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Indian Institute of Science Education & Research, Kolkata May-July

May July 2009

Mueller Imaging:
Imaging An
An approach to Detect
etect
Abnormality in
i Humaan Brain Tissues

Project Report
((13th May – 28th July 2009)
2009)

Under the humble guidance of


Dr. Asima Pradhan
Associate Professor
Department of Physics
Centre for Laser Technology
Indian Institute of Technology, Kanpur

Harsh Purwar
IInd Year Student
Indian Institute of Science Education & Research
Kolkata

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Certificate
It is certified that the work in this project report entitled “Mueller Imaging: An

approach to Detect Abnormality in Human Brain Tissues”, by Harsh Purwar has

been carried out under my supervision and is not submitted anywhere else for

publication till date.

Dated:

Place:

(Dr. Asima Pradhan)


Associate Professor
Centre for LASER Technology
Department of Physics
Indian Institute of Technology
Kanpur – 208016
Uttar Pradesh, India

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Acknowledgement
I would like to express my gratitude to my mentor honorable Dr. Asima

Pradhan for her support, guidance and motivation throughout the project. I

would also like to thank Mr. Prashant Shukla (PhD. Scholar), Jaidip Jagtap (PhD.

Scholar) and Prabodh Pandey (Final Year Student, Int. M.Sc., IIT – Kanpur) for

their help and guiding me with their rich experience in the experiments and

data analysis. I would also like to thank for the co-operation extended by all

other graduate and post-graduate students in LASER Technology Laboratory,

SL - 111, Indian Institute of Technology, Kanpur for their help and support.

I thank my parents for their moral support.

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Table of Contents
(The page numbers are indicated in the square brackets)

1. ABSTRACT [6]

2. KEYWORDS [6]

3. INTRODUCTION [6]

4. THEORY [7]
a. Stokes Vector & Mueller Matrix [7]

b. Polarization States [7]

c. Mueller Matrix [8]

d. Polarization Images [8]

e. Mueller Images [8]

f. Optical Parameters [9]

g. Decomposition Scheme [9]

5. APPARATUS & INSTRUMENTS [12]

6. DESIGNED EXPERIMENTS [14]

7. EXPERIMENTAL SETUP [14]


a. Back-scattering Mode [14]

b. Transmission Mode [15]

8. METHODOLOGY [16]

9. SAMPLE PREPARATION [17]

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10. MICROSCOPIC IMAGES OF BRAIN TISSUES [18]

11. POLARIZATION IMAGES OF BRAIN TISSUE [20]

12. DATA ANALYSIS & RESULTS [23]

13. MUELLER IMAGES OF BRAIN TISSUE [28]

14. DECOMPOSITION PLOTS FOR BRAIN TISSUE [31]

15. DISCUSSIONS & CONCLUSIONS [32]

16. PRECAUTIONS [35]

17. BIBLIOGRAPHY [35]

18. REVISED MATLAB SCRIPT [37]

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Abstract
It has been observed that in recent years, researchers have given much emphasis on exploring on
various tissues through polarized light imaging technique as a potential diagnostic tool for detecting
any kind of abnormality particularly chronic diseases such as Cancer, Tumor, etc. As we are aware
of the fact that tissues depolarize a large fraction of incident light so that the Mueller calculus lends
itself well to these applications. Cancerous tissues were well discriminated from normal tissues using

4 × 4 Mueller matrix, to give three independent parameters depolarization, diattenuation and


Mueller imaging. Analysis of 49 obtained Mueller images of samples via CCD by decomposition of

retardance in the form of three 4 × 4 matrices, containing information about the optical properties
of the tissues based on their morphology and various chemical changes that occur in cells during
different stages of chronic diseases. It was found that abnormal tissues can be identified and
distinguished from the normal tissues based on these Mueller images and various other
decomposition plots at different stages of abnormality.

Keywords:
Mueller matrix, Mueller imaging, depolarization, diattenuation, birefringence, tissue optics,
polarization imaging, retardance, Mueller decomposition.

Introduction
In general, tissues are optically inhomogeneous and absorbing media whose average refractive index
is greater than that of air. This results in partial reflection of the radiations at the tissue/air interface,
while the remaining part penetrates the tissue. Bulk scattering of radiations in tissues is a major
cause of a large fraction of its dispersion in the backward direction, whereas multiple scattering and
absorption of radiations results in LASER beam broadening and its eventual decay as it travels
through them. Cellular organelles mitochondria, nucleus etc. are the major scatterers and blood is a
major absorber in the tissues. Scattering of radiations in tissues also depends on the water content
in them (1).

 , the scattering coefficient   and the anisotropy factor . As a tissue becomes cancerous,
Biological tissues can be characterized by their optical properties, namely the absorbing coefficient

several morphological and chemical changes occur in them such as; uncontrolled growth of cells in
tissues causes the collagen fibers to breakdown, increased nucleus size & number of mitochondria
etc. These changes affect various optical properties of tissues mentioned above. Also due to
increased blood perfusion, cancerous tissue is typically higher in absorption of radiations than normal
tissue (2). In general, tissues are birefringent and therefore they randomize the incident polarized
light to a very large extent. This allows polarimetry to be used as a tool for studying the various
optical properties of tissues and distinguish them on this basis.

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Theory
Stokes Vector and Mueller Matrix
Stokes vector is a 4 element column matrix that describes the polarization state of electromagnetic

Stokes in 1852. In terms of the components of electric field ( and  ),, the stokes
radiations. The elements of a Stokes vector are called Stokes parameters. These were defined by b
George Gabriel Stokes
parameters are given by (1):

      

      

      

      

Here , ,
, are the 4 elements of Stokes vector or Stokes parameters.

