Chapter 13

Applications of Holographic Microscopy in Life Sciences

Iliyan Peruhov and Emilia Mihaylova
Additional information is available at the end of the chapter
http://dx.doi.org/10.5772/56176

1. Introduction
Imaging of microscopic objects is an essential art, especially in life sciences. Rapid progress in
electronic detection and control, digital imaging, image processing, and numerical computa
tion has been crucial in advancing modern microscopy. At present the 3D imaging of biological
samples is done by confocal microscopes. Their ability to image biological events in real time
is limited by the time necessary to capture stacks of images taken through a certain plane in
cells or tissues from which a 3D view is calculated. Digital holographic microscopy is a new
imaging technology applied to optical microscopy. The digital holographic microscopy is a
very advanced imaging technique because it yields a 3D volume image from a single image
capture.
Holography is a technique by which a wavefront can be recorded and subsequently recon
structed in the absence of the original wavefront i.e. a 3D image is observed just as if the object
was still present and being illuminated in the same way as when the holographic recording
was made [1]. In conventional holography, invented by Gabor [2], the holograms are photo
graphically recorded and optically reconstructed. Both the amplitude and phase information
of the light wave are recorded in a hologram. Because the holographic image retains the phase
as well as the amplitude information, a variety of interference experiments can be performed,
and this is the basis of many interferometric applications in metrology.
Digital holography does not require wet chemical processing of a photographic plate, although
at some expense of resolution. However, once the amplitude and phase of the light wave are
recorded numerically, one can easily subject these data to a variety of manipulations, and so
digital holography offers capabilities not available in conventional holography. The remark
able aspect of the digital reconstruction its possibility to refocus at different depths inside a
transparent object, depending on the reconstruction distance, makes this technique very
suitable for biological cells studies and could have many applications in life sciences.

2013 Peruhov and Mihaylova; licensee InTech. This is an open access article distributed under the terms of
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unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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In digital holography the reconstruction process is implemented by multiplication of the CCD

captured and PC stored digital hologram by the numerical description of the reference wave,
and convolution of the result with the impulse response function of the optical system. The
diffracted field in the image plane is given by the Rayleigh-Sommerfield diffraction formula [3]
y ( x ', y ') =

with f (x ', y ', , ) =

1
h(x ,h )r(x , m ) f ( x ', y ', x ,h )cos Qdxdy
il

(1)

exp(ik)
and = d'2 + ( x ')2 + ( y ')2

where d is the reconstruction distance, i.e. the distance measured from the hologram plane to the image plane; h(,) is the recorded hologram; r(-) represents the reference wave field;
k denotes the wave number and is the wavelength of the laser source. Due to the small angles
between the hologram normal and the rays from the hologram to the image points, the
obliquity factor cos can normally be set = 1.
Equation (1) is the basis for numerical hologram reconstruction. Because the reconstructed
wavefield (x, y) is a complex function, both the intensity as well as the phase can be
calculated [1].
Digital holographic microscopy (DHM) can provide quantitative marker-free imaging that is
suitable for high resolving investigations of transparent and reflective surfaces as well as for
fast analysis of living cells under usual laboratory conditions. One of many interesting
applications of DHM is to study cells without staining or labeling them and without affecting
them in any way.
This chapter is divided in two parts. The first part reviews the recent advances in the appli
cation of digital holographic microscopy to biological specimen. The second part of the chapter
describes the development of a digital holographic microscope at the Agricultural University
of Plovdiv and reports some of its life science applications.

