MT BGW

T HE I NFLUENCES OF THE ACTIN
C YTOSKELETON ON THE P ROPERTIES OF

THE N UCLEUS IN A DHERENT C ELL :
C OMPARISON BETWEEN 2D AND 2.5D
M ASTER T HESIS
by
Bayu Gautama Wundari
Examiner: Prof. Dr. Martin Bastmeyer

Supervisor: Dr. Franco Weth
Institute of Zoology
Karlsruhe, 30.04.2014
Declaration of Original Work

I do hereby declare that the present thesis is original work by me alone and that I have indicated
completely and precisely all aids used as well as all citations, whether changed or unchanged, of
other theses and publications.
Eigenstndigkeitserklrung
Ich versichere wahrheitsgem, die Arbeit selbststndig angefertigt, alle benutzten Hilfsmittel
vollstndig und genau angegeben und alles kenntlich gemacht zu haben, was aus Arbeiten anderer
unverndert oder mit Abnderungen entnommen wurde.
Karlsruhe, den 30.04.2014.
Unterschrift / Signature:
ii
Contents
Abstract
Introduction
Theoretical Background
2.1 Actin Stress Fibers . . . . . . . . . . . . . . . .
2.2 Cell-Matrix Adhesion Contacts . . . . . . . . . .
2.3 Microcontact Printing . . . . . . . . . . . . . . .
2.4 Image Formation . . . . . . . . . . . . . . . . .
2.4.1 Degradation Due to the Blurring Process
2.4.2 Degradation Due to Noise . . . . . . . .
2.5 Image Restoration . . . . . . . . . . . . . . . . .
2.5.1 Non-iterative Image Restoration . . . . .
2.5.2 Statistical Image Restoration . . . . . . .
2.6 Structure Tensor . . . . . . . . . . . . . . . . . .
Materials and Methods

3.1 Stamp Fabrication . . . . . . . . . . . . . . . . .
3.2 2D Environments . . . . . . . . . . . . . . . . .
3.2.1 Sample Preparations for 2D Substrates . .
3.2.2 Cell Fixation and Staining . . . . . . . .
3.2.3 Data Acquisition . . . . . . . . . . . . .
3.2.4 Image Processing for 2D Cases . . . . .
3.3 2.5D Environments . . . . . . . . . . . . . . . .
3.3.1 Sample Preparations for 2.5D Substrates .
3.3.2 Cell Fixation and Staining . . . . . . . .
3.3.3 Data Acquisition for 2.5D Cases . . . . .
3.3.4 Image Processing for 2.5D Cases . . . .
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Results and Data Analysis

4.1 Cell Nucleus and Stress Fiber Orientation . . . . . . . . . . . . . . . .
4.2 Cell Nucleus and Cell Body Area Measurements . . . . . . . . . . . .
4.3 Cell Nucleus Elongation . . . . . . . . . . . . . . . . . . . . . . . . .
4.4 Cell Volume and Surface Area . . . . . . . . . . . . . . . . . . . . . .
4.5 The Actin Cytoskeleton Supports the Cell Nucleus in 2.5D Environment
4.6 Nucleus Indentation . . . . . . . . . . . . . . . . . . . . . . . . . . . .
iii
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Contents
Discussion and Outlook
109
Summary
115
Bibliography
122
Acknowledgment
127
Contents
Abstract
The structure of the extracellular matrix (ECM) defines the cell shape which turns out to
determine the life cycle of cells. The information from cells environments are transmitted to the
nucleus by sensing the mechanical forces transmitted via the actin cytoskeleton. By defining the
shape and the size of adhesive islands, the actin cytoskeleton distribution near the nucleus and the
global distribution of the actin stress fibers seem to play a main role in defining the geometrical
aspects of the nucleus. In particular, cross-sectional area, elongation, long axis orientation,
surface area and volume will be investigated. Furthermore, the actin cytoskeleton turns out to
be the mediator in connecting all those geometrical aspect of the nucleus affecting the cell life
cycle. The actin stress fiber orientation seem to affect the long axis nucleus orientation leading
to the spindle axis orientation. It is also demonstrated that the actin stress fiber orientation gives
clue about the cell division axis on two-dimensional patterns. In this thesis, there will be also
a comparison between 2-dimensional and 2.5-dimensional environments. In 2.5-dimensional
environments, stress fibers orientate along the adhesive structures and the actin cytoskeleton is
distributed to support the floating nucleus in space. In addition, an indentation near the periphery
of the nucleus is observed.
