-filtered
Fourier transform operator of the amplitude image I d x; y, with sizes N x ; N y , where
the lower boundary of the applied bandpass filter is set to suppress the frequency parts
in the origin of the spectrum. A comparison of the different criteria on the defocusing
Figure 6
Test on algal species. (a) Modulus of the amplitude and (b) intensity of the reconstructed high-pass filtered amplitude at the best-detected focus plane (reconstruction
distance d 68 m). (c) Modulus of the refocused amplitude. (d) Unwrapped
phase map of the refocused amplitude. (e) Evolution of the criteria M H;d (proposed)
and M d (original) as a function of the reconstruction distance d. The curve exhibiting a
minimum at d 68 m corresponds to M H;d , while the one with a maximum, at the
same d, corresponds to M d . The presence of a maximum for M d shows the phase
nature of the object. Scale bar 20 m. Reprinted with permission from [34].
Copyright 2014, Optical Society of America.
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725
is shown in Fig. 7, where the processing is performed on a pancreatic tumor cell

(PaTu 8988S).
SPEC and GRA appear to be particularly applicable for digital holographic
investigations of phase objects, fulfilling the focus function criteria presented by
Groen et al. [36]: unimodality, accuracy, reproducibility, and range. Regarding the
computation time, the methods do not differ significantly; therefore the evaluation
of the appropriate algorithm can be carried out disregarding the computational
efforts. However, applying the algorithm to a specific region of interest within the
reconstructed complex object wave accelerates autofocusing in practical applications,
allowing a reduction of the computation time by approximately a factor of 12.
It is interesting to note that all the methods compared in Figs. 6(e) and 7(g) could have
several local extrema points in the searching distances range. In fact, if the latter is
incorrect, i.e., it does not contain the global extreme of the refocusing metrics, such
methods could establish a wrong focus distance. Therefore, a fundamental property
that a refocusing criterion should have is to present a single extreme in the searching
range. Recently, two refocusing methods have been proposed to be used in the same
way for both amplitude and phase objects, having the mentioned property: a measure
of image contrast based on the approximation of the Tamura coefficient (TC) [39,54],
and the Gini index (GI) [40]. These two metrics are defined in Eqs. (7) and (8),
respectively:
Figure 7
Comparison of (a), (c), (d), and (f) defocused and (b) and (e) focused (a)(c) reconstructed amplitude and (d)(f) unwrapped phase distributions of investigations of
PaTu 8988S cells in the transmission light arrangement. (g) Focus value functions
for SPEC, VAR, GRA, and LAP. Notice that the reconstruction distance is indicated
by b, while the autofocus position is given by bAF . Reprinted with permission from
[29]. Copyright 2008, Optical Society of America.
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s
fI d g
TC
;
hI d i
(7)

N
X
id k N k 0.5
GI 1 2
;
od 1
N
k1
(8)
where fg is the standard deviation operator, id vecfI d g, and od

