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

1Physics Department, Politehnica University from Bucharest, Romania

E-mails: irina.paun@physics.pub.ro, eugenia.vasile@physics.pub.ro 2Department of Life and Environmental Physics, Horia Hulubei National Institute of Physics and

Nuclear Engineering, Magurele, Romania 3Faculty 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, bio-

logical 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

1010 Mona Mihailescu et al. 2

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

3 Digital off-axis holographic microscopy 1011

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 yxA , and PS yx, 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 yxf , . In Fig. 1, it is shown a sketch with the main steps followed

1012 Mona Mihailescu et al. 4

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:

( , )d d dB A x y x y M . 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.

5 Digital off-axis holographic microscopy 1013

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

2

),(1

fyxfNN

VARyx

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],

(6) Tamura coefficient, )(

)(

I

IT

where I and I represent the

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-MB-

231 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, yx,1 and

yx,2 , are obtained, depending on the cell height yxhc , and refractive index

yxnc , in each point:

yxhnyxnyx csmc ,,2

, 11

(1)

1014 Mona Mihailescu et al. 6

yxhnyxnyx csmc ,,2

, 22

(2)

where is the laser wavelength, 1smn and 2smn 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 yxyxOPD ,2

),( 11

1

and yxyxOPD ,

2),( 2

22

,

corresponding to both wavelengths. Assuming that [48]:

– for intracellular medium: ccOHc DMCnn )()( 2 (3)

– for extracellular medium: rrDYEDYEOHsm CCnn )()()( 2 (4)

where c is a constant known as the specific refraction increment related to the

intracellular content, DMCc is the dry mass concentration, )(2 OHn is the water

dispersion, DYE is the specific refraction increment related to the dye, DYEC is

the dye concentration, r mean refractive index increments of ions and

metabolites, rC their concentration. By solving the system, for the cell height and

refractive index, the equations are [49]:

DYEDYEDYE

cC

yxOPDyxOPDyxh

)()(

),(),(),(

12

21

,

1

21

1211

),(),(

)()(),(),( sm

DYEDYEDYEc n

yxOPDyxOPD

CyxOPDyxn

.

(5)

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

7 Digital off-axis holographic microscopy 1015

computed in accordance with Lambert-Beer law, based on experimental measurements

of the incident, I0, and transmitted, I, intensities by the sample:

M

yxCyxh

I

yxIyxA c ),(),(),(

log),('0

(6)

where M is the molar mass (g/mol) and ),( yxC is the density of sample in each

point. The Eq. 1 can be written as yxhnnyxCyx csmOH ,,2

, 2

.

One can observe that, in the last two equations, the unknown values are yxhc ,

and yxC , and, by solving, both values can be provided:

),(

),(',

yxC

yxAMyxhc

,

),('

),(

2

,

yxA

yx

M

nnyxC smwater

(7)

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 high-

quality 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].

1016 Mona Mihailescu et al. 8

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 in-

focus 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:

Hb

cSMCH

2

10 (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]:

9 Digital off-axis holographic microscopy 1017

smc nn

yxDyxCMF

2

),(, (9)

where yxD , 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):

10 10

d2 2c

cS

DM s S

(10)

where is the mean PS introduced by the whole cell, cS 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

1018 Mona Mihailescu et al. 10

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 densitycS

MCHMCHSD . In

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 non-

homogeneity 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

11 Digital off-axis holographic microscopy 1019

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 V

DMnn cOHc 2 and

ffOHf DMCnn 2 , 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:

)(

)(

)(

)()( 00

0tV

tm

tV

VtVCntn

ff

cc

.

The refractive index associated with the transmembranar flux is similarly

defined and calculated as [49]:

)()(

)()()()()(

0

00

tVtV

tVtntVtntn cc

f

.

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 μm

2

hypotonic), cell volume (806±70 μm3 normal and 1419±129 μm

3 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

1020 Mona Mihailescu et al. 12

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 polypirolle-

based [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 1021

4. PHYSIOLOGICAL PARAMETERS VALUES

Table 1

Physiological parameters values

CELL TYPE PARAMETER VALUE SOURCE

red blood cells diameter mean value 7.7 ± 0.5 μm

interval 6–8 μm [60, 94]

red blood cells surface 46.7 ± 5.9 μm [60, 94]

red blood cells volume interval 30-80 fl

mean value 83.3 ± 13.7 fl

[57, 60,

94]

red blood cells MCH 29.9 ± 4.4 pg/cell [94]

red blood cells MCHC 362 ± 40 g/l [94]

red blood cells refractive index 1.418 ± 0.012 [94]

red blood cells sedimentation velocity 3.23 ± 0.07 mm/h [53]

red blood cells amplitude of membrane

fluctuation 47 nm [65]

red blood cells osmotic membrane

water permeability 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 [62]

yeast cell division dry mass concentration 0–64–0.74 pg/μm2 [69]

neuron osmotic membrane

water permeability 0.00764 cm/s

[49]

neuron volume 1671±1116 μm3 [49]

pancreatic tumor

cell refractive index

PaTu 8988T 1.38±0.016

PaTu 8988T pLXIN

E-Cadherin 1.39±0.022

[80]

pancreatic tumor

cell thickness

PaTu 8988T 23±1 μm

PaTu 8988T pLXIN

E-Cadherin 7±1 μm

[80]

MG 63 cells refractive index cytoplasmn

=1.3584±0.0073 and

nucleusn= 1.3795±0.0063

[86]

Healthy cryostat

colonic sections

and colitic

C57Bl/6

WT mice

refractive index 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 Digital off-axis holographic microscopy 1023

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