And the intensity of light by

      

connected with the Stokes vector of the incident light. If  is the initial incident Stokes vector and 
The polarization state of the scattered light in the far zone is described by the Stokes vector

is the Stokes vector describing the scattered light then we have,

    

where  is the normalized 4 × 4 scattering matrix or Mueller matrix.


matrix

Polarization States
Polarization states are various orientations of a polarizer or analyzer. There can be infinitely many
orientations and so infinitely many states. These all experiments are designed to use only seven such

example horizontal polarization state (H) should be 90°° more or


states listed below. Important point to remember is the relative differences between the states. For

less from the vertical polarization state (V).


(V). Same goes for +45 (P)
and -45
45 (M); Right circular (R) and left circular (L). The following
table lists different polarization states, and angles (in degrees) for
both the polarizers used.. Note that Polarizer (A) was used as a
polarizer and Polarizer (B) (B) was used as an analyzer. Also
remember that these states depend on the polarizer and analyzer
being used.

Figure 1:: Markings on Polarizer &


Analyzer
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Table 1: Listing various polarization states & their corresponding angles

Polarization States Polarizer (A) Polarizer (B)


Open (O) Removed Removed
Horizontal (H) 128 164
Vertical (V) 38 74
+45 (P) 353 29
-45 (M) 83 119
Right circular (R) 38 + QWP 74 + QWP
Left circular (L) 128 + QWP 164 + QWP

These angles for the two polarizers were found out by fixing one polarizer’s orientation (analyzer)
and rotating the other to get a minimum intensity of the LASER beam.

It should be clear from the table that vertical polarization state and a quarter wave plate both of
them combine to give circular polarized and we call it right circular polarized light.

Mueller Matrix
Mueller matrix is constructed as listed below. The different components of this matrix are made by
the combinations of different polarization states as stated above.

!! "!  ! #!  ! $!  %!
!"  ! ""   "  " #"    #  " %  $"  %"  $ (
 '
!#  ! "#    "  # ##    #  # %  $#  %#  $
 !$  !% "$  %  "%  $ #$  %  #%  $ %%  $$  %$  $% &

The Mueller matrix can be calculated using 16, 36, or 49 polarization images, with 36 and 49 images
corresponding to an over determined system. Here it was necessary to use more than 16 images to
reduce the error due to noise associated with the Mueller matrix calculation (2). In this experiment,
49 polarization images based system is used.

Polarization Images – raw material for Mueller matrix


In these experiments, polarization images are the images taken by the CCD for various polarization

intensity images for various states. Basically these intensity images are 512 × 768 matrix, each
states like HO, OO, VO, OV, RO, LR, etc. These polarization images may also be called as the

element storing the value of intensity falling at that particular location on the CCD’s chip. This
intensity can have values ranging from 0 to 12,000 in arbitrary units. While processing the images
and decomposing the matrix using a simple matlab script the value of each parameter is calculated
for each pixel of the images.

Mueller Images
A Mueller image is an element of the Mueller matrix. Since Mueller matrix is being calculated for
each and every pixel of the polarization images obtained, therefore each element can be obtained as

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an image of the same size i.e. 512 × 768 pixels. Each pixel of let’s say image, M11 stores the value of
the element M11 of the Mueller matrix – calculated for that pixel using the polarization images.

Optical Parameters (3)


• Depolarization:
A process which couples polarized light into un-polarized light. Depolarization is intrinsically
associated with scattering and with diattenuation and retardance which vary in space, time,
and/or wavelength. Examples of depolarizers include tissues, polystyrene microspheres,
phantoms etc.
• Diattenuation:
The property of an optical element or system whereby the intensity transmittance of the
exiting beam depends on the polarization state of the incident beam. The intensity
transmittance is a maximum (Tmax) for one incident state, and a minimum (Tmin) for the
orthogonal state. The diattenuation is defined as (Tmax - Tmin) / (Tmax + Tmin).
Any homogeneous polarization element which displays significant diattenuation and minimal
retardance is called a diattenuator. Polarizers have a diattenuation close to one, but nearly
all optical interfaces are weak diattenuators. Examples of diattenuators include the following:
polarizers and di-chroic materials, as well as metal and dielectric interfaces with reflection
and transmission differences described by Fresnel equations; thin films (homogeneous and
isotropic); and diffraction gratings.
• Polarizance:
The property of an optical element or system whereby un-polarized light is transformed into
polarized light. The polarizance is described by its magnitude (equal to the degree of
polarization of light exiting the system when un-polarized light is input) and the Stokes
vector of the output light.
• Retardance:
A polarization-dependent phase change associated with a polarization element or system.
The phase (optical path length) of the output beam depends upon the polarization state of
the input beam. The transmitted phase is a maximum for one eigen polarization, and a
minimum for the other eigen polarization. Other states show polarization coupling and an
intermediate phase.
• Birefringence:
A material property, the retardance associated with propagation through an anisotropic

of propagation with different refractive indices /0 and / . The birefringence is given by,
medium. For each propagation direction within a birefringent medium there are two modes

|/0  / |.

Decomposition Scheme for Mueller matrix


An arbitrary 4 × 4 Mueller matrix can be decomposed into three basic optical parameters namely
decomposition, retardance and diattenuation discussed in the latter half of this section. It has been
shown that any Mueller matrix can be expressed as a product of three matrices called depolarizer,
diattenuator and retarder (4). So we have,

  ∆ 3 4 ⋯ ⋯ ⋯ ⋯ 1

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The three diattenuation components 6 ⁄7, 45⁄135 and :;ℎ=/?@A= provide its complete
description. The diattenuation of a Mueller matrix described above is given by

