Download DIGITAL OFF-AXIS HOLOGRAPHIC MICROSCOPY: FROM CELLS

DIGITAL OFF-AXIS HOLOGRAPHIC MICROSCOPY: FROM CELLS
VIZUALIZATION, TO PHASE SHIFT VALUES,
ENDING WITH PHYSIOLOGICAL PARAMETERS EVOLUTION
MONA MIHAILESCU1, IRINA A. PAUN1, EUGENIA VASILE1, ROXANA C. POPESCU2,
ALEXANDRA V. BALUTA3, DIANA G. ROTARU3
1
Physics Department, Politehnica University from Bucharest, Romania
E-mails: [email protected], [email protected]
2
Department of Life and Environmental Physics, Horia Hulubei National Institute of Physics and
Nuclear Engineering, Magurele, Romania
3
Faculty of Medical Engineering, Politehnica University from Bucharest, Romania
Received November 24, 2015
Digital off-axis holographic microscopy (DoHM) is a modern technique, which
provides quantitative information about the samples in three dimensions. DoHM
allows the analysis of living cells in their growth medium, without any kind of
additional markers, leading to the values of many physiological parameters after
processing the reconstructed images. This paper is a review about the research and
development applications implying DoHM for the analysis of different biological
samples: blood cells, yeast cells, neurons, cancer cells, and osteoblasts cells. The
focus is on the values of the final physiological parameters, which can be determined
with high accuracy in marker-free conditions, at the level of the single cell, such as
refractive indices, hemoglobin content, dry mass, amplitude of the membrane
fluctuations, cells elasticity, cells dimensions, rate of sedimentation, and
transmembranar fluxes. Few aspects about the decoupling and focusing procedures
are also summarized. This review addresses to students and researchers interested in
real-time analysis of living cells in their natural environment.
Key words: digital holographic microscopy, quantitative phase imaging, biological applications, neurons, blood cells, cancer cells, refractive
index, dry mass, transmembranar fluxes, hemoglobin content.
1. INTRODUCTION
Quantitative phase imaging techniques (QPITs) are included in the field of
modern optical microscopy techniques, which provide images of the investigated
sample and, at the same time, values for: 1) dimensions in planes perpendicular to
the optical axis and 2) phase shift (PS) introduced by the sample in the optical path.
Next, from the PS values are computed: the third dimension (along the propagation
axis) and the refractive index of the sample.
Rom. Journ. Phys., Vol. 61, Nos. 5–6, P. 1009–1027, Bucharest, 2016
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Thus, QPITs are 4D imaging techniques offering answers to important
biological questions, impossible to tackle with conventional optical imaging
techniques, investigation of cells and tissues, in terms of morphology and structure
dynamics, with nanoscale sensitivity along propagation axis, over temporal scales
from milliseconds to days. The large family of QPITs includes: digital holography
with a spatially partial coherent source [1], digital in-line holographic microscopy
[2, 3], digital off-axis holographic microscopy (DoHM) [4], Hilbert phase
microscopy [5], interferometric phase-dispersion microscopy [6], Fourier phase
microscopy [7], quantitative phase microscopy [8], diffraction phase microscopy
[9], asynchronous digital holography [10], phase shifting interferometry [11],
spatial light interference microscopy [12], white light diffraction tomography [13].
In this review, we survey the applications of DoHM to investigate living
biological specimens, which end with the values of physiological relevant
parameters. DoHM is an optoelectronic technique, which allows real time
measurements, full-field (phase and amplitude) at the level of single living cell, to
obtain 3D morphological dimensions (length, width, height) and refractive index maps
(from the PS values). It is a marker free, non-invasive, non-destructive technique, with
nanometric resolution along the propagation axis. DoHM also allows full-field phase
measurements, which provide simultaneous information from a large number of
points on the sample, with the benefit to study both temporal and spatial behavior
of the investigated biological systems. In DoHM, a single hologram, recorded in
the experimental setup (no mechanical scanning needed), is used to numerically
focus on the reconstructed object image at any distance, with nanometric resolution
along the propagation axis [5, 14–16].