2. Recent advances in the application of digital holographic microscopy to

biological specimen
Kim M. K. [4] has proposed a novel digital holographic method that allows axial resolution of
objects by superposition of a number of numerically reconstructed optical diffraction fields of
digital holograms that are optically recorded with a number of wavelengths. The principle of
wavelength-scanning digital interference holography is applied to imaging of 3D objects with
diffuse surfaces, such as a biological specimen. The head of a small insect of a few millimeters
in size is imaged with 120 m axial resolution and ~20 m lateral resolution. An animated 3D
numerical model of the object surface structure is generated from the tomographic data with
good fidelity. The experiments are performed using a standard holographic apparatus.
Approximately 50 mW of ring dye laser output is expanded to about 10 mm diameter and

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spatially filtered. The object beam is apertured to about 5 mm diameter and illuminates the
sample object. The scattered light from the object is combined with the reference beam. The
magnifying lens images the optical field at infinity. The digital camera is focused at infinity,
so that it records a magnified image of the optical intensity. It is important to aperture the
object beam so that it only illuminates the area of the object that is to be imaged, otherwise
spurious scattering can seriously affect the quality of the picture.
A digital holographic microscope (DHM) in a transmission mode has been developed in
Rappaz, B. et al. [5], adapted to the quantitative study of cellular dynamics. Living cells in
culture are optically explored by calculating the phase shift they produce on the transmitted
wave front. The high temporal stability of the phase signal and the low acquisition time makes
it possible to monitor cellular dynamics processes. An experimental procedure allowing
calculating both the integral refractive index and the cellular thickness (morphometry) from
the measured phase shift is presented. Specifically, the method has been applied to study the
dynamics of neurons in culture during a hypotonic stress. Such stress produces a decrease of
the phase which can be entirely resolved by applying the methodological approach described
in the article; in fact the method allows determining independently the thickness and the
integral refractive index of cells. The phase signal depends on both the thickness and the
refractive index of the specimen. To decouple these two contributions, a procedure named
"decoupling procedure" is applied. The dynamic quantitative phase images, containing
information about both the cell morphometry and the integral refractive index, can be
unambiguously interpreted thanks to the decoupling procedure presented. Quantitatively, the
local cellular thickness measurement can be performed with accuracy of 1 m. Spatial
averaging allows measuring mean thickness of cellular regions corresponding to the area of
typical neuronal bodies, i.e. 170 m2, with an accuracy of a few tens of nanometers. On the
other hand, the spatial variations of the integral refractive index have been estimated at 0.005
and the mean integral refractive index can be measured with an accuracy of 0.0003. The cellular
refractive index is a poorly documented parameter which is related to the intracellular content
and which is relevant for the interpretation of the functional light imaging signal resulting
from a multiple scattering process in biological tissues.
Marquet, P. et al. [6] present for the first time DHM images of living cells in culture. They
represent the distribution of the optical path length over the cell, which has been measured
with subwavelength accuracy. These DHM images are compared with those obtained by use
of the widely exploited phase contrast and Nomarski differential interference contrast
techniques. The developed digital holographic microscope presents a simplified and easy-tooperate technique compared with classical interferometry. The authors show that digital
recording and numerical reconstruction of holograms, offers new perspectives in imaging,
because numerical processing of a complex wave front allows one to compute simultaneously
the intensity and the phase distribution of the propagated wave. Digital holography has made
it possible to focus numerically on different object planes without using any opto-mechanical
movement. Moreover, different lens aberrations can be corrected by a numerical procedure.
In the article for the first time absolute phase distribution images of living neurons in a culture
are obtained by use of DHM with accuracy in the 24 range. To compare the DHM with the

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standard and widely used techniques of phase contrast (PhC) and differential interference
contrast (DIC) microscopy in biology, images of living neurons obtained with PhC, DIC, and
DHM are presented.
In an article of Garcia-Sucerquia J. et al. [7] some significant characteristics of (digital in-line
holographic microscopy) DIHM and the underwater DIHM have been emphasized:
1.

Simplicity of the microscope: DIHM is microscopy without objective lenses. The hardware
necessary for the desktop version is a laser, a pinhole, and a CCD camera. For the
underwater DIHM version, they use the same elements contained in a submersible
hermetic shell.

2.

Maximum information: a single hologram contains all the information about the 3D
structure of the object. A set of multiple holograms can be properly added to provide
information about 4D trajectories of samples.

3.

Maximum resolution: optimal resolution, of the order of the wavelength of the laser, can
be obtained easily with both versions.