Contents
Contents
Contents
Chapter 1
Introduction
Cells care how big or small they are because the basic processes of cell physiology, such as flux
across membranes are dependent on cell size. Therefore, changes in volume and surface area
will be directly affecting the metabolic flux, biosynthetic capacity, and nutrient exchange [1]. It
turns out that the cell size and shape is defined by the geometry of the adhesive region. The
information of the adhesive pattern can be interpreted by the cell through the organization of
their acto-myosin traction forces and passing this signal to the nucleus via actin cytoskeleton
[2]. Thus, the actin cytoskeleton has a strong coordination with the cell nucleus. This is
proved by many studies indicating that forces coming from the environment of the cell induce
the regulation of the cells gene expression [3]. The regulation of the gene expression due to
mechanical transmission is likely to occur due to a physical connection of proteins connecting
the nuclear lamina to chromatin (such as emerin [4]) and also other proteins that connect the
nuclear lamina to the cytoskeleton (SUN-KASH bridges) [5]. Instead of osmotic pressure that
may act on the nucleus, the tension generated by the cytoskeleton would alter the shape of the
nucleus and increases the nucleocytoplasmic transport [6], however, the nucleus to cell volume
ratio (karyoplasmic ratio) has to be constant for unknown reason [7]. Physical conformation of
the nucleus or the disrupted volume ratio is often associated with cancer cells. For example, the
nuclei of epithelial cells enlarge dramatically and become hyperchromatic (they stain abnormally
darkly with a contrast dye as a result of changes in their chromatin content) when they become
cancerous [8]. Furthermore, the nuclear volume changes might be affecting the concentration of
nuclear proteins, DNA, and RNA, and then it eventually would disturb the activities of RNA and
DNA polymerases [9].
In order to study the correlation between the actin cytoskeleton and the nucleus, it is important
to design an experiment that can mimic the in situ environments of cells. In situ, within
organ or tissues, cells behave totally different than in classical culture conditions. The cell
microenvironments, for instance the extracellular matrix (ECM) and the neighbouring cells,
provide a variety of cues (represented in red color in figure 1.1) for cell morphogenesis, ranging
from geometrical constraints to biochemical signalling and mechanical resistance.
However, when cells are cultured on flat, homogeneous and isotropic substrates, they will
completely lose those information and form a large number of random shape deformation since
they actively assemble and disassemble their cytoskeleton during migration. For this reason,
micropatterning techniques are used in order to engineer the environment that can mimic specific
7
Chapter 1. Introduction
Figure 1.1: In situ vs in vitro cell microenvironment [10].

microenvironmental cues. One important aspect about the physiological ECM network is that it
is fibrillar and heterogeneous, i.e. the ECM network does not completely surround the cells,
consequently there will be some free contact surfaces on cells. These circumstances can be
closely mimicked by using micropatterns of various shapes like a V shape with variety of angles
[10]. Cells plated on micropatterned substrates precisely adapt their cytoskeleton architecture
to the geometry of their microenvironment by remodelling of actin and microtubule networks
which further impacts cell migration, growth, and differentiation [2, 10]. And it has been
demonstrated that the geometry of the ECM plays many essential roles in the morphogenesis
of cells: in determining cell polarities, actin cytoskeleton distribution, cell shape and spindle
positioning [11, 2, 12].