The success of digital holographic techniques began with the change of the
recording media: from classical holographic plates to modern sensor of CCD or
CMOS cameras [17]. Although the resolution of the digital sensors is still far from
the resolution of holographic plates, they have the advantage of digitalizing
images, which are transferred to a computer as an array of numbers, allowing
further numerical operations to find the reconstructed images. Next, the hidden
morphological and structural parameters of the samples can be computed. This
change in recording media, also allows fast speed holograms recording, which
implies: (1) minimum isolation from mechanical vibrations and (2) the ability to
track fast processes. Due to digital processing of the holograms, aberration
compensation is possible [18, 19].
Although other reviews appeared in the literature, the majority of them are
only addressing principles and methods of DoHM [20–22], or are focusing on other
specific applications, like: three dimensional profiling and tracking [23], imaging
of complex fluids [24], MEMs/ MOEMs device inspection [25]. However, there
are several recent reviews that follow biological applications of DoHM [26, 27].
Here we emphasize the link between holographic images and parameters with
clinical relevance. Because there are many commercial softwares used for the
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reconstruction of the holograms, this review will focus on what is possible to do on
the reconstructed images, starting from the values contained in the PS maps, which
cannot be obtained in classical optical microscopy, or in electron microscopy.
This paper is organized as follows. The basic principles of general
holography and associated processes from the experimental setup and numerical
image reconstruction to the phase shift value maps are presented in Sec. 2. The
specific procedures for focusing and decoupling are also explained in Sec. 2 on the
basis of some new approaches used by different research groups. In Sec. 3, a
survey of the DoHM applications on different cell types is presented, along with
results for parameters correlated to the cells behavior, in situations which otherwise
cannot be investigated, or are investigated using marker-based techniques. Useful
values for these parameters are tabulated in Sec. 4.
2. FROM VISUALIZATION TO PHASE SHIFT VALUE MAPS
DoHM implies all steps from classical holography: recording and
reconstruction of the hologram. The recording step is experimental, based on the
Mach-Zehnder interferometer, because it offers flexibility in the geometrical
arrangement [28]. For biological samples, which are transparent for visible
wavelengths, the transmission geometry was chosen. One microscope objective is
needed in the object beam, in order to magnify the investigated samples, which
have dimensions in the micrometers range. To match the wavefront curvature for
both beams on the CCD sensor, another identical microscope objective must be
inserted in the reference beam (or other similar optical components to expand it).
After the fascicles passage through the second beam splitter, they are offset by an
angle, so that the hologram contains fringes and the interfringe can be changed for
needs in the experimental setup. Also, in the hologram are present the diffraction
maxima and minima coresponding to the sample details.
The experimentally recorded holograms are numerically converted to Fourier
domain in order to obtain their angular spectrum [29, 30]. Due to the off-axis
configuration, in the Fourier domain is possible to obtain separately the +1, 0 and
the –1 orders (in the digital in-line holographic microscopy, all these are
overlapped) and only the +1 order is considered further, because it corresponds to
the real image. Thus, the twin image is removed and the image of the reconstructed
object is more accurate. It is obtained by simulating the backward propagation
using Fresnel transform or Fresnel-Fourier transform [31], at a given distance [32,
33]. As a result, it is obtained an array of complex numbers containing separately
the amplitude Ax, y  and PS  x, y  as images of the sample [34]. Latter on,
when we will generally talk about both cases (amplitude or phase), we will denote
the matrix as f x, y  . In Fig. 1, it is shown a sketch with the main steps followed
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from the experimental hologram, to values of different parameters, in the general
case where both amplitude and phase informations are relevant.
Usually, the resolution in the transversal dimension is slightly under 1μm,
depending on the used wavelength. To improve it, few methods were proposed,
starting from the changes in the experimental setup, using time and angular
multiplexing [35], or using a dynamic phase grating [36].