4.

Simplicity of sample preparation: this is mainly true for biological samples, because no
sectioning or staining is required, so that living cells and specimens can be observed in
depth. Indeed, for the underwater DIHM, there is no sample preparation at all, and realtime information of living organisms can be retrieved.

It is shown [7] that high-resolution tracking of many particles in 4D can be obtained from just
one difference hologram. Since resolution of the order of a wavelength of the light has been
achieved with DIHM, tracking of organisms as small as bacteria is possible, as would be the
motion of plankton in water or, at lower resolution, the trajectories of flying insects. DIHM
can also be used on macroscopic biological specimens, prepared by standard histological
procedures, as for a histological section of the head of the fruit fly, Drosophila melanogaster. Such
images reveal the structure of the pigmented compound eye and different neuropile regions
of the brain within the head cuticle, including the optic neuropiles underlying the compound
eye. DIHM, with its inherent capability of obtaining magnified images of objects, unlike
conventional off-axis holography, is therefore a powerful new tool for a large variety of
research fields.
The optical arrangement described by Parshall D. et al. [8] provides a straightforward means
for high-resolution holographic microscopic imaging. There is no need for elaborate processing
such as magnification by using a reconstruction wavelength that is compared with the
recording wavelength, which inevitably introduces aberration, or using an aperture array in
front of the camera and scanning it to artificially increase the CCD resolution. Also the phase
image appears less noisy than the amplitude image. The amplitude image reflects the intensity
variations in the reference wave, whereas the phase noise comes mostly from the quality of
the optical surfaces in the imaging system. The former is much more difficult to control. The
phase images have less noise than the amplitude image, and one can readily discern the index
variation over the nucleus as it is done for an onion cell.

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In this article [8] a number of experimental results has been presented that reveal the effec
tiveness of digital holography in high-resolution biological microscopy. In particular, phaseimaging digital holography offers a highly sensitive and versatile means to measure and
monitor optical path variations. The authors have offered biological microscopy by twowavelength phase-imaging digital holography and proposed its extension to three-wave
length phase imaging for longer axial ranges with undiminished resolution.
Jeong, K. et al. [9] show that coherence-gated digital holography detects motility as deep as 10
optical thickness lengths inside tissue. This opens prospects to use motility as a contrast agent
when imaging at depths inaccessible to conventional motility assay approaches. Coherencegated digital holography is an interferometric imaging approach that measures motility with
displacement sensitivity below a fraction of a wavelength, over a macroscopic lateral field of
view up to 1 mm. Motility at depth appears in real-time holograms as dynamic speckle.
Furthermore, the authors define a motility metric based on the coefficient of intensity variance
per pixel that becomes a novel imaging contrast agent. The authors demonstrate that the
motility metric enables direct visualization of the effect of cytoskeletal anti-cancer drugs on
tissue inside its natural three-dimensional environment, allowing measurements of tissue and
cellular response to drugs. The reconstructed en face image of the rat tumor spheroid is striking.
The rat osteogenic sarcoma tumor spheroids are grown in vitro in a rotating bioreactor. The
spheroids may be grown up to several mm in diameter, and thus are large enough to simulate
the thickness of different mammalian tissue. As tumor spheroids are cultured, they undergo
cell apoptosis and necrosis in their center and so consist of an inner necrotic core with low
activity and an outer shell with a thickness of 100 to 200 m of viable proliferating cells with
high motility. The speckle images of the tumor spheroids shimmer due to motility in tissue,
and statistical properties of the dynamic speckle are obtained by capturing successive images
at a fixed depth.
A method of quantitative phase microscopy with asynchronous digital holography has been
suggested by Chalut K. J. et al. [10]. An essential requirement that must be met to apply a phase
microscopy system to imaging the dynamics of live cells is that the system can acquire
quantitative phase images of the sample at a high rate (>100 Hz). Although modern CCDs are
capable of >100 Hz image acquisition rates, multiple interferograms are often necessary to
extract the phase information, which reduces the acquisition rate considerably. Additionally,
if multiple inteferograms are used, they must be recorded fast enough so that instabilities in
the system and the dynamics of the cells themselves do not vary appreciably during acquisi
tion. The authors [10] demonstrated that the system is capable of obtaining quantitative phase
measurements on millisecond time scales. The inclusion of acousto-optic modulators in each
arm of the interferometer permits to use phase-shifting interferometry. The system is innova
tive in the field of digital holography, because the phase shift is easily evaluated, which greatly
simplifies the experimental setup. In addition, the algorithm requires only two phase-shifted
interferograms, compared to the usual 4 interferograms required in most phase shifting
algorithms. A potential increase in speed can be realized by utilizing frame transfer CCD
devices, which can record two images on a microsecond time scale. By transferring frames
without reading them out, the latency between two interferograms is greatly reduced and the