1
B C0

 0D

 0E

⋯ ⋯ ⋯ ⋯ 2
00

BI 1 0
Similarly diattenuation vector is given by

FBG HBEJ K  H0D K ⋯ ⋯ ⋯ ⋯ 3


B3 00 
0E

And 4 × 4 diattenuation matrix is given by


1 FGM
B
4  L O ⋯ ⋯ ⋯ ⋯ 4
FBG N4

where N4 is given by
1 0 0
P
N4  1  B H0 1
 FG B
0K  Q1  P1  B R B FGM ⋯ ⋯ ⋯ ⋯ 5
0 0 1

matrix (Eq. – 4) was calculated for each pixel of cropped polarization images (100 × 100 pixels).
The value of diattenuation (Eq. – 2), diattenuation vector (Eq. – 3) and diattenuator or diattenuation

The values of diattenuation for 10000 pixels are shown in the form of a figure (later). So, we are
now left with,

4S0  ∆ 3  T ⋯ ⋯ ⋯ ⋯ 6

⇒ NT  N∆ N3 ⋯ ⋯ ⋯ ⋯ 7

Let V0 , V and VD be the eigenvalues of NT NT M . So from Eq. – 7, N∆ has PV0 , PV and PVD as
eigenvalues. After calculating the eigenvalues of NT NT M we evaluate the following expression,

S0
N∆  ±XNT NT M  YPV0 V  PV VD  PVD V0 Z [
× XYPV0  PV  PVD ZNT NT M  PV0 V VD [ ⋯ ⋯ ⋯ ⋯ 8

If the determinant of NT is negative then minus sign is applied otherwise positive. Now to get ∆
from N∆ we first calculate polarizance vector and polarizance matrix using,

1 0
#FG  HD0 K ⋯ ⋯ ⋯ ⋯ 9
00 
E0

#FG  NB
FG
#FG∆  ⋯ ⋯ ⋯ ⋯ 10
1B 

Now we write ∆ as a 4 × 4 matrix in terms of #FG∆ and N∆ as below,

1 F0GM
∆  L O ⋯ ⋯ ⋯ ⋯ 11
#FG∆ N∆

The value of depolarization power or simple depolarization is also calculated and is shown as a figure
(later) using,

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|=:∆   1|
∆ 1  ⋯ ⋯ ⋯ ⋯ 12
3

Now again we pre-multiply T (Eq. – 6) by ∆S0 to get,

3  ∆S0 T ⋯ ⋯ ⋯ ⋯ 13

The value of retardance was calculated as,

=:3 
%  cos S0 L  1O ⋯ ⋯ ⋯ ⋯ 14
2

After calculating the values of all the parameters for each pixel of the cropped polarization images,
these values are plotted or represented in the form of images of the same size and orientation.

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Apparatus & Instruments Required


• Ne LASER ( λ =632.8
He-Ne 632.8 nm,, 20 mW)
mW

• Charge Coupled Device (512 512 × 768


pixels): A charge-coupled
charge coupled device (CCD) is an
analog shift register that enables the
transportation of analog signals (electric
(electri
charges) through successive stages
(capacitors), controlled by a clock signal.
Today, they are most widely used in arrays of
photoelectric light sensors to serialize parallel Figure 2: A He - Ne LASER
analog signals. "CCD" refers to the way that
the image signal is read out from the chip. Under the control of an external circuit, each
capacitor can transfer its electric charge to one or another of its neighbors. CCDs are used
in digital photography, digital photogrammetry, astronomy (particularly in photometry),
sensors, electron microscopy, medical
fluoroscopy, optical and UV spectroscopy, and
high speed techniques such as lucky imaging. In
a CCD for capturing images, there is a
photoactive region, and a transmission region.
An image is projected by a lens on the
capacitor array (the photoactive region),
causing each capacitor to accumulate an
electric charge proportional to the light
intensity
ensity at that location. A one
one-dimensional
dimensional
array, used in line-scan
line scan cameras, captures a
single slice of the image, while a two- two
dimensional array, used in video and still
cameras, captures a two-two-dimensional
dimensional picture
corresponding to the scene projected onto the
focal plane of the sensor. Once the array has
been exposed to the image, a control circuit
causes each capacitor to transfer its contents

Figure 3:: A 512 X 768 pixels Charge Coupled to its neighbor. The last capacitor in the array
Device (CCD) dumps its charge into a charge amplifier, which
converts the charge
charge into a voltage. By repeating this process, the controlling circuit converts
the entire semiconductor contents of the array to a sequence of voltages, which it samples,
digitizes and stores in some form of memory.

• Computer: A computer is also required which


which is to be connected to the CCD by a serial
or parallel port cable for recording various polarization images.

• Lenses: Three lenses, two bi-convex


bi convex of
of focal lengths 8 - 10 cm & 7 – 9 cm having diameter
around 5 cm and a plano-convex
plano convex lens of focal length 10 – 15 cm having diameter 5 – 6 cm.

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• Polarizer & Analyzer

• Coherence Scrambler (or diffuser): A coherence scrambler is an optical instrument


that diffuses or spreads out or scatters light in some manner, to give soft light of nearly
uniform intensity. Optical diffusers use different methods to diffuse light and can include
ground glass diffusers, Teflon diffusers, holographic diffusers, opal glass diffusers, and greyed
glass diffusers. I used glass diffuser. Ideally a diffuser should not change the wavelength or
frequency of the light.

• Quarter Wave Plates (QWP): A wave plate or retarder is an optical device that alters
the polarization state of a light wave travelling through it. A wave plate works by shifting the
phase between two perpendicular polarization components of the light wave. A typical wave
plate is simply a birefringent crystal with a carefully chosen orientation and thickness. The
crystal is cut so that the extraordinary axis or "optic axis" is parallel to the surfaces of the
plate. Light polarized along this axis travels through the crystal at a different speed than light
with the perpendicular polarization, creating a phase difference. When the extraordinary
index is smaller than the ordinary index the extraordinary axis is called the "fast axis" and
the perpendicular direction in the plane of the surfaces is called the "slow axis". Depending
on the thickness of the crystal, light with polarization components along both axes will
emerge in a different polarization state. For instance a quarter-wave plate creates a quarter
wavelength phase shift and can change linearly polarized light to circular and vice versa. This
is done by adjusting the plane of the incident light so that it makes 45° angle with the fast
axis. This gives ordinary and extraordinary waves with equal amplitude.