Fig. 1 – General sketch with the steps from the experimental hologram to physiological parameters.
2.1. PROCEDURES TO FIND THE FOCUSED IMAGE
As a consequence of using scalar diffraction theory in Fresnel approximation,
the numerical reconstruction is sensible to the distance where the focused object
image is formed, but it does not provide any criterion to find the distance where the
reconstructed image is focused. DoHM permits subsequent numerical focusing by
varying of the propagation distance. Determining the optimal propagation distance
for a sharply focused image is of particular importance.
First, a criterion to detect the focusing plane, based on the analysis of the
amplitude images [37], was proposed, using the invariant properties of the energy
E and complex amplitude under the propagation: the effective propagated
amplitude modulus has a global lower bound which is independent of d:
B 

A( x, y )dxdy  M d . This integrated amplitude is minimum for pure
amplitude object and maximum for pure phase object, when the focusing distance,
d, is reached. Different details are visible at different heights (Fig. 2).
Fig. 2 – Images reconstructed at two distances, from the same hologram, to visualize details
from different heights in a MG63 cell and surrounding environment.
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Other criterias are based on:
(1) an algorithm to maximize the sharpness metric related to the sparsity of
the signal’s expansion in distance-dependent wavelet-like Fresnelet bases [38],
(2) analysis of the gray value distribution; sharp structures in a focused image
result in a higher contrast than in a smooth defocused image. The image contrast is
statistically quantified by the variance (VAR) of the histogram of the gray level
VAR 
1
NxNy
 f ( x, y)  f
2
where Nx and Ny are the image dimension and f
is the mean value calculated on the whole image (amplitude or phase) [39],
(3) squared gradient algorithm (SGA) and Laplacian filtering algorithm [40],
(4) integrated modulus amplitude in the case of amplitude object [41],
(5) high-pass filtered complex amplitudes with the aim of obtaining
minimum values for both types of objects when the focusing plane is reached [42],
 (I )
(6) Tamura coefficient, T 
where  I  and  I  represent the
 (I )
image gray-level standard deviation and mean, respectively. I represents the values
of every pixel from the region of interest of the reconstructed images (which are
converted in gray level images) [43, 44]. It has the intrinsic advantage of finding a
single focus-value without ambiguity, in the entire reconstruction volume, by
finding the distance where the calculated coefficient, T, for that image is minimal.
Using this capability of DoHM to track cells in 3D environment with
quantitative information on all axes, a simple method to measure cell motility was
developed and tested on many cellular lines [45]. The information between the
cells volume and their speed was correlated for L929, L56Br-Cl and MDA-MB231 cell lines.
2.2. PROCEDURES TO DECOUPLE INFORMATION ABOUT HEIGHT
AND REFRACTIVE INDEX
In given experiments, the values for cell height and refractive index in each
point (x, y), strating with the PS values, are needed separately. A method which can
be applied for cells attached on substrates [46] implies two holograms recording of the
same cell surrounded succesively by two media with slightly different values for
refractive indices. After the reconstruction process, two PS maps, 1 x, y  and
2 x, y  , are obtained, depending on the cell height hc x, y  and refractive index
nc x, y  in each point:
2
nc x, y   nsm1 hc x, y 
1 x, y  
(1)

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 2 x, y  
2
6
nc x, y   nsm2 hc x, y 
(2)

where  is the laser wavelength, n sm1 and n sm2 are the refractive indices for each
surrounding medium. The PS is calculated for a beam which travels through the
cell and one which travels outside the cell (very close to its edge). The solutions of
this system provide the height values and the refractive index values in each point.
At the end, we can conclude that values for length, width, height (3D
morphological distances 3D-MD) and the refractive indices are available from the
reconstructed images, being used as an indicator of physical density or chemical
concentration (mainly of protein content), which are the starting point for further
calculations of different parameters with biological relevance. The living cells
morphology was observed with 40 nm resolution for height and half micrometer
for transversal dimensions [4, 47].