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quantitative phase imaging frame rate is then nearly the frame rate of the camera, of the order
of 100 Hz. It is demonstrated, with a red blood cell sample, and a smooth muscle cell sample,
that this system is capable of obtaining quantitative phase images of live cells.
Kemper, B. & von Bally, G. [11] carried out an analysis of living pancreas tumor cells (Pa
tu8988T) to reveal the prospective of digital holographic microscopy for the visualization of
drug induced morphology changes. The authors exposed the tumor cells to an anticancer drug
(Taxol). Digital holograms of selected cells were recorded continuously every 120 s over 16 h
in a temperature stabilized environment with an inverse digital holographic microscopy setup.
DHM has clearly shown that Taxol first induces morphological changes as cell rounding that
effects an increase in cell thickness. Afterward, for all the specimens, the final cell collapse is
detected precisely by a significant decrease of the phase contrast. The authors also demonstrate
that the subsequent numerical focus adjustment reduces unwrapping artifacts that are caused
by diffraction patterns in the defocused phase contrast images. For investigation of suspension
cells this feature is of particular advantage because cells in different focal planes can be
investigated by the evaluation of a single captured hologram. The results show that digital
holographic phase contrast microscopy can be applied for quantitative long-term observation
of living cells. The studies show new ways for marker-free dynamic monitoring of cell
morphology changes to access new parameters, e.g., for quantitative observation of the timedependent reactions of cells to drugs. In addition, for investigation of cells, the scattering
properties of the cell culture medium and the optical quality of the cell handling equipment
(e.g., glass carrier, coverslip, or Petri dishes) must be considered. In summary, the presented
results demonstrate that digital holographic microscopy can be applied for noncontact,
marker-free, and quantitative phase contrast imaging. The method allows a high resolution
multifocus reconstruction of amplitude and phase data from a single recorded digital holo
gram. It enables hologram capture time in the millisecond range. The hologram acquisition
rate is limited by the digital recording device.
A phase-imaging technique to quantitatively study the three-dimensional structure of cells is
presented by Khmaladze A. et al. [12]. The method, based on the simultaneous dual-wave
length digital holography, allows for higher axial range at which the phase imaging can be
performed. The technique is capable of nanometer axial resolution. The method compares
favorably to software unwrapping, as the technique does not produce non-existent phase
steps. Curvature mismatches between the reference and an object beam is numerically
compensated. The 3D images of SKOV-3 ovarian cancer cells are presented. The measurements
of the optical thickness of cells can then be performed. One also needs to make an assumption
of the cells refractive index, which is taken to be 1.375. While it may not be possible to precisely
determine the refractive index of the cell at each individual point, this number is always close
to the refractive index of water and unlikely to deviate by more than a few percent. As a result,
the accuracy and the level of details of the dual-wavelength images of cells, presented here,
are superior to what has been previously demonstrated. In comparison to the software
unwrapping, dual-wavelength optical unwrapping method is advantageous, as it requires no
intensive computation procedures and can handle complex phase topologies. The proposed
method of curvature correction is simple and effective to easily implement the experiment