As outlined above that the cell shape is determined by the adhesive pattern on which the cell
adheres that can be interpreted through the actin cytoskeleton. It turns out that one response that
cells give to these changes is by altering the spatial coordination of the cell-division axis with
cellular polarity and/or with the position of neighbouring cells which is crucial for embryonic
development, organogenesis and tissue homeostasis [13]. The former understanding of cell
division axis is based on Hertwig rule stating that the tendency of cells to divide is along their
long axis [14, 15]. However, recent understanding of cell division is based on the cortical cues
together with the retraction fibers that have been shown to be able to guide spindle orientation
[12]. And the cortical cues have connection with the location of the adhesive sites.
The discussion above shows how close the connection between the actin cytoskeleton with the
nucleus is. However, it is also important to show that the physical connection between them
alters the geometrical aspect of the nucleus in terms of cross-sectional area, elongation, long
axis orientation, surface area and volume. Therefore, the main goal of this thesis is to show
the changes of the nucleus geometry by comparing cells cultured in two-dimensional (2D) and
2.5-dimensional (2.5D) environments and trying to explain the effects to the cell life cycle.
In chapter 2 of this thesis, the principles of the actin stress fibers and cell-matrix contacts are
introduced as they are the main cell components that are measured. Furthermore an explanation
about the soft lithography and image processing methods that are used for the quantification
are given: the structure tensor and the image restoration. Chapter 3 contains the materials and
methods which were used for the experiments and the image processing tasks. The results and
the data analysis are given in chapter 4. Chapter 5 gives a discussion about the results and the
outlook. Finally, in chapter 6 the summary of the thesis is given.
10
11
12
Chapter 2
Theoretical Background
The actin cytoskeleton plays a key role in supporting the live of animal cells. It maintains a large
number of cellular processes within the cells including polarity establishments, morphogenesis,
and motility. The actin cytoskeleton also acts as the mediator that helps the cell to communicate
with its environment by changing its mechanical properties to carry the mechanical forces
coming from the environment to the cell nucleus. To modulate their mechanical properties, actin
filaments have capabilities to organize into a variety of architectures generating a diversity of
cellular organizations including branched or crosslinked networks in the lamellipodium, parallel
bundles in filopodia, and antiparallel structures in contractile fibers [16]. In this thesis, the actin
distribution and the stress fiber orientation are considered to be the main actor that links the cell
environment to the nucleus. This connection is described in the geometry changes of the nucleus
such as elongation, long axis orientation, area, volume, and surface area.
2.1
Actin Stress Fibers
As one of the actin cytoskeleton architecture, actin stress fibers are the major mediators for
cell contraction. They are composed bundles of 10-30 actin filaments [17]. The bundles are
tightened with the help of actin-crosslinking proteins like -actinin, fascin, espin, and filamin
[18]. -actinin crosslinking proteins held the actin filaments in parallel arrays as displayed in
figure 2.1a.
In order to enable the sliding contraction between each block of bundled actin filaments, it turns
out that they should have polarity to its successive blocks so that the myosin motors which
are interconnected within the bundles can slide between the bundles to cause contraction or
retraction. This polarity is represented in terms of a plus (barbed) end and a minus (pointed)
end and the myosin motors move towards the barbed end. The distribution of polarity could be
uniform or random [17, 18].
Furthermore, the actin stress fibers also play an important role in mechanosensing. Contractile
forces generated by stress fibers regulate the assembly and dynamics of focal adhesions. Cells
assemble stress fibers only when they encounter mechanical stress (force). Most animal cell
types form stress fibers and focal adhesions which are aligned along the major cell axis when
they are grown on a rigid matrix. When they are grown on compliant matrix, focal adhesions
are smaller and stress fibers are poorly aligned. This is demonstrated in figure 2.2. Cells on soft
13
14
Chapter 2. Theoretical Background
(a) Models of stress fibers structure and contractility [18]
(b) Model of stress fibers formation [18]
(c) Types of stress fibers in cultured animal cells [19]
Figure 2.1: Actin stress fibers structures
substrates have a diffuse cytoskeleton, composed of near random arrangements of actin filaments.