• Mounts for lenses, polarizers, QWP, CCD, tissues etc.

• Polystyrene microsphere stock solution (Mean Diameter: 0.64 mm)

• Solid epoxy and titanium-oxide tissue phantom

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Designed Experiments
Exp. – 1

• To compare the values of the optical parameters (depolarization, diattenuation and


retardance) obtained for a solid epoxy & titanium oxide phantom and determine the role of
a diffuser (coherence scrambler) in the experiments done using a LASER beam having
speckles in backscattering mode.

Exp. – 2

• To study the effect of scattering on optical parameters by taking various solutions of


polystyrene microspheres (scatterers) of different scattering coefficients and calculating
Mueller matrix for them from the polarization images obtained as data from the CCD and
decomposing it to give the values of the parameters.
• To determine whether this approach is sensitive for small changes in the concentration
(number) of scatterers in a medium. Is it possible to distinguish them using this approach?

Exp. – 3

• To distinguish between the various stages of abnormality in brain tissue slides obtained from
a local medical college by comparing the various parameters calculated from the polarization
images obtained from the CCD.
• To correlate the values of the optical parameters with the actual microscopic structures of
the tissues using Mueller imaging.

Experimental Setup
Back-scattering Mode (Exp – 1 and 2):
The setup for various experiments described above based on the backscattering mode
was laid out in the following manner-

• Helium Neon LASER was first aligned on an optical bread board and its beam was made
horizontal up to a distance of a few meters. The height of the LASER beam from the bread
board was adjusted according to the height of the other mounts and CCD.
• Then the LASER beam was expanded using an objective lens of a compound microscope.
• The expanded beam was then made parallel using a bi-convex lens (A) and a plano-convex
lens (B) of focal lengths 8 – 10 cm & 7 – 9 cm respectively. Lens (B) was placed at a distance
so that the image formed by the Lens (A) was at its focus and the rays emerging out of this
two lens system were parallel.

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• This parallel beam was made to fall on the iris and its hole was adjusted according to the
need of the experiments.
• A polarizer and a quarter wave plate follows iris and were placed on the board such that the
beam falls at the center of them.
• Liquid samples were filled in a cuvette and mounted on adjustable stands in front of the
beam. The solid samples were mounted directly. The LASER beam was incident normally to
the samples.
• The light scattered from these samples was collected using a biconvex collecting lens of large
aperture (diameter) having short focal length. This collecting lens was placed as close to the
sample as possible so as to collect maximum scattered light. The sample should also be
oriented such that the speckles are reflected back and are not collected by the lens.
• The converged light (through lens) was focused on the CCD’s chip used to record intensity
profile.
• Another polarizer (as analyzer) and a quarter wave plate were placed between CCD and
collecting lens and their positions were marked on the board. These were used for
constructing various polarization states.
• The CCD was connected to the computer for recording the various polarization images for
analysis.

Diffuser
He-Ne LASER Lens A Lens B Mirror

Polarizer

CCD

Polarizer QWP

QWP

Collecting Lens

Sample

Transmission Mode (Exp. – 3):


The setup for various experiments described above based on the transmission mode
was laid out in the following manner-

• Helium Neon LASER was first aligned on an optical bread board and its beam was made
horizontal up to a distance of a few meters. The height of the LASER beam from the bread
board was adjusted according to the height of the other mounts and CCD.

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• This aligned LASER beam was then made to fall on the iris and its hole was adjusted
according to the need of the experiments.
• A polarizer and a quarter wave plate follows iris and they are placed on the board such that
the beam falls at the center of both of them.
• The samples were mounted directly on an adjustable mount. The LASER beam was incident
normally on the samples.
• The transmitted light is allowed to fall on the CCD’s chip used to record intensity profile.
• In between the CCD and the collecting lens another polarizer (or analyzer) and a quarter
wave plate was placed and their positions were marked on the board. These polarizers and
QWP’s will be used for constructing various polarization states.
• The CCD was finally connected to the computer with a serial port cable provided with it.

Above basic setup was used for most of the experiments done in back-scattering and transmission
mode after desired modifications/adjustments such as adding an appropriate ND filter in front of the
LASER beam if the incoming intensity saturates the CCD.

A coherence scrambler (diffuser) was used and placed in front of the LASER to study its effect in
Exp. – 1.

ND Filter QWP Sample QWP


He-Ne LASER

Polarizer Polarizer CCD

Methodology
• Intensity of the source i.e. He – Ne LASER has to be made uniform as much as possible
before starting the experiments. A coherence scrambler (diffuser) may be used for this
purpose. Formation of any kind of patterns due to dust particles, scratches on the optical
instruments or due to the interference of the speckles (5; 6) produced by the LASER should
be avoided.
• A CCD controlled by a software called MaxImDL was used for measuring intensity profile
and it was cooled to -20° C before taking the images.
• The liquid/solid samples (according to the experiment) were mounted and illuminated.
• The exposure time of the CCD was adjusted before starting the experiment by recording
OO polarization images so that the maximum intensity in this state is below its saturation
level.
• A reference box (set wise) was drawn on the image window and its co-ordinates were
noted. This helps in monitoring even a small shift in the bright spot of the LASER beam
during the experiment. This reference box may be required to be changed in subsequent set
of images.
• The various polarization states like VO, HO, LO, MH, PV, etc were constructed manually by
rotating the polarization axis of polarizer and analyzer in specific angles mentioned above.
• The 49 polarization images of a set were recorded and saved in .tif format, maintaining the
same exposure time and its position marked by reference box.