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without the microscope objectives in the reference arms of the Michelson interferometer. This
greatly simplifies the optical setup and makes it much easier to achieve the initial adjustments
of the apparatus. Simultaneous dual wavelength setup utilized together with the angular
spectrum algorithm provides an easy way to acquire single frame images in real time, which
can be used to study cell migration.
Langehanenberg P. et al. [13] propose autofocusing in digital holographic phase contrast
microscopy on pure phase objects for live cell imaging. Common passive optical autofocus
techniques are based on axial scanning of the image space by mechanical adjustment of a lens
element or a stage to find the maximum image definition. In digital holography, this scanning
process is performed numerically by variation of the propagation distance in the convolutionbased propagation. The main task in passive autofocusing is the determination and maximi
zation of the image sharpness. Pure phase objects with negligible absorption such as technical
reflective specimens or biological cells are sharply focused at the setting with the least contours
in the amplitude distributions. In contrast to the bright-field case, in digital holography this
setting is of particular interest, as the amplitude and phase distributions are accessible
simultaneously, and the focal setting with the least-contrasted amplitude image corresponds
to the best-resolved structures in the quantitative phase contrast distribution. Four numerical
methods are compared in order to identify best autofocus method for application in digital
holographic microscopy.
Remmersmann C. et al. [14] present research for the optimization of a temporal phase-shifting
(TPS) - based digital holographic microscopy setup. In order to enable a phase-shift-dependent
investigation a variable three-step algorithm is applied. First, the phase error of the recon
structed object wave is evaluated theoretically. In a second step the results obtained from the
calculations are compared to the measured phase noise. Finally, the applicability for noise
reduction is demonstrated by quantitative phase contrast imaging of a pancreas tumor cell
sample. Theoretical and experimental investigations on phase errors in temporal phaseshifting-based digital holographic reconstruction have been performed in order to minimize
the noise within the reconstructed object wave. Coherent as well as partially coherent light
sources were applied and compared. The application example of LED and laser-based digital
holographic microscopy on fixed pancreas tumor cells demonstrates that disturbances in the
reconstructed amplitude and phase distributions due to multiple reflections within the
experimental setup can be effectively reduced by partially coherent light sources.
Choi Y. S. and Lee S. J. [15] apply digital holographic microscopy (DHM) for three-dimensional
volumetric measurement of red blood cells in motion. Currently, various particle image
velocimetry (PIV) measurement techniques have been applied to numerous hemorheological
studies. Standard PIV methods provide two-dimensional (2D) planar information confined in
a thin depth of field. Holography is capable of recording 3D volumetric field information in a
single hologram. The recent development of digital holography enables the volumetric
measurement of particle fields without the use of any chemical or physical processes. In this
technique, a digital hologram of the particles distributed in a flow is directly recorded digitally.
The 3D flow information can be subsequently obtained through the numerical reconstruction
and the particle tracking procedure. The authors applied DHM to measure the 3D motion of