In contrast, cells on stiff substrates contain many stress fibers, aggregations of actin , and other
proteins that slowly contract under the influence of nonmuscle myosin II [19].
2.2. Cell-Matrix Adhesion Contacts
15
Figure 2.2: Effect of substrate stiffness on stress fibers. Left figure shows fibroblast cells plated
on a rigid (2 MPa) substrate display thick and well aligned stress fibers. Right figure shows cells
plated on a compliant (5 kPa) substrate display thinner and poorly oriented stress fibers [19].
Formation of Actin Stress Fibers
Actin stress fibers are formed into at least four different categories in accordance with their
morphology and association with focal adhesion and briefly shown in figure 2.1c [19]:
1. Dorsal stress fibers
Dorsal stress fibers are anchored to focal adhesions at their distal ends. They typically
do not contain myosin II, and therefore it cannot contract. However, dorsal stress fibers
appear to serve as a platform for the assembly of other types of stress fibers, as well as to
link them to focal adhesion.
2. Transverse arc stress fibers
This kind of stress fibers have a curved shape bundles displaying a periodic -actininmyosin pattern which is typical for contractile actomyosin bundles. They do not directly
attach to the surrounding environment, but they could transfer contractile force to the
connected dorsal stress fibers and therefore the forces still can be transferred to the
surrounding.
3. Ventral stress fibers
The most distinguished characteristic of these fibers is that they have attachments to focal
adhesion at both ends. Ventral stress fibers are often located at the posterior part of cells
where occasional contraction cycles promote rear constriction and facilitate cell movement.
4. The perinuclear cap stress fibers
These stress fibers are located above the nucleus and have the function to regulate the
shape of the nucleus in interphase of cells. They also have a function to convey force from
the environment to the nucleus and therefore acting as a mechanotransducer. Some certain
stress fibers also have a connection to the nucleus through specific membrane proteins to
stabilize the position of the nucleus and to regulate cell movements.
2.2
Cell-Matrix Adhesion Contacts
Cells can attach to their ECM with the help of matrix adhesion contacts on their cell surface
which contains integrin receptors as their main transmembrane proteins. The assembly of
16
cell-matrix adhesions are initiated at protruding cell edges, and the adhesions to the ECM
is initiated within lamellipodia or filopodia at the cell periphery [20]. The outer parts of the
matrix contacts (extracellular domains) bind to specific ECM components (like fibronectin) with
the help of distinct integrin heterodimers (like v 1 ). Whereas the inner parts (cytoplasmic
tails) connects the F-actin cytoskeleton with the help of adaptor proteins (figure 2.3a). The
physical connection of the F-actin cytoskeleton with the ECM regulates the adhesion by dynamic
assemblies of structural and signalling proteins that couple the cytoskeleton and the ECM [21].
There are 4 phases of matrix contacts maturation that can be distinguished, namely nascent
adhesions, focal complexes, focal adhesions (or focal contacts), and fibrillar adhesions (figure
2.4). Adhesion of a protruding cell edge is initiated by the nascent adhesion whose size is
smaller than 0.25 m. A subpopulation of the nascent adhesions disassembles within minutes as
the leading edge advances. Whereas the remainder grow and mature into focal complexes whose
size is about 5 m and mature further into focal adhesions [21].
The characteristics of focal complexes are described by their size which is small (<0.5m
diameter) and they are dynamics (figure 2.3a and 2.4). The ECM proteins which bind to these
complexes are recognized primarily by vitronectin receptor v 3 via their RGD peptide motifs
[22].