vecfOx; y; z dg, in which vecfg is an operator that creates a column vector from
a matrix, 1 is the l 1 -norm, and id k are the sorted entries of vector id in ascending
order, for k 1; ; N.
These two methods are successfully applied for the calculation of focus distance in the
case of both amplitude and phase objects. In particular, GI is originally used as a
measure of signal sparsity. However, there is empirical evidence that confirms that
sparsity metrics lead to effective image contrast measures. In fact, a sparsity-based
refocusing approach has already been proposed in [41], in which a sharpness metric,
related to the sparsity of the holograms expansion in distance-dependent wavelet-like
Fresnelet bases, is used. An interesting comparison, among TC criteria, GI, and
energy-based methods [32], is investigated by Memmolo et al. in [40]. In particular,
focus distance searching of the hologram of a mouses cell is investigated, demonstrating the accuracy of compared metrics. We report these results in Fig. 8.
Figure 8
Comparing GI, TC, and energy metrics for the hologram of a mouses cell. In (a) we
show the values given by the three metrics for different values of reconstruction distance. In (b) we show the amplitude reconstruction, calculated at a distance corresponding to the extreme point of GI (black circles), while in (c) the corresponding
phase reconstruction is reported. Reprinted with permission from [40]. Copyright
2014, Optical Society of America.
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727
Notice that the computational cost of these focus plane searching methods rapidly
increases as the resolution of the system becomes higher, since the object image
has to be numerically reconstructed from the hologram. To reduce the computational
time, we mention two interesting approaches, one based on redundant data elimination
by the critical resampling of the complex reconstruction [37], enabling real-time refocusing with up to 12 speed-up factors in comparison to the classical approaches,
and the other investigating the behavior of the sharpness curves of the object images
obtained from the scaled holograms, demonstrating that it is possible to estimate the
autofocus distance accurately with a speed improvement proportional to the square of
the scaling ratio [44]. Finally, refocusing approaches have been demonstrated in the
case of multi-wavelength DH [35,42,45], lensless DH [38], and optical scanning
holography [43].
3.2. Methods for Transverse Particle Localization
Once one has calculated the focus planes of particles in the imaged FOV for each
frame in a time-lapse holographic sequence, the corresponding complex wavefronts
can be reconstructed by numerically calculating the diffraction Fresnel propagation
integral. From this, the quantitative phase-contrast maps (QPMs) can be retrieved and
the transverse localization of particles may be possible. The most well-known method
of transverse particle tracking is based on the calculation of the cross correlation between two subsequent images [49,50]. In particular, the movement of the particle from
a generic image k of the sequence to the next (k 1) is measured by calculating the
displacement of the maximum value of the correlation from the position of the particle
in the image k. Therefore, the movement of each particle can be tracked individually
throughout the entire series. An example of the application of this approach in DH to a
L929 cell motility study is reported in Fig. 9.
Despite the fact that the cross-correlation method is the most used method, some
drawbacks can be identified. In particular, this approach works efficiently when
the motion is mostly (locally) translational between frames, and when there are
no large photometric variations. Also, a high image noise level and significant
morphological feature variation between frames can corrupt the particle position calculations. Therefore, several other approaches have been proposed in the literature. It
is possible to identify two categories of methods, which we name single image positioning (SIP) and multiple image positioning (MIP). In the SIP approaches, particle
locations are calculated by exploiting their morphological features, which are typically
obtained by image segmentation methods. The most used are the maximum phase
values [51], the centroid [52], and the center of mass (or weighted centroid) [53].
Instead, the MIP methods are based on the calculation of suitable parameters that
consider at least two subsequent images to be compared. An example of a MIP technique is described in [54], where the minimum boundary filter (MBF) is proposed to
be robust with respect both to the noise and to the morphological variations of
particles in time. Obviously, the above cited cross-correlation method belongs to
the MIP category.
The maximum phase values approach has been specifically designed for the study of
cell migration [51]. In fact, the maximum phase value corresponds to the position of
the nucleus in the cells body, which is directly associated with its movement, making
this method robust to the morphological changes that do not affect the nucleus.
However, it is strongly dependent on the QPM noise level, which can randomly
modify the maximum values position. This limitation is partially overcome by using
the centroid and the center of mass techniques. Both are based on the preliminary
identification of a region of interest (ROI) containing the particle to be tracked; then
an automatic ROI segmentation is applied. Most approaches are based on the Otsu
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filter [99], the anisotropic diffusion filter [25], the watershed method [100], and pattern recognition in DH [8082]. The output of this procedure is a binary image where
all background pixels are set equal to zero. Obviously, the remaining pixels, whose
value is 1, represent the area covered by the particle, whose centroid is chosen as the
particle position. Instead, by using the retrieved binary image as a filter for the initial
ROI to identify the particle area, we can calculate its center of mass, which is defined
as the particle position.
Notice that the accuracy of these two methods is strongly influenced by the segmentation step, which is related to the morphological variation of the particle in time. In
fact, especially in the case of biological samples, when a modification of the cells
shape occurs, the segmented binary image changes and consequently its centroid or
weighted centroid. In 3D environments, cells possess a spindle-like morphology in
which highly dynamic membrane processes are continuously projected out the cell
body in order to scan the pericellular environment without really moving around,
hampering the correct assessment of cell position. A hybrid technique, which tries
to combine the advantages of the above methods, is the MBF [54]. It is described
by the following steps:
Consider the ROIs containing the cell under analysis in two subsequent frames,
labeled k and k 1.
For both ROIs, one computes binary images, by a segmentation method, and
weighted centroids.
Translate the (k 1)-th binary image on that kth by superimposing the corresponding weighted centroids.
The MBF is obtained by filtering the result of the last superimposition. The centroid of this new image is the estimated position of the cell in the frame k 1.
It has been demonstrated that the MBF approach is very suitable for particles with
highly morphological variations during their motion [54]. However, it is worthwhile
to underline that it is not possible to determine which strategy is the best among all the
aforementioned methods, since the exact position of particles to be tracked should be
known. However, an interesting investigation in which this technique appears to be the
Figure 9
Reconstructed digital holographic phase image showing the tracking of L929 cells for
14 h: (a) tracking of a cell population; (b) 3D rendering showing the tracking of a
single L929 cell. The images were captured and the cells were analyzed at 10%
20% confluence. Scale bar in A represents 50 m. Reprinted, with permission of
Formatex Research Center, from Ref. [49].
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most robust with respect to image noise and morphological changes is addressed in
[54], and we report a comparison in Fig. 10. In this example, four methods are used to
track the cell in Fig. 10(a): the maximum phase values [51], the centroid [52], the
weighted centroid [53], and the MBF [54]. Note how the cell presents a strong morphological variation between a frame and the next one. The considered cell was
tracked by using the TC method as a refocusing strategy [39,54], and in Figs. 10(b)
and 10(c), a comparison of the 2D trajectories of the cell, obtained by the four considered methods, is reported. Observing Fig. 10, it is possible to say that the maximum
phase value method is very unstable compared to the others. Considering the stability
as a performance parameter, the weighted centroid method and the MBF appear to be
the most efficient. However, a morphological variation, between the current frame and
the previous one, produces very different results in the estimation of the positions
Figure 10
Comparison among the maximum phase values [51], the centroid [52], the weighted
centroid [53], and the MBF [54]. (a) reports the strongly morphological change of the
tracked cell between two subsequent frames. In (b) the color dots indicate the positions estimated using the four methods. The in-focus distance, calculated by the TC
method [39,54], is reported on the top of (b). (c) reports the trajectories of the four
methods estimated up to 25 frames. (b) and (c) are reprinted with permission from
[54]. Copyright 2012, Optical Society of America.
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among the different methods up to 10 m. Since MBF is an algorithm built specifically to take into account the morphological changes, it seems like an effective
tracking strategy.
Finally, in order to highlight the incredible development of a holographic 3D particletracking framework for the characterization of cell migration and motility, we report
the main results obtained for the investigation of human sperm motility, revealing the
rare helical trajectories of such cells within a large volume. This phenomenon was
observed by the Ozcan group in [95], where an efficient 3D tracking approach in
a dual-view and dual-color lens-free DH setup was implemented. In particular, the
2D centroid position of the sperm head projection in the vertical channel was directly
used as the sperms transverse coordinate, while the precise axial coordinate of the
sperm was calculated by dividing the distance between its vertical and oblique projection centroids by the tangent of the oblique illumination angle in water [95]. In
Fig. 11 some trajectories of human sperm swimming patterns are reported.
Figure 11
Four major categories of human sperm swimming patterns. (a) Typical pattern.
(b) Helical pattern. (c) Hyperactivated pattern. (d) Hyperhelical pattern. The inset
in each panel represents the front view of the straightened trajectory of the sperm.
The arrows indicate the directions of the sperms forward movement. The time position of each track point is encoded by its color (see the color bar). The helices shown in
(b) and (d) are both right-handed. Reprinted with permission from Su et al., Proc. Natl.
Acad. Sci. USA 109, 1601816022 (2012) [95].
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731
Further, Fig. 12 shows the investigation of the use of a classical digital holographic
microscope, with partial spatial coherent illumination, for the automated detection and
tracking of spermatozoa [96].
3.3. Special Case: Methods for Simultaneous 3D Coordinates Recovery
The approaches described in the above paragraphs are based on the separation of the
complex wavefront information into amplitude and phase, and the analysis of the lateral displacement can be made only after refocusing. Furthermore, the efficiency of
the refocusing affects the accuracy of the employed transverse localization method. To
overcome this limitation, one-shot holographic 3D particle-tracking approaches have
been proposed in recent years [88,89,101104]. For these methods, no decoupling
between the contribution of amplitude and phase is needed, but the 3D particle
positions are retrieved simultaneously in a single step.
In particular, Javidi and coworkers [88,89] propose a 3D region tracking method based
on a maximum a posteriori tracker and adapt it to digital hologram sequences. Griers
group reports an interesting comparison between two complementary approaches to
analyze holographic data recorded by using an in-line interferometer [101]. The first is
based on a fitting method, where each image is fitted to predictions of the LorenzMie
theory of light scattering [105,106]. The second approach is based on phenomenological interpretation of the scattered light field reconstructed with Rayleigh
Sommerfeld backpropagation. The comparison is made for measurements on
micrometer-scale colloidal spheres as a test case, demonstrating that these two approaches agree quantitatively, except for a systematic offset in the measured axial
positions, which can be accounted for quantitatively using LorenzMie optics.
Basically, the LorenzMie theory of light scattering provides a wealth of information
with outstanding resolution, but it is computationally very expensive and works well
only for particles whose light-scattering properties are completely described by the
theory.
Instead, 3D particle tracking obtained with RayleighSommerfeld backpropagation
provides volumetric reconstructions of particles, promising high-speed model-free
measurements of multiple objects positions and motions. The images in Fig. 13
are the normalized images of a 1.51 m diameter polystyrene sphere in water and
the corresponding pixel-by-pixel nonlinear least-squares fit. The quality of the fit
can be seen in the azimuthally averaged results reported in the plot.
Figure 12
Multiple sperm cell tracking. (a) Transversal and (b) reconstructed three-dimensional
path. Scale bar is 20 m; data were acquired over is 11 s. Reprinted with permission
from [96]. Copyright 2014, Optical Society of America.
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Instead, the example in Fig. 14 shows a typical volumetric reconstruction for a 1.5 m
diameter polystyrene sphere in water [Fig. 14(a)], and a collection 1.58 m diameter
silica spheres in water [Fig. 14(b)] obtained by implementing the Rayleigh
Sommerfeld backpropagation approach.
Finally, a comparison between these two methods is reported in Fig. 15, where
Fig. 15(a) shows the trajectory of a 1.5 m diameter polystyrene sphere diffusing
in water analyzed with both LorenzMie fitting and RayleighSommerfeld backpropagation. The two approaches agree quantitatively on the particles in-plane position,
Figure 13
LorenzMie particle tracking and characterization. The upper image is the normalized
hologram of a 1.51 m diameter polystyrene sphere in water. The lower image is a fit
to the LorenzMie theory. The solid curve is the azimuthal average of the measured
intensity around the center identified by the fit. Dashed curves indicate the azimuthal
standard deviation of the holograms values, and indicate the measurement error. Plot
points show the corresponding radial profiles of the fit. Error bars on the fit values are
smaller than the plot symbols. Reprinted with permission from [101]. Copyright 2010,
Optical Society of America.
Figure 14
3D tracking results by using RayleighSommerfeld backpropagation. (a) Volumetric