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• A background image was also recorded by blocking the main LASER source to correct the
intensity of OO polarization state later on, during image processing and cropping. Further
background correction also plays an important role in cropping the images by the algorithm
designed by me.
• These images were then processed and cropped by using a simple matlab script mentioned
at the end of this report.
• Images were then analyzed and decomposed according to the above mentioned
decomposition scheme to give the values of the three parameters – depolarization,
diattenuation and retardance for every pixel of the cropped images.

Sample Preparation
Solid Phantom (Exp. – 1)
The epoxy and titanium oxide solid phantom was prepared in my laboratory by Mr. Prashant Shukla
(PhD. Scholar) under the guidance of Dr. Asima Pradhan (Associate Professor, Department of
Physics, IIT – Kanpur) and was used as a sample in this experiment. The main reason behind this was
that it was a solid phantom and could be mounted directly in front of the LASER beam. In
backscattering mode to study the effect of speckles produced by the LASER it was necessary to
collect the specular reflection. While in case of a liquid medium speckles are reflected by the
container’s surface (cuvette) and not from the sample placed inside it. Therefore it was essential to
use some solid as a sample and mount it directly in front of the beam. However, there is no specific
reason for using this same solid phantom and so it could be changed. (Results for different phantoms
might be different).

Polystyrene Microspheres (Exp. – 2)


The polystyrene microspheres used were bought from Bangs Laboratories, Inc. and their detailed
specifications are –

• Mean diameter of microspheres: 0.64 mm

Density of Solid Polymer: 1.05 g/ml


• Buffer: DI water

Number of microspheres per ml: 6.972 × 1000


Scattering coefficient (calculated from Mie Theory): 381 mm-1



Four samples of different scattering coefficients (  3, 4, 6, 10 mm-1) were made by diluting the
above stock solution according to the fundamental dilution formula (0 0    ).

Brain Tissue (Exp. – 3)


The samples of brain tissues were obtained from a Sanjay Gandhi Postgraduate Institute of Medical
Sciences (SGPGIMS), Lucknow.

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Microscopic Images of Brain Tissues


Brain Tissue (Exp. – 3)
The microscopic images of these tissues were taken by a compound microscope at 5X zoom.

Slide ID Slide Picture

14 wks

Microscopic
Image (5X)
P#: 1 P#: 2 P#: 3 P#: 4

17 wks

Microscopic
Image (5X)
P#: 1 P#: 2

20 wks

Microscopic
Image (5X)
P#: 1 P#: 2 P#: 3

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24 wks

Microscopic
Image (5X)
P#: 1 P#: 2

28 wks

Microscopic
Image (5X)
P#: 1 P#: 2 P#: 3 P#: 4

33 wks

Microscopic
Image (5X)
P#: 1 P#: 2 P#: 3 P#: 4

36 wks

Microscopic
Image (5X)
P#: 1 P#: 2 P#: 3

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Polarization Images of Brain Tissue

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Figure 4: Polarization Images of Brain Tissues (Slide ID: 33 wks; Position # 2)

Above polarization images are centered and cropped to size 100 × 100 pixels using a small matlab
script. The depolarization scheme is then applied to all 10,000 pixels to give depolarization,
retardance and diattenuation values for all 10,000 pixels. These values are then scaled properly and
mapped to produce following plots.

22 | S u m m e r P r o j e c t R e p o r t
Indian Institute of Science Education & Research, Kolkata May-July 2009

Data Analysis & Results


The most critical part of any experiment is the analysis of its data and reaching to a definite
conclusion. Analysis of data in such bio-physical experiments is even more important as it may lead
to very important and significant conclusions. Data analysis in these experiments, where data is in
the form of images was done in the following manner.

• The obtained images were processed and cropped up to the bright spot’s size (100 × 100
pixels).
• These cropped images were then decomposed to give diattenuation, depolarization and
retardance values for each pixel.
• The values of these parameters were averaged over all the pixels and plotted against other
quantities.
• The Mueller images were also calculated and are compared. These images were normalized
in two different ways before comparing –
1. Normalized with respect to first element of Mueller matrix (M11)
2. Normalized with respect to the maximum intensity in that particular image.
• The decomposition plots were averaged either column wise or row wise and corresponding
values were plotted for comparison between two or more samples and distinguishing one
sample from the other. Similar treatment was also done with the Mueller images.

Solid Phantom (Exp. – 1)

Table 2: Listing average values of the three parameters, average being calculated over 100 X 100 pixels for
the two sets.

Mean over `aa × `aa pixels


Particulars
0.8955 ± 0.018 0.1795 ± 0.004 2.0919 ± 0.042
Depolarization Diattenuation Retardance

0.8256 ± 0.016 0.2138 ± 0.004 2.1514 ± 0.043


With diffuser
Without diffuser

Figure 5: Depolarization Plots – without diffuser (left) & with diffuser (right)

23 | S u m m e r P r o j e c t R e p o r t
Indian Institute of Science Education & Research, Kolkata May-July 2009

Figure 6: Diattenuation plots – without diffuser (left) & with diffuser (right)

Figure 7: Retardance plots – without diffuser (left) & with diffuser (right)

Polystyrene microspheres (Exp. – 2)

Table 3: Listing average values of the three parameters, average being calculated over 100 X 100 pixels for
various sets. Exposure time of CCD was 0.1 sec.

Scattering Coeff. Mean over `aa × `aa pixels


(bc ) (mm-1)
Solution ID
0.9114 ± 0.018 0.0271 ± 0.001 2.7613 ± 0.055
Depolarization Diattenuation Retardance

0.9256 ± 0.019 0.0417 ± 0.001 2.5750 ± 0.052


Sol – 1 3.0

0.9373 ± 0.019 0.0220 ± 0.000 2.8883 ± 0.058


Sol – 2 4.0

0.9593 ± 0.019 0.0311 ± 0.001 2.6838 ± 0.054


Sol – 3 6.0
Sol – 4 10.0

Scattering coefficient was calculated using Mie calculator (7) based on the Mie Theory for scattering
of light in a highly turbid media.