For technical reasons, the described procedure is not suitable for fast processes;
another approach is to record the same hologram at two different wavelengths, when
adding a highly dispersive agent (dye) to the surrounding medium. From the
hologram reconstruction, two matrices are available, having values for optical path
difference OPD1 ( x, y ) 

1
1 x, y  and OPD 2 ( x, y )  2  2 x, y  ,
2
2
corresponding to both wavelengths. Assuming that [48]:
– for intracellular medium: nc ( )  nH 2O ( )   c DMCc
(3)
– for extracellular medium: nsm ( )  nH 2O ( )   DYE ( )C DYE   r Cr (4)
where  c is a constant known as the specific refraction increment related to the
intracellular content, DMCc is the dry mass concentration, n H 2O ( ) is the water
dispersion,  DYE is the specific refraction increment related to the dye, C DYE is
the dye concentration,  r mean refractive index increments of ions and
metabolites, C r their concentration. By solving the system, for the cell height and
refractive index, the equations are [49]:
hc ( x, y ) 
OPD1 ( x, y )  OPD 2 ( x, y )
,
 DYE (2 )   DYE (1 )C DYE
nc1 ( x, y)  OPD1 ( x, y)
 DYE (2 )   DYE (1 )C DYE
OPD1 ( x, y)  OPD 2 ( x, y)
(5)
 nsm1 .
Another approach is to use intensity measurements [50]. The absorbance in each
point, A'(x, y), for a liquid solution in terms of molar extinction coefficient,  , can be
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computed in accordance with Lambert-Beer law, based on experimental measurements
of the incident, I0, and transmitted, I, intensities by the sample:
 I ( x, y )    hc ( x, y )  C ( x, y)
 
A' ( x, y)   log
(6)
M
 I0 
where M is the molar mass (g/mol) and C ( x, y) is the density of sample in each
2
point. The Eq. 1 can be written as  x, y  
  C x, y   nH 2O  nsm hc x, y  .

One can observe that, in the last two equations, the unknown values are hc x, y 
and C x, y  and, by solving, both values can be provided:
M  A' ( x, y)
n water  n sm
, C  x, y  
hc x, y  
   ( x, y )
  C ( x, y)
(7)

2  M A' ( x, y )
3. FROM PHASE SHIFT VALUE MAPS TO PHYSIOLOGICAL PARAMETERS
Generally, biological specimens, such as living cells and their intracellular
constituents, are mostly transparent in the visible range (they are phase objects) and
therefore problematic for conventional bright-field microscopy. For this reason, in
medical laboratories, routine analysis are based on the chromatographic agents or
fluorescent markers (for example, Papanicolau test for cervical cells, peripheral
blood smear, histopathological evaluation of biopsy samples, microscopic
examination of bacteria, etc.). To avoid these external interventions, different
standard techniques were developed: phase contrast microscopy, differential
interference contrast, which gives a 3-D perception of the object, but only the
information about dimensions in planes transversal to the propagation axis.
Because cameras and detectors can only measure intensity, interferometric methods
are employed to obtain the phase information [1–13]. In 2005, Marquet et al. from
Ecole Polytechnique Fédérale Lausanne, Switzerland claimed the first DoHM
images of cells in natural environment [4]. These first results, illustrating highquality images of live neurons, demonstrated the potential of DoHM to become an
useful tool in cell biology, involving living specimens, being a label-free,
minimally invasive, and highly sensitive method to visualize and measure subtile,
fast changes in the physical and physiological states of cells and tissues in specific
processes [51].
1. Blood cells. For blood cells (BCs) analysis in DoHM, a simple procedure
is usually used: a droplet of the harvested blood is sandwiched between cover slips,
with no additional preparation [52].