Focal adhesions (FA) are sites where cells in culture adhere strongly to the underlying ECM
via specific members of the integrin family of ECM receptors [23]. FA contains a high level
of tyrosine-phosphorylated proteins such as vinculin, talin, paxillin, tensin, the tyrosine kinase
FAK, and many others compared with other cell compartments and as a consequence of that, FA
has function both as adhesion and signal transduction organelles, informing cells about the state
of the extracellular matrix (ECM) [24, 23]. The focal adhesions then either disassemble within
the cell body within 10-20 minutes [25, 21], or mature further into stable fibrillar adhesions that
do not promote migration but are involved in ECM remodelling [26, 21].
The detail process of the adhesion maturation is not well defined yet. However, a brief process
can be sufficiently depicted in figure 2.5. The microcluster of integrin which is less than 10
micromolecules is believed to be the initial stage of the focal adhesions [21]. Then cells enhance
the affinity of integrin extracellular heads for the ECM (integrin activation) at their surface by
signalling from within the cells (inside-out signals) [27]. Inside-out activation of the integrin
heterodimer involves conformational changes of its ectodomain that are triggered by binding of
adaptor proteins talin and kindlin to its cytoplasmic tails [22]. In lamellipodium, this microcluster
of activated integrin move retrogradely together with F-actin [20]. The activated integrins then
either bind to the ECM or associate with the dynamic actin at the leading edge [21]. The
integrin binding to the ECM promotes the recruitment of paxillin to integrin clusters and once the
mechanical stable contacts are established, the adhesions will grow further into focal adhesions
[22].
The adhesion maturation may occur when the nascent adhesions are stimulated mechanically
[28]. The promotion of the adhesion growth due to the tension are thought as the results of
the conformational changes that unmask the protein binding sites and therefore increase the
protein binding probabilities when the tension is applied [29]. The binding of new proteins to
2.2. Cell-Matrix Adhesion Contacts
(a) Formation of focal complexes
17
(b) Formation of focal adhesions (or focal

contacts) which mature from focal complexes
(c) Fibrillar adhesions are pulled out from

focal contacts in the process of extracellular
fibronectin matrix assembly
Figure 2.3: Schematic representation of matrix adhesion contacts commonly found in fibroblasts
cultured on 2D substrates. Nascent adhesions are not shown here [22].
Figure 2.4: Schematic diagram of morphological phases of adhesion maturation. Curved black
arrows show the adhesion turnover after a certain amount of time [21].
the unmasked sites results in the focal adhesions growth and reinforces the linkage between the
ECM and the cytoskeleton [21].
18
(a) Side view schematic. (1) The lamellipodium (LP) drives nascent
adhesions assembly at the leading edge cell. (2) Nascent adhesions is
getting mature by recruiting adaptor proteins (such as talin and kindlin,
results in the enhancement of the linkage strength to the F-actin) and
ECM molecules (results in the increased size of the adhesion spots).
(3) The adhesion spots further mature which occurs within the lamella
(LM).
(b) Top view schematic. TA denotes transverse arcs which are parallel
to the cell edge and the LP-LM boundary. SF denotes stress fibers
which are anchored in focal adhesions and in perpendicular to the cell
edge.
Figure 2.5: Schematic diagram of adhesion assembly stages [20].
2.3
Microcontact Printing
Microcontact printing (P) is one of the best methods to create a micrometer or submicrometer
sized pattern on a variety of substrates [30]. It is done by coating molecular ink to an
elastomeric stamp having a raised relief pattern. Then by bringing the raised areas of the stamp
into contact to the substrate surface, the ink then will be transferred from the stamp to the
substrate and form self assembled monolayers (SAM).
According to [30], there are 3 kinds of stamp deformations that result the most undesired
consequences:
1. Lateral sticking of high aspect ratio plates
This kind of deformation occurs when the capillary forces due to retained liquids on the
plates are strong enough to cause them to come into contact. Once contact occurs, plates
may stick to each other as a result of surface adhesive forces.
2.4. Image Formation
19
2. Buckling with high aspect ratio plates

Buckling might happen if the aspect ratio (h/2a) is too large so that the plates can collapse
when loaded or even under their own weight.