reconstruction of the scattered intensity due to a single colloidal sphere, colored by
intensity. (b) Volumetric reconstructions of 22 individual 1.58 m diameter silica
spheres organized in body center crystalline lattice with holographic optical tweezers
in distilled water. Reprinted with permission from [101]. Copyright 2010, Optical
Society of America.
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as is shown by the differences histogram in Fig. 15(b). Figure 15(c) shows the
distribution of differences of the axial positions measured with the two methods
as a function of the axial position of the LorentzMie estimate. Finally, Fig. 15(d)
highlights the dependence on both composition and size for colloidal silica and
polystyrene spheres of varying radii.
A similar approach to that reported in [101] is proposed by Finks group in [102],
where it has been demonstrated that a 3D volumetric deconvolution, applied to
the reconstructed wavefront, permits to resolve all particles simultaneously in 3D.
This concept was preliminarily investigated by the same author in [107], where it
has been demonstrated that all the positions of particles distributed in 3D can be retrieved at once from a holographic reconstruction, obtained by 3D volumetric deconvolution with the point-spread function of the optical system. In [102], the same
paradigm is applied to obtain the positions of an ensemble of moving particles. In
Fig. 16, the reconstructions of three consecutive holograms before and after
deconvolution are shown.
Each hologram in the experimental sequence was resampled with 200 200 pixels
and reconstructed at a series of distances from the hologram, ranging from 1 to 7 mm
with a step width of 0.3 mm to obtain a total of 200 reconstructions. Thus, from each
hologram, the reconstructed 3D volumetric complex-valued field, sampled with 200
200 200 pixels, is obtained (see middle column of Fig. 16). Then, the algorithm of
instant 3D volumetric deconvolution, proposed for the first time in [108] for tracking
Figure 15
Comparison of LorenzMie and RayleighSommerfeld particle-tracking algorithms.