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To study the red BCs rate of sedimentation, a physiological solution
containing (mM): NaCl 145, glucose 10, morpholinoethane sulfonic acid/Tris_hydroxymethyl_aminomethane _MES/Tris_10, pH 7.4 at room temperature, was
prepared [53]. In such conditions, the sedimentation velocity was determined to be
3.23 ± 0.07 mm/h. These are possible due to the capability of DoHM to record the
unfocused holograms without mechanical scanning and then to reconstruct the infocus cell image using adequate software. This procedure is faster than the classical
procedures. Another advantage is the fact that, by using DoHM, the shape and
dimensions of the investigated cells are available from the same image. The
assumption of optical homogeneity of red BC was used [54, 55] and justified by
the known fact that red BCs content mainly consists in hemoglobin solution; they
represent a particular type of structure without nuclei and organelles.
Using the refractive index of the cell and the surrounding plasma of 1.40 and
1.34, respectively [56], a highly dynamic process of hemoglobin flow out of the
cell during hemolysis was investigated with subnanometer path-length sensitivity
at the millisecond time scales and measurements revealed that the cell volume
decreased by 50% in less than 4s [57].
The PS values, available from the reconstructed images, allow calculating
other clinically important red BCs parameters, including the mean corpuscular
volume (MCV), the mean corpuscular hemoglobin concentration (MCHC), as a
ratio between mean corpuscular hemoglobin (MCH) and MCV, where MCH is:
MCH 
10      S c
2  Hb
(8)
and  Hb is the hemoglobin refraction increment (1.96x10-3 dl/g at wavelength 633
nm) and Sc is the projected area.
Parameters, such as red BCs radius, height, volume, refractive index, shape,
gradient from the weight center and hemoglobin content are important
characteristics to identify their type, function after storage, membrane fluctuation,
membrane permeability [58–61].
The red BCs membranes are a composite of a fluid lipid bilayer and a
triangular network of semiflexible filaments (spectrin). By measuring the area of
the projected surface, cell volume and mean corpuscular hemoglobin variations at
different osmolality, it was possible the computation of the shear modulus (μN/m)
in the interval 100–800 mOsm [62]. By simultaneously computing, for the same
population, of few parameters: the area of the projected surface, cell volume,
sfericity coefficient (the ratio between the red BCs height on ridge and in
concavity) [63], information about the red BCs elasticity on three axis were
statistically correlated.
As a consequence of these elastic properties, red BCs show spontaneous cell
membrane fluctuations (CMF) [64, 65]:
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Digital off-axis holographic microscopy
CMF x, y  
D ( x, y )  
2  nc  nsm 
1017
(9)
where D x, y  is the deviation phase map expressed in degrees (computed from a
reference value). CMF having medium fluctuation amplitude of 47 nm, are
heterogeneously distributed on the cellular surface and seem to correlate with the
biconcave equilibrium shape of erythrocytes. For ethanol-fixed red BCs, an
amplitude much smaller, of 5 nm was observed [65].
The capability of DoHM for three-dimensional tracking was extensively used
in hematology to measure the blood flow with high spatial and temporal resolutions
in a volume, to characterize red BCs trajectories and their 3D velocity profile
[66, 67].
Yi et al. [68] described a procedure to automatically test and compare the red
BCs characteristics for new and stored samples, which included steps like image
binarization, generation and combination between the internal and external
markers, application of watershed algorithm, a procedure which currently requires
a time-consuming manual examination by skilled personnel.
These red BCs’ biophysical parameters, noninvasively monitored by DoHM,
are clinically relevant parameters that can be used as diagnostic tools (e.g. involved
in the anemia classification). Also, a promising direction in the study of white
blood cells [52] is to replace the classical procedure used in differential white
blood count, which is based on chromatographic agents.