3. Roof collapse of low aspect ratio plates (raised regions of the stamp that have the pattern)
Roof collapse might happen if the aspect ratio is too small so that the recessed area of the
stamp and the raised area can be deformed and make a contact with the substrate surface.
(a) Stamp with rectangular cross section
(b) Buckling
(c) Lateral Collapse
(d) Roof Collapse
Figure 2.6: Micropattern stamp and its failure modes [30]
It has been shown that if the aspect ratio is too high, the plates can collapse when loaded or even
under their own weight (buckling).
2.4
Image Formation
Generally, people are familiar with an image as a recorded picture captured by an optical
imaging system such as a camera, a microscope, a telescope, etc. That recorded picture contains
information about the physical object that is being observed. Therefore, speaking formally, an
image can be defined as a signal carrying information about a physical object [31]. The image
received by any imaging system (a microscope in particular), however, does not resemble the
whole object and all original information contained in it due to the diffraction limit. Moreover,
there is still another strong factor which is adding to the degradation: noise. The degradation
then can be caused by two main sources as described in [31]:
1. Degradation because of the process of image formation
The associated degradation is called blurring
20
2. Degradation because of the process of image recording.

The associated degradation is called noise
Based on the degradation definition above, the schematic of the image formation path where
the signal is coming from the object until being recorded can be clearly depicted in figure 2.7.
By definition, the image is considered as a noise-free (but blurred) image after passing through
the imaging system. Then, after being recorded by the recording system, noise is added to the
image signal resulting a noisy image.
In this thesis, the imaging is considered as incoherent because in fluorescence microscopy there
is no fixed phase relationship exists among the fluorescent molecules that compose the object
[32].
Figure 2.7: Image formation pathway from the object until being recorded [31].
2.4.1
Degradation Due to the Blurring Process
As outlined previously, blurring is a degradation due to the diffraction limit problem (frequency
limited problem) of the imaging system in collecting all the information contained in
the object. It is also important to point out that the blurring process happens during the
imaging process and before detection by the sensor. Hence the formed image is a noiseless image.
It turns out that the diffraction limit is not the only main cause of the degradation. It
should be pointed out that other degradation such as the motion which is happening during
capturing the image would also contribute to the degradation. Other examples are aberrations
produced by the lens and atmospheric disturbances. In order to simplify the problem,
21
the degradation which are considered in this thesis are only due to the diffraction limit
problem and can be modelled by the point spread function (PSF) of the optical system
(in this case is the microscope). The lens is also assumed to be free of manufacture
defects. Motion blurring can be neglected since the microscope is firmly embedded to its
stage. Whereas the atmospheric disturbances can also be neglected because the temperature
is relatively constant and the distance between the object and the optical system is relatively short.
The diffraction limit problem cannot be avoided, even if the lenses of the optical system have
been produced perfectly in such a way that no aberration takes place. The Diffraction limit
problem comes from the failure of the optical system to collect all the frequency information
contained in the object. In order to understand which frequency information the object has, it is
helpful to understand Fourier optics in which the signal is analyzed in the frequency domain.
From this point of view, an image is considered as a superposition of harmonic functions of
different frequencies.
To understand Fourier optics, one should start from the Fourier series. According to the Fourier
theorem, any periodic signal can be expressed as a weighted linear combination of harmonic
functions (sine and cosine in particular) having different frequencies. Non-periodic signals
however can be expressed as the integral of sines and cosines multiplied by a weighing function
as long as the area under the curve of that signal is finite. Fourier series are given mathematically
by equation 2.1.
g(x) =
=
X
n=0
an cos
2nx
2nx X
bn sin
+
n=0
an cos kn x +
n=0
bn sin kn x
n=0
where
g(x) = signal in spatial domain
= spatial period
kn = spatial frequency
2
=
an = Fourier coefficient
(2.1)
/2
2 Z
=
g(x) cos kn x
/2
bn = Fourier coefficient
/2
2 Z
=
g(x) sin kn x
/2
Consider as a simple example the expansion of a unit-step function into Fourier series depicted
in figure 2.8. Notice that as the expansion takes more and more terms into account, the expanded
signal is approaching the original signal. It can be explicitly stated that the expanded signal
22
which has more terms contains more higher frequency information than the one that has less
terms. Signals that have less terms only contains lower frequency information of the original
signal.