24 | S u m m e r P r o j e c t R e p o r t
Indian Institute of Science Education & Research, Kolkata May-July 2009

Mean Depolarization
0.99
Mean over 100 X 100 pixels

0.98
0.97
0.96
0.95
0.94
0.93
0.92
0.91
0.9
0.89
0.88
0 2 4 6 8 10 12
Scattering Coefficient

Plot 1: Mean depolarization versus scattering coefficient

Mean Diattenuation
0.045
Mean over 100 X 100 pixels

0.04
0.035
0.03
0.025
0.02
0.015
0 2 4 6 8 10 12
Scattering Coefficient

Plot 2: Mean diattenuation versus scattering coefficient

Mean Retardance
3
Mean over 100 X 100 pixels

2.9
2.8
2.7
2.6
2.5
2.4
2.3
2.2
0 2 4 6 8 10 12
Scattering Coefficient

Plot 3: Mean retardance versus scattering coefficient

25 | S u m m e r P r o j e c t R e p o r t
Indian Institute of Science Education & Research, Kolkata May-July 2009

Brain Tissues (Exp. – 3)

Table 4: Listing average values of the three parameters, average being calculated over 100 X 100 pixels for
various sets. Exposure time of CCD was 1 sec.

Slide Position Mean over `aa × `aa pixels


ID # Depolarization Error Diattenuation Error Retardance Error
1 0.7313 0.007 0.0644 0.001 0.3384 0.015
2 1.1581 0.007 0.0651 0.001 0.3347 0.023
14 wks
3 0.7497 0.007 0.0638 0.001 0.333 0.015
4 0.7496 0.007 0.063 0.001 0.3333 0.015
1 0.6708 0.006 0.0558 0.001 0.2765 0.013
17 wks
2 0.6714 0.006 0.0632 0.001 0.2782 0.013
1 3.1036 0.007 0.0743 0.001 0.3426 0.062
20 wks 2 3.0882 0.008 0.0673 0.001 0.3786 0.062
3 3.0754 0.005 0.0725 0.001 0.2620 0.062
1 0.6245 0.006 0.0685 0.001 0.3038 0.012
24 wks
2 0.6386 0.005 0.0931 0.002 0.2343 0.013
1 0.7564 0.006 0.0647 0.001 0.3133 0.015
2 0.7739 0.006 0.0550 0.001 0.2890 0.015
28 wks
3 3.1146 0.006 0.0641 0.001 0.3214 0.062
4 3.1085 0.006 0.0785 0.002 0.2871 0.062
1 0.7248 0.006 0.0712 0.001 0.3106 0.014
2 0.6401 0.006 0.0527 0.001 0.2818 0.013
33 wks
3 0.8756 0.006 0.0668 0.001 0.2858 0.018
4 0.6097 0.005 0.0517 0.001 0.2342 0.012
1 3.1079 0.007 0.0667 0.001 0.3353 0.062
36 wks 2 0.4284 0.007 0.0651 0.001 0.3485 0.009
3 0.5301 0.006 0.0408 0.001 0.2973 0.011

26 | S u m m e r P r o j e c t R e p o r t
Indian Institute of Science Education & Research, Kolkata May-July 2009

Mean Depolarization
0.4
Mean over 100X100 pixels

0.35

0.3

0.25

0.2
10 15 20 25 30 35 40
Slide ID (wks)

Plot 4: Mean depolarization versus Slide ID

Mean Diattenuation
0.1
Mean over 100X100 pixels

0.09
0.08
0.07
0.06
0.05
0.04
0.03
10 15 20 25 30 35 40
Slide ID (wks)

Plot 5: Mean diattenuation versus Slide ID

Mean Retardance
3.5
Mean over 100 X 100 pixels

3
2.5
2
1.5
1
0.5
0
10 15 20 25 30 35 40
Slide ID (wks)

Plot 6: Mean retardance versus Slide ID

27 | S u m m e r P r o j e c t R e p o r t
Indian Institute of Science Education & Research, Kolkata May-July 2009

Mueller Images of Brain Tissue


Following are the normalized Mueller images (normalized with respect to M11) for the brain tissue.
(Slide ID 33 wks, position 2)

Figure 8: Normalized (with respect to M11) Mueller images for Brain tissue (Slide ID: 33 wks; Position # 2)

28 | S u m m e r P r o j e c t R e p o r t
Indian Institute of Science Education & Research, Kolkata May-July 2009

Table 5: For comparing the normalized (with respect to maximum intensity in that image) Mueller images
M11 (only) for various sets.

Normalized Mueller Image (M11)


Silde ID
Position 1 Position 2 Position 3 Position 4

14 wks

Avg. 0.4921 0.5053 0.4407 0.4876

17 wks

Avg. 0.5067 0.4509

20 wks

Avg. 0.3742 0.4396 0.4351

24 wks

Avg. 0.4327 0.4589

28 wks

Avg. 0.4898 0.4821 0.4795 0.4925

33 wks

Avg. 0.5002 0.5233 ± 0.15 0.4726 0.4918

29 | S u m m e r P r o j e c t R e p o r t
Indian Institute of Science Education & Research, Kolkata May-July 2009

36 wks

Avg. 0.6179 0.5903 0.5702

Average M11 Intensity


0.65
Mean over 100 X 100 pixels

0.6
0.55
0.5
0.45
0.4
0.35
0.3
10 15 20 25 30 35 40
Silde ID (wks)

Plot 7: Average Intensity of M11 versus Slide ID

30 | S u m m e r P r o j e c t R e p o r t
Indian Institute of Science Education & Research, Kolkata May-July 2009

Decomposition Plots for Brain Tissue


2-D decomposition plots of depolarization, diattenuation and retardance for a brain tissue slide
(Slide ID 33 wks, position 2) is shown below

Figure 9: Depolarization (top left), Retardance (top right) and Diattenuation (bottom) plots for brain tissue
(Slide ID: 33 wks; Position # 2)

Note the common pattern in all the three plots. This common pattern is the diffraction pattern
formed due to speckles of the LASER beam. This was confirmed after recording & analyzing the
LASER spot (or oo polarization state’s image) and Mueller images (Figure 8).