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human red blood cells (RBCs) in a microtube flow. DHM requires only a pair of particle
hologram images to get complete 3D flow information and this is of great advantage in motion
analysis of individual blood cells. The viability and uncertainty of the established DHM system
in the detection of 3D RBC position were evaluated by a planar test target. The position in
depth of a RBC was located by applying focus functions that quantify the sharpness of its
reconstructed image. Five focus functions were evaluated to find the suitable function that
provides minimum uncertainty. Finally, the sample trajectories as well as the 3D velocity
profiles of RBCs inside the microtube flow are presented and the measurement uncertainties
are discussed.
Warnasooriya N. et al. [16] captured pictures of gold nanoparticles in living cell environments
using heterodyne digital holographic microscopy. With recent developments in the fields of
nanotechnology and modern optical microscopy, the use of nanometric particles as biomarkers
in biological specimens has been rapidly increased. The paper describes an imaging micro
scopic technique based on heterodyne digital holography where subwavelength-sized gold
colloids can be imaged in cell environments. Surface cellular receptors of 3T3 mouse fibroblasts
were labeled with 40 nm gold nanoparticles, and the biological specimen is imaged in a total
internal reflection configuration with holographic microscopy. Due to a higher scattering
efficiency of the gold nanoparticles versus that of cellular structures, accurate localization of
a gold marker is obtained within a 3D mapping of the entire samples scattered field, with a
lateral precision of 5 nm and 100 nm in the x,y and in the z directions respectively, demon
strating the ability of holographic microscopy to locate nanoparticles in living cell environ
ments. However, in order to apply these techniques to biological specimens, important issues
must be considered. In biological samples, the particle holographic signal is superimposed
with the light scattered by cell refractive index fluctuations, which yields a speckle field. This
paper studies the possibility of 3D holographic imaging in a biological context. Since the cellscattered speckle field cannot be avoided, it is important for future cell labeling applications,
to scale the particle signal with respect to the scattered speckle. The authors show that the
amplitude of the 40 nm gold particle signal is much larger than the cell-scattered field. NIH
3T3 mouse fibroblasts are used (quoted as 3T3 cells) with integrin surface receptors labeled
with 40 nm gold particles. Streptavidin-coated gold nanoparticles were attached to the cell
surface integrin receptors via biotin and fibronectin proteins. Fibronectin proteins were labeled
with biotin. The illumination source is a single-mode near infrared laser diode. A polarizing
beam splitter cube (PBS) is used to split the original illumination laser light into two beams, a
reference beam and an object illumination beam forming the two arms of a Mach- Zehnder
interferometer. A CCD camera detects the interference pattern (hologram) and sends it to a
computer. The hologram is then reconstructed numerically. Using a parabolic approximation
for the local field, the location of the gold particle can be calculated by fitting the data points
that are above half maximum. The accuracy of the measurement made by this method is 5
nm in the x and y directions. The authors show that the acquisition of a single image is sufficient
to localize in 3D the nanoparticle within a 90 micrometer thick sample, with localization
accuracy similar to that obtained in conventional light microscopy. This method provides
significant progress towards the development of 3D microscopy in living cell environments,

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CCD sensor

Object

Pinhole

Polariser

Laser

Figure 1. Optical setup of the digital in-line holographic microscope

since the 3D reconstruction of such a thick sample by conventional light microscopy would
require the acquisition of a stack of hundreds of slices.
The traction force produced by biological cells has been visualized by Yu X. et al. [17].
Quantitative phase microscopy by digital holography (DH-QPM) has been utilized to study
the wrinkling of a silicone rubber film by motile fibroblasts. Surface deformation and the
cellular traction force have been measured from phase profiles in a direct and straightforward
manner. DH-QPM is shown to provide highly efficient and versatile means for quantitatively
analyzing cellular motility. The traction force has been measured as ~4 103 dyn/cell based
on the degree of wrinkling determined from phase information. Fourier transformation and
the angular spectrum methods were applied to the complex hologram obtained to calculate
the phase-contrast, dark-field, Zernike and differential interference contrast (DIC) images. The
basic principles of DH have been applied to quantitative imaging of wrinkles on silicone rubber
due to cell adhesion and motility. The approach is sensitive to cellular forces and it can detect
and quantify variations in force within the adhesion area of a cell over time. DH-QPM is shown
to be an effective approach for measuring the traction forces of cells. A time-lapse phase movie
of the migration of cells was recorded every 3 min over a period of 2 hours. The traction force
for NHDFs is a factor of five smaller than for chick heart fibroblast cited in literature. This is a
substantial achievement in the quantitative profiling of substrate deformation and wrinkling
under cellular traction force achieved by the quantitative phase microscopy of digital holog
raphy.

3. Digital in-line holographic microscopy for life science applications at the

Agricultural University Plovdiv
A digital in-line holographic microscope (DIHM) was developed at the Agricultural University
of Plovdiv. The light source is 20 mW He-Ne laser. The emerging spherical wave illuminates
the object, and the hologram is recorded on a CCD sensor and stored in a computer.