(a) Trajectory of a colloidal silica sphere at 1/30 s intervals obtained with the two
strategies. Each point indicates the position of the sphere in one holographic snapshot
as estimated by the LorenzMie (LM) and RayleighSommerfeld (RS) approaches.
(b) Distribution of differences x and y in the in-plane position estimated by the two
strategies. (c) Mean difference in the axial position as a function of the LorenzMie
estimate, obtained from 10,000 measurements. (d) Mean difference in axial position as
a function of sphere radius for polystyrene (PS) spheres (circles) and silica (SiO2 )
spheres (squares). Solid curves are predictions of LorenzMie theory. Reprinted with
permission from [101]. Copyright 2010, Optical Society of America.
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of few objects in the scene, is applied to each volumetric reconstruction to achieve the
3D positioning of particles (right column of Fig. 16). We note that the methods proposed in Refs. [101,102] have another common aspect: both need a preliminary hologram normalization step (see Fig. 13 and left column of Fig. 16), necessary to mitigate
the speckle and the obscured fringes due to all other surfaces and particles along the
optical train, in order to efficiently compute the volumetric reconstructions.
Also, our group has recently proposed two techniques for single-shot holographic 3D
tracking. The first is based on a compact twin-beam DH [103], which can be
adopted as a flexible diagnostic tool in microfluidic platforms with twofold functionality, i.e., the 3D tracking of particles, and at same time, quantitative phase imaging of
tracked micro-objects by interference microscopy, without changing the configuration. The optical configuration is sketched in Fig. 17(a). The particles are loaded
in a chamber of about 5 mm 5 mm 0.3 mm, assembled by using two cover
glasses (0.15 mm thick) with a double-sided tape spacer (0.3 mm thick). Two beams,
coming from the same DPSS laser (532 nm, 250 mW), enter, slightly off-axis, into the
microscope objective (oil immersion Nikon 100, 1.2 NA). Theoretically, the two
beams have the same focal plane, but they produce aberrations resulting in some focus
shift.
When particles flow in the intersection of the two beams, two projections are imaged
by the CCD. The separation between these two shadows is a function of the 3D position of the particle, as shown in the illustration in Fig. 17(b). In particular, the distance
between the two images furnishes the axial position, while their middle point is the
Figure 16
Three successive holograms out of the sequence and their three-dimensional

reconstruction before and after applying the three-dimensional volumetric
deconvolution are shown. The area of the reconstructed volume amounts to
625 m 626 m 6000 m. Reprinted with permission from [102]. Copyright
2014, Optical Society of America.
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735
transverse position. Obviously, when the particle transits in the image plane, the two
projections converge into one image, as shown in Fig. 17(c).
In Fig. 18 the 3D trajectories of three particles simultaneously transiting through the
imaged volume are shown.
The second technique, proposed by our group in 2014 [104], is based on the maximization of the enhanced correlation coefficient (ECC). The ECC was proposed for
the first time in [109] as a performance criterion for the image alignment problem,
which is achieved by maximizing the ECC, by using either direct search or gradientbased algorithms. In [104] we extend this concept for wavefront alignment, demonstrating that ECC maximization can be successfully applied to complex fields in order
to measure, simultaneously, the displacement in 3D of moving particles. The ECC is
defined in Eq. (9) as
t
a b
ECCfA; Bg Re
(9)
;
ab
where Refg is the real part operator, a vecAm and b vecBm are the vectorization of images Am and Bm obtained from A and B subtracting the corresponding
arithmetic mean, is the Euclidean norm, and the superscripted quantities, t and
*, are the transpose and conjugation operators, respectively. Considering a time
sequence of recorded holograms and a generic frame k, let C k be the numerical complex reconstruction of the imaged particle, for which we know its 3D position
Figure 17
Twin-beam DHM for 3D particle tracking. (a) Illustration of the optical arrangement,
(b) simulated interference between two laser beams, and (c) three frames from a recorded sequence of the optical axis motion of a particle. Each subfigure is reprinted
with permission from [103]. Copyright 2011, Optical Society of America.
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pk xk ; yk ; zk , and C k1 be the sequent one, reconstructed at the same optical distance zk . The 3D particles coordinates in the frame k 1; pk1 xk1 ; yk1 ; zk1 ,
are obtained by solving the following maximization problem:
popt arg max ECCfC k ; T C k1 ; pg
pk1 pk popt ;
(10)
where T ; is a 3D translation of the complex wavefield C k1 , i.e., propagating

C k1 from zk to zk z and shifting it by x; y. Essentially, the solution of
the optimization problem in Eq. (10) furnishes the 3D position of particles in the frame
k 1 by aligning two subsequent complex fields (k and k 1), i.e., calculating the
3D displacement of the same particle in two subsequent instants of time. This strategy
was tested on living cells (i.e., NIH 3T3 mouse fibroblast) flowing into a microfluidic
channel. An example of the application of this method is shown in Fig. 19 for one of
the cells tracked in the experiment. In particular, two QPMs of the same cell in two
subsequent instants of time, visualized in the same ROI, are shown in Figs. 19(a) and
19(b); then the first and last aligned complex wavefronts are reported in Figs. 19(c)
and 19(d), highlighting the initial and final ECC values (in the top) as well as the
calculated 3D displacements (in the inset white boxes).
A comparison of such an approach with a classical holographic tracking method,
based on the separation of the amplitude and phase information from the complex
wavefront, is reported in Fig. 19(e), where the TC metric [39,54] is used as the refocusing criterion and the weighted centroid (WC) [53] as a method for transverse
coordinates calculation. Finally, Fig. 19(f) shows the results of the simultaneous
tracking of multiple cells and their calculated trajectories.
Figure 18
Recording sequence of random motion of particles highlighted by green, blue, and red
circles, and the corresponding 3D trajectories. Adapted from Ref. [103].
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4. HOLOGRAPHIC TRACKING FOR MICROFLUIDICS PARTICLE DYNAMICS