2. Yeast cells. The quantitative-PS map, associated with a living cell, is
linked to the cell’s dry mass density, i.e., its non-aqueous content. Thus, DoHM
has the ability to quantify cell growth with femtogram sensitivity and without
contact [69]. In 2009, Rappaz et al. studied the dry mass production during the cell
cycle in wild type yeast cells, exploiting the relationship proved more than 50 years
ago [48] between the PS and dry mass (DM) of cell, as an indicator of protein
production (proportional with the refractive index):
DM 
10 
2  

Sc
 ds 
10 
  Sc
2  
(10)
where  is the mean PS introduced by the whole cell, S c is the projected cell
surface and  is a constant known as the specific refraction increment related to
the intracellular content (usually 1.8-2.1 x 10-3 m3/kg when considering a mixture
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Mona Mihailescu et al.
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of all components of typical cell [48]). The DM (nonaqueous material) is defined
as the weight of the cell when water has evaporated and which mainly depends on
protein concentration. Monitoring the evolution of yeast cells using DoHM [69],
the stages of the Sc and DM in the cell growth and division has been highlighted: 1)
Sc linearly increases till 20 min before division, then stagnation followed by linear
increasing, 2) DM concentration abrupt increasing during last 20 min before
division, constant within 40 min after division [70].
Using the same expression for dry mass, in the case of red BCs, the specific
refraction increment,  , is associated with the hemoglobin content. Its value for
wavelengths around 633 nm is 0.00196 dl/g [46, 71]. In order to evaluate the RBC
properties during storage on long terms, an important parameter, which includes
the hemoglobin concentration and also the information about morphological
changes, is mean corpuscular hemoglobin surface density MCHSD  MCH . In
Sc
the case of stored RBCs, [59], it was observed that: 1) the Sc area is constant during
the first 30 days and then decreases, 2) the  values are constant during the first
30 days and decrease after this period, 3) the MCHSD is constant during the first 30
days and increases after. For all these three parameters measured during storage
interval, the standard deviation increases, which shows an increasing nonhomogeneity in RBCs population and hence the possibly of altering their
functionality, substantially changes.
3. Neurons. The mechanisms involved in the glutamate neurotransmitter
and glutamate N-Methyl-D-aspartate (NMDA) receptors, at single neuron level, is
interesting in the understanding of the synaptic plasticity and memory functions
[72]. By real time monitoring of the absolute cell volume using DoHM, the steps
associated with water influx in the cell (dilute intracellular content and decrease the
PS) and outflow (concentrate the intracellular content and increase the PS) were
highlighted on primary mouse cortical neurons in culture 1) biphasic, 2) reversible
decrease, and 3) irreversible decrease responses [73]. These indicate, respectively,
a low level, a high level, and an “excitotoxic” level of NMDA activation.
Moreover, furosemide and bumetanide, two inhibitors of sodium-coupled and/or
potassium-coupled chloride movement, strongly modify the PS, suggesting an
involvement of two neuronal co-transporters, NKCC1 (Na_K_Cl) and KCC2
(K_Cl), in the genesis of the optical signal.
In a recent study, Pavillion et al. [74] correlated the calcium dynamics
with phase measurements, in order to evaluate the neuron viability [75, 76, 49].
The osmotic membrane water permeability was calculated starting from values
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measured in the reconstructed object images from experimental holograms, and its
value is 0.00764 cm/s [49].
Equations similar with 3 and 4 can be written nc  n H 2O   c
DM
and
V
n f  nH 2O   f DMC f , which characterize the refractive index of the cell and of
the surrounding media. In the condition of transmembranar water fluxes, an
accumulation of DM appears in the cells, whose behavior in time leads to changes
in the cell refractive indices, which can be written as:
nc (t )  nc 0 
 f C f V (t )  V0    m(t 0 )
V (t )

V (t )
.
The refractive index associated with the transmembranar flux is similarly
defined and calculated as [49]:
n f (t ) 
nc (t )  V (t )  nc (t 0 )  V (t 0 )
.