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DIHM was applied to visualise live algae cells of two different species (Pseudokirchneriella
subcapitata and Chlorella vulgaris) without any preliminary preparation. Digital reconstruction
of the recorded interference patterns is performed using the HoloVision 2.2 software [18].
Figure 2 and Figure 3 show the holograms and the reconstructed intensities to represent the
object. Four wavefront intensities of each digital hologram (2a and 2b) are reconstructed at
different consecutive planes with the distance between them changing by 2 m. The recon
structed intensities illustrate the possibility of observation of different layers in a live cell
obtained from one digital hologram only. In that way many cuts of one live object can be done
and observed from one hologram of the whole object.
These experiments illustrate the capability of DHM for non-invasively visualizing and
quantifying biological cells and tissues. Thats why DHM can be successfully used for:
cell counting
measuring cell viability directly in the cell culture
label-free viability analysis of adherent cell cultures etc.

Figure 2. Images of algae Pseudokirchneriella subcapitata: a) digital hologram; b-e) the wavefront intensity at four
consecutive planes with the distance between them changing by 2 m; f image from electron microscope

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Clearly, DIHM is capable of visualising live cells with dimensions 5 10 m without any
preliminary preparation. It can be applied to dynamic quantitative visualisation of live cell
deformations to study their interactions with other particles as well as the surrounding
environment. This makes the DIHM a valuable technique for many life science applications.
Further development of the technique is envisaged in order to overcome the limited pixel
resolution of a CCD sensor, which is the major drawback of DIHM at present.

Figure 3. Images of algae Chlorella vulgaris: a) digital hologram; b-e) the wavefront intensity at four consecutive
planes with the distance between them changing by 2 m; f image from electron microscope

Figure 4 and Figure 5 present digital holograms of algae cells Pseudokirchneriella subcapitata and
Chlorella vulgaris taken at different stages of their life cycle and the reconstructed intensities of
these holograms. The cells morphology is visible on the images showing the reconstructed
intensities.
These experiments illustrate the capability of DHM for:
label free morphology analysis of cells
label free studies of cell division and migration
label-free analysis of subcellular motion in living tissues etc.

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By combining several images reconstructed from the same digital hologram, but at different
focal planes, an increased depth of field can be obtained, which is vastly superior to the depth
of field achieved with traditional light microscopy.

(a)

(b)

(c)

(e)

(d)

(f)

Figure 4. Images of algae Pseudokirchneriella subcapitata, approximately 10 m in a sickle: a) digital hologram of two
days old cells; b) the wavefront intensity of a) c) digital hologram of 4 days old cells; d) the wavefront intensity of c) e)
digital hologram of 9 days old cells; f) the wavefront intensity of e).

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(a)

(b)

(c)

(e)

(d)

(f)

Figure 5. Images of algae Chlorella vulgaris, approximately 3 m in diameter: a) digital hologram of two days old cells;
b) the wavefront intensity of a) c) digital hologram of 4 days old cells; d) the wavefront intensity of c) e) digital holo
gram of 9 days old cells; f) the wavefront intensity of e).

4. Conclusion
DIHM imaging is very advanced method because digital holography yields a 3D volume image
from a single interferogram capture. This makes the development of a dynamic microscope
capable of fast 3D imaging an achievable objective.

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The attractive features of DHM are: a very high acquisition rate (limited only by the video
acquisition frequency), monitoring of physiological and pathological activity of cell and tissue
culture, non contact, non destructive, marker free in vivo imaging.
DIHM is capable of label free morphology analysis of cells and label free studies of cell division
and migration. It is a very attractive technique for application in biological research and in the
agricultural science. Other life science and medical applications are also envisaged.
Further development of this technique will involve the use of lasers with shorter wavelength
and CCD cameras with higher resolution. These developments will allow the application of
DIHM for study of cell features having dimensions below 100 nm.
The work on the development of the Digital In-line Holographic Microscope was financially
supported by the Agricultural University of Plovdiv.