Figure 10: LASER Spot (Non-uniform intensity distribution)

31 | S u m m e r P r o j e c t R e p o r t
Indian Institute of Science Education & Research, Kolkata May-July 2009

Discussions & Conclusions


• The use of diffuser (coherence scrambler or spinning ground glass) was found to be
important for the experiments done in the backscattering mode. Comparing the above plots
(Figure 5, Figure 6, Figure 7), it is very clear that there is more uniformity in the values of all
the three parameters when diffuser was used. This is only possible if the uniformity of the
initial LASER spot has changed. And a diffuser actually does that. As mentioned earlier that a
diffuser improves the uniformity in the intensity distribution of the LASER beam (from
Gaussian to uniform) and also by removing the speckles produced by the LASER. This is
even clearer from the following plot.

Figure 11: Average Depolarization – averaged through column

The above figure represents the average values of depolarization – calculated for the set of
data taken, without using a diffuser and using a diffuser. The spread in the average values of
depolarization is less using a diffuser. This is because the incident light is now more uniform
than the LASER beam.

• Different solutions of polystyrene microspheres of varying scattering coefficients are easily


distinguishable using this approach of decomposing the Mueller matrix to give complete
information about depolarization. The above plot (Plot 1) for mean depolarization verses
scattering coefficient shows an increase in the value of depolarization as the concentration
(or number) of scatterers in the solution increases (although not very sensitive). This is
because more the light scatters more is its depolarization. Diattenuation is also found to be
very sensitive but not consistent (Plot 2) whereas retardance (Plot 3) was neither very
sensitive nor did it gave consistent results in this case.

• Brain tissues can also be distinguished from each other on the basis of cell density or
scatterer density. More is the concentration of the cells in a particular region more are the

32 | S u m m e r P r o j e c t R e p o r t
Indian Institute of Science Education & Research, Kolkata May-July 2009

number of scatterers, absorbers, etc in that particular region. Talking simply if at a particular
location there is a very high density of scatterers like mitochondria, etc which happens in
cancer and other chronic diseases then a similar pattern should be shown by the values of
depolarization as well (concluded by Exp. – 2). From the plots (Plot 4, Plot 5 & Plot 6) it is
shown that these parameters are very sensitive and tissues can be distinguished on this basis.
Tissues can also be distinguished on the basis of the Mueller images. Following is a plot of
average of Mueller element (M43), average taken over each column of the Mueller image M43.

According to the above plot which shows a clear cut distinction in the values of the Mueller
element M43 compared between the various tissue slides. These can be divided into two
major groups based on this plot. Similar are the two following plots for M24 and M34, which
also shows that there is a range in which the values of these elements lie.

33 | S u m m e r P r o j e c t R e p o r t
Indian Institute of Science Education & Research, Kolkata May-July 2009

Any abnormality in the tissue’s morphology shows an abnormal increase or decrease in the
values of these parameters at that particular location. The curves for 36 wks (all positions)
all have the minimum value for this element (M24) and all lie close to each other. Similarly the
curves for 20 wks position 1 and 2 lie close to each other while the same for position
number 3 is quite high and might indicate some change in tissue morphology. This should be
compared with the histopathology report (yet to come) and then infer the final results.

Comparing the above graphs with the microscopic images of the tissues (shown above),
shows that the values being indicated by the above graphs for the various sets of data match
with them and hence, this approach can be used to detect severe abnormalities in the live
systems and should be further developed.

34 | S u m m e r P r o j e c t R e p o r t
Indian Institute of Science Education & Research, Kolkata May-July 2009

Precautions
• The background intensity and thermal noise of CCD should be lesser than the LASER
intensity (signal). It is strongly recommended to do the experiments in a completely dark
room.
• Before starting the experiment, initial spot’s size and its uniformity should be checked. There
should be no or very little variation in intensity.
• All optical instruments such as lenses, polarizers, quarter wave plate, CCD etc should be
free from any scratches or dust particles. Acetone or alcohol may be used for cleaning
purposes. Dust or scratch produces diffraction patterns due to which intensity varies.
• Don’t look at the LASER beam not even it reflection with the naked eyes.
• Wear gloves when dealing with cancerous tissues or toxic chemicals.
• In case of polystyrene microspheres solution (Exp. – 2) specular reflection in back-scattering
mode should not be taken into account. The speckles generally are of lesser intensity (than
main LASER beam) and get reflected from the walls of cuvette and so carry no information
about the sample filled inside it.

Bibliography
1. Tuchin, Valery. Optical properties of tissues with strong (multiple) scattering. Tissue Optics: Light
Scattering Methods and Instruments for Medical Diagnosis. Second. 1, pp. 3-108.

2. Gupta, Vivek. Mueller matrix in optical imaging for cervical cancer detection. Department of Physics,
Indian Institute of Technology. Kanpur : s.n., 2009. pp. 1-6.

3. Reference Page. Axometrics Web Site. [Online] http://www.axometrics.com/reference.htm.

4. Interpretation of Mueller matrices based on polar decomposition. Lu, Shih-Yau and Chipman,
Russell A. 5, Alabama : Optical Society of America, May 1996, Opt. Soc. Am. A, Vol. 13, pp. 1106-
1113.

5. Size determination by use of two-dimentional Mueller matrices backscattered by optically thick random
media. Dillet, Jerome, et al. 19, s.l. : Optical Society of America, February 23, 2006, APPLIED
OPTICS, Vol. 45, pp. 4669-4678.