Figure 2.8: Expansion of a unit-step function using Fourier series [33].

A lens actually performs a Fourier transform to the objects to be imaged. And people can observe
the image with the help of an objective lens which perform an inverse transform. Images with
higher frequency components and lower frequency components are compared in figure 2.9. From
that comparison, one can immediately distinguish the differences of the high-pass and low-pass
filtered image. When the image is low-pass filtered, only the lower frequency components of the
object are allowed to pass the imaging system. The lower frequency components correspond to
areas with little variation in intensities (like background). This results a blurry image denoting
that the finer structure are lost and therefore the resolution is decreased. In the image that has
been high-pass filtered, the finer details of the image look more prominent (high contrast) but
halos are observed around the edges.
The description of images in the frequency domain has been explained briefly. Now it comes to
the point of giving the reason why the image is degraded. Recall in the previous section that the
blurring process is defined as the degradation that occurs during the process of image formation
in the optical imaging system before being recorded by the recording system and caused mainly
by the limitation of the imaging system to collect all the information contained in the object. This
limitation however caused by the finite dimension of the entrance pupil of the imaging system
(spatial limitation) to let all the incoming signal in the form of optical wave passing through the
system. This situation is explained beautifully by Goodman, [35] (see figure 2.10) and will be
repeated briefly here.
(a) Original image without frequency components

distortion
(c) High-pass filtered image
23
(b) Low-pass filtered image
(d) Band-pass filtered image
Figure 2.9: Image comparison showing how the image looks like when the frequency
components are distorted [34]
Figure 2.10: A generalized model of an imaging system [35].

The imaging system in figure 2.10 is assumed to be able to produce real images in space. The
black box in the system is a lumped system containing a set of lens system. The most important
parts of the black box are the terminal input and output that are represented as the entrance and
exit pupils. To represent imaging system as a black box, one needs to know the mapping between
an input distribution (object) and an output distribution (image of the object) by neglecting the
complexity of how the imaging system is built.
Diffraction comes to play in the passage of light through the black box. From the fact that
the pupils are limited in size, there will be some information of the object that cannot be
transferred completely to the image plane (u-v plane in the figure 2.10). This is the main reason
of diffraction limit. This situation further gives bad impact to the resolution of the output.
24
Ernst Abbe (1873) gave an explanation about this limited resolution during his studies of
coherent imagery with a microscope which is depicted in figure 2.11 for the case of an object
that is a grating with several orders. He gave his famous formula showing the connection
between a wavelength and the numerical aperture NA (equation 2.2).
d=
2N A
(2.2)
Figure 2.11: Diffraction limited according to Abbe theory [35].
Figure 2.11 shows that due to the limited size of the lens, not all diffraction order can be
collected by the lens. These uncollected information is in the higher order of the diffraction
pattern and corresponds to the high-frequency components of the object.
This limitation also plays a role in biological imaging. The size of biological samples are in
the range of micrometers and nanometers, therefore diffraction can also play a role during the
imaging of the sample. Accordingly, the limited resolution can be explained in the same way as
before. And this causes the image that is formed by an imaging system looks blurry before being
recorded by the recording system (see figure 2.7).
How blurry the image formed by the imaging system is, is determined by the so called as point
spread function (PSF). This function describes how a point object in the object plane will look
like in the image plane. Thus, it specifies the behaviour of the imaging system. Additionally,
the image will be also affected by noise. An example of PSF is given in figure 2.12. The lateral
bandwidth of the PSF is around three times smaller than the axial bandwidth, meaning that the
lateral resolution is roughly three times better than the axial one.