Author details
Iliyan Peruhov and Emilia Mihaylova
Department of Mathematics, Informatics and Physics, Agricultural University Plovdiv,
Bulgaria

References
[1] Jones, R, & Wykes, C. Holographic and Speckle Interferometry, Cambridge University
Press, Cambridge, UK (1989).
[2] Gabor, D. Nature 161, 777-778 (1948).
[3] Palacios, J, Ricardo, D, Palacios, E, Goncalves, J, & Valin, R. De Souza, 3D image re
construction of transparent microscopic objects using digital holography, Optics
Communications 248, 41-50 (2005).
[4] Kim, M. K. Tomographic three-dimensional imaging of a biological specimen using
wavelength-scanning digital interference holography, Optics Express 7 (9), 305-310
(2000).
[5] Rappaz, B, Marquet, P, Cuche, E, Emery, Y, Depeursinge, C, & Magistretti, P. J.
Measurement of the integral refractive index and dynamic cell morphometry of liv
ing cells with digital holographic microscopy, Optics Express 13, 9361-9373 (2005).
[6] Marquet, P, Rappaz, B, & Magistretti, P. J. Digital holographic microscopy: a nonin
vasive contrast imaging technique allowing quantitative visualization of living cells
with subwavelength axial accuracy, Optics Letters 30, 468-470 (2005).

Applications of Holographic Microscopy in Life Sciences

http://dx.doi.org/10.5772/56176

[7] Garcia-sucerquia, J, Xu, W, Jericho, S. K, Klages, P, Jericho, M. H, & Kreuzer, H. J.

Digital in-line holographic microscopy, Applied Optics 45 (5), 836-850 (2006).
[8] Parshall, D, & Kim, M. K. Digital holographic microscopy with dual-wavelength
phase unwrapping, Applied Optics 45 (3), 451-459 (2006).
[9] Jeong, K, Turek, J. J, & Nolte, D. D. Volumetric motility-contrast imaging of tissue re
sponse to cytoskeletal anti-cancer drugs, Optics Express, 15 (21), 14057-14065 (2007).
[10] Chalut, K. J, Brown, W. J, & Wax, A. Quantitative phase microscopy with asynchro
nous digital holography, Optics Express 15 (6), 3047-3052 (2007).
[11] Kemper, B, & Von Bally, G. Digital holographic microscopy for live cell applications
and technical inspection, Applied Optics 47 (4), AA61(2008). , 52.
[12] Khmaladze, A, Kim, M, & Lo, C. M. Phase imaging of cells by simultaneous dualwavelength reflection digital holography, Optics Express 16 (15), 10900-10911 (2008).
[13] Langehanenberg, P, Kemper, B, Dirksen, D, & Von Bally, G. Autofocusing in digital
holographic phase contrast microscopy on pure phase objects for live cell imaging,
Applied Optics 47(19), DD182 (2008). , 176.
[14] Remmersmann, C, Stwald, S, Kemper, B, Langehanenberg, G, & Von Bally, G.
Phase noise optimization in temporal phase-shifting digital holography with partial
coherence light sources and its application in quantitative cell imaging, Applied Op
tics 48 (8), 1463-1472 (2009).
[15] Choi, Y. S, & Lee, S. J. Three-dimensional volumetric measurement of red blood cell
motion using digital holographic microscopy, Applied Optics 48 (16), 2983-2990
(2009).
[16] Warnasooriy, N, Joud, F, Bun, P, Tessier, G, Coppey-moisan, M, Desbiolles, P, Atlan,
M, Abboud, M, & Gross, M. Imaging gold nanoparticles in living cell environments
using heterodyne digital holographic microscopy, Optics Express 18 (4), 3264-3273,
(2010).
[17] Yu, X, Cross, M, Liu, C, Clark, D. C, Haynie, D. T, & Kim, M. K. Measurement of the
traction force of biological cells by digital holography, Biomedical Optics Express 3
(1), 153-159 (2012).
[18] Oysen Skotheim and Vegard LTuft, HoloVision 2.2.1 software package for numeri
cal reconstruction and analysis of digitally sampled holograms, Norwegian Universi
ty of Science and Technology, Norway (2001).

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