4.1. Optical Tweezers for Particle Manipulation and Tracking
Optical tweezers (OT) is a recent technique that exploits the forces exerted by a laser
light to manipulate mesoscopic objects with high spatial resolution and without
mechanical interactions. The first demonstration of particle trapping by a single beam
was given by Ashkin and coworkers in 1986 [60,61]. A strongly focused laser beam
exerts a recoil force along the optical axis able to push objects toward the focus; such
force is not negligible for objects in the micro- and nano-meter ranges. Stable trapping
is established when this optical gradient force dominates the radiation pressure so that
the trapped particle experiences a restoring force as applied by a spring; i.e., the motion of the trapped particle is overdamped. Today, OT is recognized as a valid and
high-throughput investigation tool in experiments where no direct contact is the
key requirement [6,7]. In order to achieve quantitative measurements, the tracking
of the trapped particle is fundamental. The positions and displacements of the probe
allow us to evaluate the forces exerted on it in elastic, viscous, and viscoelastic regimes. Hundreds of applications have been demonstrated in the last 20 years, where
trapped particles are used as passive probes, for example, in the case of rheological
measurements, or active probes, as in the case in which an intentional stress needs to
Figure 19
(a), (b) Two subsequent QPMs of motile cell. In (c) their superimposition has been
reported [the red and the green cells are the same in (a) and (b), respectively], and in
the upper part the initial values of the ECC and the initial displacements are shown. In
(d) the final output of ECC maximization with the corresponding ECC value (top) and
final displacements (top right) is shown. (e) Comparison of 3D positions of cell calculated by ECC maximization (blue line) and classical approach (TC+WC) (cyan
line). (f) In the top, a QPM with three cells, and in the bottom, the corresponding
trajectories. The blue line in (b) is the same as in (a). All displacement units are
expressed in micrometers. Adapted from Ref. [104].
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be applied. Accuracy in measurement position goes down to a few nanometers, while

the retrievable forces are in the piconewton range. At these scales single-molecule
and single-cell biophysics are among the most interested research fields in OT
[16,24,110112]. A powerful example is reported in Fig. 20, where a functionalized
trapped bead is anchored to a microtubule in order to quantitatively measure the
strength of its elongation usually involved in a mitosis mechanism.
The subsequent improvement in OT is due to the development of light-shaping techniques. The engineering of intensity and phase profiles of laser light through, for example, diffractive optical elements has renewed the OT potentiality passing from
single to multiple beams [68,113]. In particular, holographic OT (HOT) realized
by means of spatial light modulators has manifold applications such as the creation
of 2D and 3D arrangements of particles or cells [114], the possibility to rotate objects
under investigation [115], and the multiple pointwise sampling of viscosity
properties [116].
Figure 20
Recording microtubule dynamics with tension applied by an optical trapping-based

force clamp. (a) Schematic showing experimental geometry and force clamp operation. A polystyrene bead (blue) is held by an optical trap (orange). Dam1 complex
(green) on the bead surface mediates attachment to the tip of a dynamic microtubule
(red). A portion of the microtubule (dark red) is anchored to the coverslip. As the
microtubule grows and shortens, the coverslip is moved via computer to keep a fixed
separation (x) between the bead and trap, thereby maintaining a constant level of
tension. (b) Three (of N 298) representative records showing position against time
for tip-attached beads under tension. Increasing position represents bead movement
away from the anchored portion of the filament during microtubule. Reprinted by
permission from Macmillan Publishers Ltd.: Franck et al., Nat. Cell. Biol. 9, 832
837 (2007) [110]. Copyright 2007.
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4.1a. Back-Focal-Plane Displacement Detection
The most widespread method for tracking in OT is back-focal-plane (BFP) displacement detection. The intensity distribution of light scattered by the trapped particle is
detected by a quadrant photodiode in the BFP of the lens that is collimating the trapping laser light. In 1998 Gittes and Schmidt [62] demonstrated that the angular intensity shift in the BFP can be used to measure the motion of the trapped object and
consequently the forces exerted on it. The model identifies the intensity shift as the
first-order interference in the far field between the light that passes through the focus
and the light scattered by the trapped particle. The success of the BFP detection in OT
is due to the high lateral resolution that is in the nanometer range at bandwidths of
hundreds of kilohertz and consequently the possibility to measure the forces exerted at
the macromolecular level. Many applications of BFP tracking have been developed in
recent years [117120], and the brightness and effectiveness of the technique that
combines OT with BFP are well-recognized, as testified by the most recent high-impact papers [121,122]. Moreover, over the past few years, several improvements in
this detection scheme have been shown, for example, by Farr et al. [123]. The main
drawback of BFP tracking is the difficulty in simultaneously tracking more than two
probes. Even though the extension of the technique has been validated in some
particular cases [124,125], it is not the best-suited displacement detection mechanism
in a multi-trapping configuration. In order to perform accurate tracking of multiple
particles, as in the case of HOT setups, video-based algorithms are implemented for
simultaneously measuring the forces exerted on the probes in various sample sites.
From the experimental point of view, the quadrant photodiodes are replaced with
CCD or high-speed CMOS cameras. Today, commercially available CMOS cameras
allow sampling in the kilohertz regime, being comparable with the photodiodes
response [126128].
4.1b. Holographic Tracking in Optical Tweezers
The main drawback of the video-based sensing in OT is the difficulty in tracking particles along the axial direction. This limitation is completely removed when digital
holography (DH) is used as the imaging tool. The autofocusing property, described
in the previous sections, allows nanometer resolution positioning by means of the
numerical backpropagation of the wavefront in the in-focus plane. Besides complete
3D tracking, DH is particularly suited in bio-inspired experiments because of the
retrieval of the quantitative phase-contrast maps in the entire FOV.
Tracking algorithms usually employed for HOT applications calculate the axial position by exploiting the autofocusing criterion, while the position in the transverse plane
is measured by evaluating the position of the maximum in the phase signature of the
probe [129] (see Fig. 21 for an example). In contrast to the methods described in the
previous paragraphs, a novel holographic approach, for the accurate 3D dynamic
position tracking of multiple trapped probes, is proposed in Ref. [130]. This method
takes advantage of the so-called lateral shear interferometry (LSI), where the lateral
displacement of the wavefront under test allows us to obtain interference between the
original and the laterally shifted wavefronts. The basic concept of LSI has been transferred to DHM to compute a wavefront difference by single-image processing, avoiding the undersampling issues and circumventing the QPI reconstructions that occur in
the classical holographic particle-tracking procedure. Therefore, the main advantage
consists of performing fast and accurate particle tracking through the direct and
numerical subtraction between two experimental complex wavefields at two subsequent instances of time, tk and tk1 . If the interval from the frame k to the frame
k 1 is sufficiently small compared to the object velocity, it is possible to detect its
transverse motion due to the spatial superimposition of the same object that occurs
between the two frames [see Fig. 22(a)]. Mathematically, let W k and W k1 be the two
740
Review
complex wavefronts; the lateral displacements of imaged particles are calculated as the
argument of their ratio:
argfW k1 W k g W a1 X a2 Y a3 :
(11)
Equation (11) is valid in an internal region of the trapped particles, indicated by a