V (t )  V (t 0 )
Using the decoupling procedure [77], it was determined: the swelling factor
1.76±0.31, projected surface area (187±41 μm2 normal and 200±43 μm2
hypotonic), cell volume (806±70 μm3 normal and 1419±129 μm3 hypotonic). As a
consequence of water influx, the refractive index decreases from 1.3847±0.0003 to
1.3645±0.0003.
4. Cancer cells. Human breast adenocarcinoma cell line, MCF-7 were
studied using DoHM, determining the cells height (approx. 12 μm) and its
refractive index, as a map highlighting the cell morphology including the cell body,
protrusions and lamellipodia, the refractive index of the nucleus, having grater
values than the refractive index of the surrounding cell material [78].
In G3S2 cells derived from human breast carcinoma were observed changes
of the cells dry mass within a deliberately chosen interval showing its motility and
its non-uniform spatial distribution, having high values around the whole cell
border, rather than normal central compactness [79].
Investigations of living pancreas tumor cells (Patu8988T) were carried out
[80] to find the influence of protein content in the refractive index and height
values. The same team, using DoHM, investigated drug-induced changes in
pancreas tumor cells [80]. The same cell line was investigated in terms of PS
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values, at different time intervals, after Taxol addition in the culture media,
showing that it firstly induces morphological rounding and increase in cell height.
The final cell collapse is precisely detected by a significant decrease of the PS [81]
or a decrease of five times in area being highlighted after 500 min, accompanied by
a sharp peak of the volume increasing twice from 8000 to 16000 μm3 [82, 83].
It is crucial to understand the cellular mitosis in cancer diseases. Using
DoHM, it was clearly established that the area decreases for the mother cell, but its
height increases, while the volume is approximately constant before division; after
division, both mother and daughter cells area increases, while their height
decreases [82].
A cytotoxicity assessment was successfully demonstrated on HeLa cells,
using DoHM, which can highlight the morphological and local biomolecules
(proteins and nucleic acids) [84], in good agreement with the classical analysis.
5. Osteoblasts MG63 cells. Using DoHM, long-time observations are
possible for cells in natural environment on different substrates: flat polypirollebased [85], or 3D micropatterned scaffolds with different geometries [86]. Using
the decoupling procedure, were calculated: the height of the 3D micropatterned
scaffolds of 10 – 20 μm (measurements unavailable using atomic force microscopy
which is limited at 10 micrometers in depth), the height of the polymeric flat
substrates (under 1 micrometer range), the refractive index of the polymeric
materials and of the cells (separated on cytoplasm and nucleus regions) [87]. The
observations during several days are possible, because in DoHM the cells are
investigated in their natural environment. Regular placing of the structure walls
tends to be a guiding model for the cells spatial orientation, observations important
in tissue engineering design.
Other studies on cells-substrates interaction were possible using DoHM: red
blood cells and HT-1080 fibrosarcoma sedimentation on collagen substrates [53],
stem cells on 3D micropatterned polymeric scaffolds obtained using matrix assisted
pulsed lased evaporation [88], cells motility in interaction with microfibers [89],
cancer cells cultures in matrix gels as scattering media [90], cells in flexible
substrate which distortions measure their traction force values [91].
6. Tissues. From the PS values of cryostat colonic sections of 7 μm constant
thickness and colitic C57Bl/6 WT mice, it was concluded: the refractive indices
values for healthy tissue are in the range from 1.375 to 1.382, while the lowest
refractive index value for colitis altered tissue (1.354) is well correlated with the
refractive indices of cells with high water content [92].