6. Interpreting Mueller images of tissues. Smith, Matthew H. Alabama : SPIE, 2001, SPIE, pp. 82-89.

7. Prahl, Scott. Mie Scattering Calculator. [Online] Oregon Medical Laser Center, 2007.
http://omlc.ogi.edu/calc/mie_calc.html.

8. Measurement and calculation of the two-dimensional backscattering Mueller matrix of a turbid medium.
Cameron, Brent D., et al. 7, Texas : Optical Society of America, April 1, 1998, OPTICS
LETTERS, Vol. 23, pp. 485-487.

35 | S u m m e r P r o j e c t R e p o r t
Indian Institute of Science Education & Research, Kolkata May-July 2009

9. Diffuse backscattering Mueller matrices patterns from turbid media. Lian-Shun, ZHANG, et al. 6,
China : Chinese Physical Society and IOP Publishing Ltd., January 23, 2006, CHIN. PHYS. LETT., Vol.
23, pp. 1603-1606.

10. Bladwin, Angela M. Mueller matrix imaging for skin cancer detection. Biomedical Engineering,
Texas A & M University. Texas : s.n., 2004. pp. 1-135, Thesis.

11. Mueller decomposition images for cervical tissue: Potential for discriminating normal and dysplastic states.
Shukla, Prashant and Pradhan, Asima. 3, s.l. : Optical Society of America, February 2, 2009,
OPTICS EXPRESS, Vol. 17, pp. 1600-1609.

12. Hahn, Brian D. and Valentine, Daniel T. Essential Matlab for engineers and scientists. Second.
s.l. : Elsevier Ltd., 2007. p. 428. ISBN 13: 9-78-0-75-068417-0; ISBN 10: 0-75-068417-8.

13. Wikipedia. [Online] http://en.wikipedia.org.

36 | S u m m e r P r o j e c t R e p o r t
Indian Institute of Science Education & Research, Kolkata May-July 2009

Revised Matlab Script for better results:


Add the following script/code to a new .m file and execute it. The depolarization, diattenuation and
retardance plots will be plotted and saved automatically. Remember to place the new .m file
containing the following script/code in the same directory in which finalchz_mod2.m file is saved and
you may have to change the directories accordingly. Appropriate changes were also made in the
finalchz_mod2.m script file.

close all
clear
wdir=input('Enter the location of original images: ','s');
fname=['oo';'oh';'ov';'op';'om';'ol';'or';'hh';'ho';'hp';'hm';'hr';'hl';'vv';'vo';'vp';'vm';'vr';'vl';'pp';'pl';'ph';'pr';'pv'
;'po';'mm';'mh';'mv';'mr';'ml';'mo';'rr';'rh';'rv';'ro';'rp';'rm';'rl';'ll';'lp';'lm';'lh';'lv';'lo';'lr';'hv';'vh';'mp';'pm'];
[w,e]=size(fname);
hlim=100;
wlim=100;
ch=input('Do you want to crop the images(Y/N)? ','s');
if ch=='Y' || ch=='y'
fprintf('\nEnter the dimensions of the box...\n');
tl=input('Top left coordinates of the box: ');
rb=input('Right bottom coordinates of the box: ');
cd(wdir);
mkdir new_data
bg=imread('bkg.tif');
z=1;
while z<=w-4
im=imread(fname(z,:),'tif');
I=im-bg;
J=double(I);
maxI=max(J(:));
M=(J/maxI)*255;
M=uint8(M);
thres=graythresh(M);
bw=im2bw(M,thres);
bw=bwareaopen(bw,5);
bw=imfill(bw,'holes');
[B,L]=bwboundaries(bw);
[p,q]=size(B);
if p==0
fprintf('BAD IMAGE...%s.tif\n',fname(z,:));
else
stats=regionprops(L,'Centroid','Area');
for t=1:p
A(t)=stats(t).Area;
end
[val,ind]=max(A);
cord=stats(ind).Centroid;
I=imcrop(I,[round(cord(1,1)-wlim/2),round(cord(1,2)-hlim/2),wlim-1,hlim-1]);
cd new_data
imwrite(I,[fname(z,:),'.tif']);
cd ..
fprintf('%s.tif done...\n',fname(z,:));

37 | S u m m e r P r o j e c t R e p o r t
Indian Institute of Science Education & Research, Kolkata May-July 2009

clear A
end
z=z+1;
end
clear B z L t bw p q thres M maxI cord val ind e J im I stats
cd(wdir)
for z=w-3:w
I=imread(fname(z,:),'tif');
I=I-bg;
[u,v]=size(I);
for i=1:u
for j=1:v
if I(i,j)<0
I(i,j)=0;
end
end
end
J=imcrop(I,[(tl(1)+(rb(1)-tl(1))/2),(tl(2)+(rb(2)-tl(2))/2),wlim-1,hlim-1]);
cd new_data
imwrite(J,[fname(z,:),'.tif']);
cd ..
fprintf('%s.tif done...\n',fname(z,:));
end
clear tf rb I bg J z u v
end

ndir=[wdir,'\new_data'];
ch=input('Do you want to check the images(Y/N)? ','s');
if ch=='y' || ch=='Y'
cd(ndir);
for i=1:w
im=imread(fname(i,:),'tif');
if rem(i,12)==1
figure;
end
if rem(i,12)==0
j=12;
else
j=rem(i,12);
end
subplot(3,4,j);imagesc(im);axis off;colorbar;
title(fname(i,:));
end
end
char=input('Do you want to decompose the Mueller images (Y/N)? ','s');
if char=='y' || char=='Y'
cd 'C:\Users\Harsh Purwar\Documents\MATLAB'
finalchz_mod2
end
cd 'C:\Users\Harsh Purwar\Documents\MATLAB'
clear

38 | S u m m e r P r o j e c t R e p o r t

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