black box in Fig. 22(b), and transversal displacements (xk ; yk ) can be found by
fitting W with a plane, i.e., by calculating the coefficients a1 ; a2 , and a3 , neglecting
the contribution of higher-order aberrations:
xk a1 R;
yk a2 R:
(12)
Figure 21
(a), (b) White light images and (c), (d) DH in microscopy phase images of the PaTu
8988T cell. Particle A is moved in the lateral y direction. (e) Cross sections along the
continuous red line and dashed green line, respectively, through the phase images in
(c) and (d). The dynamic lateral tracking of the particle that is obtained from the spatial
coordinates of the point with maximum phase contrast in the phase images is illustrated in (b). (f) 3D trajectory of the particle obtained by combining the lateral
and axial trajectories. . Barroso et al., Small 9, 885893 (2013). [129]. Copyright
Wiley-VCH Verlag GmbH & Co. KGaA. Reproduced with permission.
Review
741
In Eq. (12), R is the curvature radius of the trapped particle. In the ideal case of spherical particles and noiseless images without aberrations, this method exactly furnishes
the displacements (xk ; yk ) as Eq. (11) has verified with equality. This holographic
tracking method was implemented with the aim to study the Brownian motion of
trapped micro-beads when chemically attached to a suspended cell, as shown in
Figs. 22(c)22(e).
First, the displacements of the optically trapped beads are measured to prove the
ability of the method in detecting the Brownian motion confined in the potential created by the HOTs. Then, the effect induced by the cell on the beads movement is
monitored. These results are reported in Fig. 23, where Figs. 23(a) and 23(b) show the
transversal displacements of the trapped beads, before the cell approaches, for
the bead on the left [Fig. 23(a)] and right [Fig. 23(b)], respectively. Figures 23(c)
and 23(d) report the transversal displacements computed after chemical bonding
of the two beads with the cell membrane. The main results achieved in this investigation regard the observation of a damping of the beads movement, as highlighted in
Fig. 23(e), which is related to the change of the mechanical properties of the cell [130].
Notice that the beads position along the optical axis is calculated by the TC refocusing criterion [39,54].
4.2. Holographic Particle Image Velocimetry
In particle image velocimetry (PIV) [131,132], the fluid motion is made visible by
adding small tracer particles, and from their positions at two or more instances of
time, it is possible to retrieve the flow velocity field. One of the first applications
of the PIV theory was laid out by Adrian, who described the expectation value of
the autocorrelation function for a double-exposure continuous PIV image
[133,134]. With the development of microfluidic devices, the classical PIV needs
to be revised for these small domains due to the low Reynolds number or the relative
importance of surface forces. In this scenario, we refer to micro-particle image
Figure 22
(a) Schematic drawing of the intrinsic lateral shear for a micro-bead illuminated by
coherent light. (b) W calculated for two trapped micro-beads. (c)(e) show QPIs of
trapped beads, beads approaching a cell, and beads attached on the cell surface.
Adapted with permission of the Royal Society of Chemistry from Ref. [130].
742
Review
velocimetry (PIV) as a quantitative method that is used to characterize the performance of such microfluidic systems with spatial resolutions better than 1 m [135].
Therefore, in its current form, PIV means the accurate, quantitative measurement of
fluid velocity vectors at a very large number of points simultaneously [132]. The PIV
Figure 23
Transversal displacements of (a) left and (b) right beads trapped by the HOT in
Fig. 22(c). Transversal displacements of (c) left and (d) right beads corresponding to
Fig. 22(e). (e) Horizontal displacement of the bead in (d) versus time. Reproduced
from Ref. [130] with permission from the Royal Society of Chemistry.
Review
743
technique, today commonly used in microfluidics, was introduced by Santiago et al.