13
Digital off-axis holographic microscopy
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4. PHYSIOLOGICAL PARAMETERS VALUES
Table 1
Physiological parameters values
CELL TYPE
PARAMETER
red blood cells
diameter
red blood cells
surface
red blood cells
volume
red blood cells
red blood cells
red blood cells
red blood cells
MCH
MCHC
refractive index
sedimentation velocity
amplitude of membrane
fluctuation
osmotic membrane
water permeability
red blood cells
red blood cells
VALUE
mean value 7.7 ± 0.5 μm
interval 6–8 μm
46.7 ± 5.9 μm
interval 30-80 fl
mean value 83.3 ± 13.7 fl
29.9 ± 4.4 pg/cell
362 ± 40 g/l
1.418 ± 0.012
3.23 ± 0.07 mm/h
47 nm
SOURCE
[60, 94]
[60, 94]
[57, 60,
94]
[94]
[94]
[94]
[53]
[65]
0.0052 cm/s
[49]
red blood cells
mean corpuscular
hemoglobin surface density
0.70 ± 0.11 pg/ μm2 after 8 days
and 1.28 ± 0.26 pg/μm2 after 57
days of storage
[59]
red blood cells
shear modulus
6–12 μN/m
[62]
red blood cells
cytosol viscosity
1–12 mPa.s
yeast cell division
dry mass concentration
0–64–0.74 pg/μm2
neuron
osmotic membrane
water permeability
0.00764 cm/s
neuron
volume
1671±1116 μm3
pancreatic tumor
cell
refractive index
pancreatic tumor
cell
thickness
MG 63 cells
refractive index
PaTu 8988T 1.38±0.016
PaTu 8988T pLXIN
E-Cadherin 1.39±0.022
PaTu 8988T 23±1 μm
PaTu 8988T pLXIN
E-Cadherin 7±1 μm
ncytoplasm
Healthy cryostat
colonic sections
and colitic
C57Bl/6
WT mice
refractive index
nnucleus
=1.3584±0.0073 and
[62]
[69]
[49]
[49]
[80]
[80]
[86]
= 1.3795±0.0063
1.375-1.382, compared
with 1.354
[92]
1022
Mona Mihailescu et al.
14
5. CONCLUSIONS
Digital holographic microscopy is a marker-free technique which can be used
to analyze living biological samples (transparent in visible light) in their natural
environment (beside the electron microscopy where only deshidrated cells can be
visualized).
The progress introduced by the DoHM technique is the fact that quantitative
information is available in all three dimensions. Besides the classical optical
microscopy (phase contrast, differential interference contrast), which is capable to
visualize the cells in 3D but without any value along the propagation axis, in
DoHM, the phase shift values provide information about the height of the cell and
about its refractive index, as a map in each point (x, y). These values are then used
to compute many interesting parameters in biology or medicine.
Another advantage of DoHM is the fact that in the experimental setup, no
scanning is required, besides techniques like: atomic force microscopy or confocal
microscopy. One hologram, acquired in fraction of seconds, contains all the
information about the sample. These allow fast processes analysis which monitors
live biological specimens.
Altogether, these three advantages can be found only at phase imaging
techniques based on interference or holography which leads to new important
information about cell, impossible to track using other conventional microscopic
techniques.
This paper contains a short review of the principal algorithms employed in
the focalization process necessary in the reconstruction stage and in the decoupling
procedures for height and refractive index. But the focus is to point out as many
parameters available after processing the digital images, which are not available
using other microscopic techniques.
In this review, we demonstrated the capability of the DoHM as a marker-free
technique which crosses the borders of simple imaging technique and delivers
values about many morphological and structural parameters at the level of single
cell, such as 3D dimensions, projected surface, volume, eccentricity, refractive
index, dry mass, transmembranar fluxes, mean corpuscular volume, amplitude of
the cell membrane fluctuations, cell elasticity, rate of sedimentation, and viability
rate. These are arguments for researchers from biology, medicine, environment,
biochemistry, biophysics, and material science, to use DoHM in the analysis of
their complex samples.
Acknowledgement. This work was supported by a grant of the Romanian Authority for
Scientific Research and Innovation, CNCS-UEFISCDI, project number PN-II-RU-TE-2014-4-2534
(contract number 97 from 01/10/2015).
15
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1023
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