in [136] and used epi-fluorescent illumination and correlation methods to calculate the
speed of the tracer particles. Afterward, other processing methods were developed,
such as correlation averaging, which is able to reduce the influence of Brownian motion, low seed particle concentrations, and low-quality images [137,138]. The extension of PIV into 3D space has been achieved by a number of techniques including
stereoscopic particle tracking [139], defocusing digital particle image velocimetry
(DDPIV) [140], and forward scattering particle image velocimetry (FSPIV) [141].
Figure 24
Vortex shedding cycle recorded and reconstructed by holographic PIV. The vortex
structures are represented by the vorticity isosurface at suitable threshold.
Reprinted with permission from [144]. Copyright 2005, Optical Society of America.
744
Review
However, the stereoscopic approach can only measure particles in a thin volume,
DDPIV is restricted in the particle density and image volume size that can be simultaneously mapped, and FSPIV is only applicable to a microscopic FOV and cannot
handle a large number of particles [142]. On the other hand, DH appears to be potentially the best contender as the next-generation technology for holographic diagnostics of flow field measurement [63,64,143,144]. The challenging problems in fluid
dynamics concern complex 3D nonstationary flows, and holographic imaging is one
of the keys to measuring all spatial components of the velocity vector, allowing 3D
Figure 25
3D velocity results by Wu et al. (a) 3D position and velocity, (b) velocity distribution,
and (c) velocity distribution along the radial direction in the pipe. Reprinted with
permission from [146]. Copyright 2015, Optical Society of America.
Review
745
flow characterization in the PIV paradigm. This specific field is referred to as holographic PIV (HPIV). Recent progress in holographic velocimetry has been truly powered by technological advancements, and a sophisticated HPIV apparatus has been set
up in different parts of the world to yield impressive results [63]. Specifically, HPIV
provides the metrology for displacement mapping in flow diagnostics, exceeding the
performance of ordinary PIV by two orders of magnitude in sensitivity [63]. In particular, digital HPIV (DHPIV) revolutionizes flow measurement science by providing
an effective 3D velocimetry tool in complex flows [64]. Typically, two approaches
have been adapted for measuring the displacement of particles from reconstructed
digital holograms of seeded flows: 1) tracking of individual particles and 2) correlation-based analysis [63,64,143,144]. However, correlation-based methods, applied to
raw images, usually do not work well in HPIV due to speckle noise in the hologram
reconstructions [64]. Instead, in the first case, all of the holographic 3D tracking methods reported in this review should be used, improving the accuracy in velocity extraction, limited only by the precision of particle detection. Several research
groups work on this topic worldwide; in particular, Pu and Meng propose an off-axis
DHPIV system called Gemini [50]. In [144] they achieve the goal of both temporally
and spatially resolved flow measurements, also demonstrating the temporal potential
of HPIV with a phase-locked measurement of a flow passing a wall-mounted tab.
They reveal a sequence of a vortex shedding process in a complete cycle. An example
of their results is reported in Fig. 24.
More recently, Wu et al. provide interesting applications of HPIV by implementing a
micro in-line DHPIV system, applied to investigate the 3D flow field in straight and
Y-junction micro-channels [145]. In particular, they propose a new processing algorithm based on the combination of integrated cross-correlation and nearest neighbor
matching algorithms, taking advantage of information from both the reconstructed
particle field and the hologram fringe patterns. In 2015 the same group reported
the investigation of the gassolid pipe flow by DHPIV, where particle size, 3D position, and velocity are measured simultaneously [146]. In Fig. 25 we show one of the
results reported in [146].
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Pasquale Memmolo received a bachelors degree in computer engineering in 2005 and a Masters degree in communication engineering in 2008 at the Universit degli Studi di Napoli Federico
II (Napoli, Italy), working on numerical and optical multiplexing
and de-multiplexing methods in digital holography. In 2011, he received a Ph.D. in electronic and telecommunication engineering,
with a thesis titled Compressed sensing: a new framework for
signals recovery and its application in digital holography. Dr.
Memmolo is currently Post Doc at the Center for Advanced Biomaterials for Health
Care@CRIB, Istituto Italiano di Tecnologia, Napoli, Italy, and he is a collaborator
at the CNR Institute of Applied Sciences and Intelligent Systems, Pozzuoli, Italy.
Dr. Memmolo is coauthor of about 40 publications in peer-reviewed journals and more
than 70 proceedings and conference papers. His research interest is in the fields of digital
holography, microfluidics, opto-fluidics, digital image processing, and computer vision. He is also a referee for several peer-reviewed journals (OSA, Elsevier, Wiley,
IEEE) and Editor of Mathematical Problems in the Engineering Journal (Hindawi).
Lisa Miccio received the Ph.D. degree from the University of
Florence in 2010; her main thesis activity was on the investigation
of the photorefractive and pyroelectric properties of ferroelectric
crystals to realize integrated optical micro-devices. She is currently
a research scientist under contract of the Italian National Research
Council (CNR). Her research interests are in the field of coherent
light imaging, particularly devoted to the integration of label-free
methods in microfluidic lab-chip devices for bio-photonics application. She is coauthor of about 45 contributions in peer-reviewed journals, five book
chapters, and more than 30 oral presentations at international conferences. Dr. Miccio
has collaborated and published papers with outstanding scientists, students, and scholars both from Italian and international research institutions. She has been a supervisor
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of many Ph.D. students. Moreover, she is a referee for peer-reviewed journals such as
the OSA, Elsevier, Wiley, and IEEE journals.
Melania Paturzo is a research scientist at the CNR Institute of
Applied Sciences and Intelligent Systems. Her research interest
is in the field of optics. Her research activities concern the investigation of material properties by means of interferometric techniques, the fabrication of periodically poled lithium niobate
samples by the electric field poling process, the development of
optical techniques to obtain super-resolution in digital holographic
microscopy, holographic display, quantitative phase-contrast
microscopy for cell investigation, and optofluidics. She is author and/or coauthor
of more than 40 papers in international peer-reviewed journals and of more than
50 conference papers.
Giuseppe Di Caprio received his M.S. in physics in 2007 and his
Ph.D. in Novel Technologies for Materials, Sensors, and Imaging
in 2011, both from the University of Naples Federico II. During
his Ph.D. program, he spent a period at the Universit Libre de
Bruxelles under the supervision of Prof. Frank Dubois. After three
years as a post-doctoral researcher at the Rowland Institute,
Harvard University, he is now a research associate at the
Harvard Medical School, Department of Cell Biology. His research interests include lattice light sheet microscopy, quantitative phase microscopy,
3D cell imaging, and microfluidics for the development of organ-on-chip.
Giuseppe Coppola received the Electronic Engineering degree,
summa cum laude, and Ph.D. degree from the University
Federico II, Napoli (Italy), in 1997 and 2001, respectively.
Currently, he is a senior researcher at the Institute for
Microelectronics and Microsystems of the Italian National
Research Council (CNR). Since 2004, he has been leader of
the optoelectronic Lab of that institute. From 2005 to 2012, he
was a contract professor at the University of Calabria. His main
research interests concern the design fabrication and characterization of silicon-based
optoelectronics devices, and contactless interferometric characterization techniques,
such as digital holography. On these topics he is a tutor for post-doc students,
Ph.D. students, and several graduating students. During his research activity, he
has collaborated on several research projects financed by the CNR and public and
external organizations. He has published more than 170 papers in peer-reviewed journals and conference proceedings.
Paolo A. Netti received his Ph.D. in Chemical Engineering in
1994 from the University of Naples Federico II. During his doctoral studies he has worked at the IRC in Biomedical Materials at
the University of London and later for his post-doctoral stage
joined Harvard University for more than four years. He is the coordinator of the Center for Advanced Biomaterials for Health Care
IIT@CRIB and a full professor of Material Science at the
University of Naples Federico II. His research interest mainly focuses on the elucidating the complex interplay between materials properties and biological entities such as cells and biomolecules. His activities span from the basic
understanding of the cells regulatory events occurring at the cellmaterial interface
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to the technological development of a novel class of biomaterials able to elicit and

direct specific cell fate by encoding multiple arrays of biological active moieties. He
has served on several European Scientific Commission panels for defining a viable
European roadmap for the development of novel biomaterials platforms (VII framework program), he is a panel member of the ERC advanced grant evaluation committee, and he has also served as a scientific tutor of several research platforms
from the Italian Minister of Research and University (Program FIRB by MIUR).
Pietro Ferraro received the doctor of physics degree, summa cum
laude, from the University of Napoli Federico II, Italy, in 1987.
Soon after, he joined Aeritalia-Alenia Aeronautics (the major
Aerospace company in Italy) as a researcher to develop applied
research in Optical Non-Destructive Testing of carbon fiber materials. In 1993 he joined the Consiglio Nazionale delle Ricerche
(CNR) Optics Group at the Institute of Cybernetics, Pozzuoli
(Napoli), Italy, as an Associate Researcher to develop interferometric and holographic methods for testing and characterization of optical components and materials. In 2001 he joined as a Researcher the CNRInstitute of
Microelectronics and Microsystems, Napoli. In 2003 he joined the National
Institute for Applied Optics (INOA) as a Senior Research Scientist. Since 2005 he has
been Head of the Research Line and Group on behalf of CNR in Optical diagnostics,
Interferometric and Microscopy. Currently, he is Director of the Institute of Applied
Sciences and Intelligent Systems (ISASI-CNR) in Napoli. He has worked in a number
of areas in optics research, including laser interferometry; holography for the optical
characterization of materials, components, and devices, including MEMS, MOEMS,
waveguides, etc.; optical fiber sensors; holographic lithography; and micro- and nanostructuring of ferroelectric optical crystals (LiNbO3) for application in photonics. He is
now pursuing interests in micro- and nano-structuring advanced ferroelectric photonic
crystals and materials, poling, photorefractivity, quantitative phase-contrast microscopy by digital holography for in vivo cell investigation, and 3D imaging and display
with either visible or IR laser light. In addition he is leading his group in optical microdevices and instrumentation development based on LiNbO3 such as phase-array,
point-diffraction-interferometer, and microfluidic structures. He has published more
than 150 conference proceedings, including more than 20 invited international
conference papers and presentations. He has completed three book chapters and is
currently editor of a book titled Micro-/Nanoengineering and Characterization of
Ferroelectric Crystals for Photonic Applications, Springer (Germany). He has been
Editor of 10 Special Issues published in Optics and Lasers in Engineering (Elsevier)
and in Journal of Optics A (Institute of Physics). He has 12 patents, some of which
have been licensed by industry. He is a Topical Editor of Optics Letters, a member of
the Editorial Board of the Optics and Lasers in Engineering Journal (Elsevier), a
member of the Int. Editorial Board of the Measurement & Science Technology
Journal (Institute of Physics), and Editor of Research Letters in Optics (Hindawi).
He is active as a reviewer for major journals in optics/photonics. He has chaired three
international conferences, two of which for the European Optical Society (EOS).
Moreover, he has served on the program committees and chair sessions of more than
20 international conferences on optics/photonics, imaging, and information systems
sponsored by the SPIE. He has been elected to the grade of Fellow of SPIE (2008),
elected to the grade of Senior Member of IEEE (2007), and since 2002 has been on the
board of the Italian Chapter of IEEE-LEOS. Since 2007 he has been an elected
member of the presidential board of the Italian Society of Optics and Photonics
(SIOF-EOS). He is member of OSA.

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Vol. 7, No. 4 / December 2015 / Advances in Optics and Photonics

Recent advances in holographic