The behaviour of the imaging system in describing the object is mathematically given in equation
25
Figure 2.12: PSF of 200nm bead taken with Confocal Laser Scanning Microscope
(LSM510,Zeiss) at KIT, Institute of Zoology. The protocal for taking the PSF measurement is
based on [36].
2.3 and a schematic representation is given in figure 2.13.
g(x, y) =
Z Z
f (x0 , y 0 )h(x, y; x0 , y 0 )dx0 dy 0
where
g(x, y) = image function in the image space (x,y)
h(x, y; x0 , y 0 ) = point spread function (PSF)
f (x0 , y 0 ) = object function in the object space (x,y)
(2.3)
Figure 2.13: A Model of the image degradation and restoration process (adapted from [37]).
The equation 2.3 points out that the response g(x,y) in the image plane (x,y) of the imaging
system is given as the result of a weighted sum of all input points described by f(x,y) in the
object plane. It means that the output located at (x,y) in the image plane is affected by all points
26
in the object plane weighted with the PSF h(x, y; x0 , y 0 ). In describing the image of the object,
most imaging systems obey 2 important rules which are of primary importance in simplifying the
imaging process both analytically and numerically. Those rules are linearity and shift-invariance
(or space-invariance) rules. Under those two rules, then the imaging equation 2.3 becomes:
g(x, y) =
Z Z
f (x0 , y 0 )h(x x0 , y y 0 )dx0 dy 0
(2.4)
It is clear from equation 2.4 that a large spreading of the PSF would contribute to a severe
blurring of the image since many points in the object plane contribute to one point in the image
plane. Consequently, the resolution of the image is reduced. Besides blurring, other factors
contributing to influence the degradation are the background originating from autofluorescence,
scattering and offsets in the detector gain.
2.4.2
Degradation Due to Noise
Noise discussed in this section is caused by photodetectors (the camera of the microscope). It is
obvious that noise reduces the quality of the detected signal. Therefore, a good modelling of
noise is one important step to improve the quality of the observed image. There are however,
two types of noise in terms of physical nature: quantum noise (or shot noise) and thermal noise.
Quantum noise results from the statistical nature of a quantum event due to particle characteristic
of photons. Whereas thermal noise, also known as Johnson noise or Nyquist noise in electronics
and photonics, is the consequence of thermal fluctuations and is directly associated with thermal
radiation [38]. Quantum noise is of importance in this thesis. Thus, equation 2.4 can be further
refined:
g(x, y) =
Z Z
f (x0 , y 0 )h(x, y; x0 , y 0 )dx0 dy 0 + n(x0 , y 0 )
(2.5)
where
n(x0 , y 0 ) = noise function
The photons emitted by the object are not distributed uniformly in time, but arrive at the detector
randomly in time. This random behaviour causes the optical signal and the number of photons
received in a given time interval to fluctuate around the average values which are characterized
by the Poisson statistics. Therefore, the first contribution of noise to the observed image is due
to the radiation emitted by the object in a Poisson process. The Poisson distribution is given by
equation 2.6. For low intensities, the expected value is near to one, and therefore the fluctuation
is greater at this intensity level, causing the intensity changes from one pixel to the next.
p(n) =
e n
n!
where
= Expected value of the count
n = Number of particles
(2.6)
2.5. Image Restoration
27
The second contribution comes from the background emission due to the auto-fluorescence of
the medium embedding the sample. This emission is again following Poisson distribution [39].
Other sources of noise, for instance, dark current, electronic noise, and by reflections of the
excitation light are also following Poisson distribution [40].
2.5
Image Restoration
Because degradation due to blurring and noise affects the accuracy of quantitative measurements
of the image, the distortion of the image signal needs to be fixed. Image restoration refers to the
attempt of giving solutions to this problems.
Again, coming back to the imaging equation 2.4, provided that the knowledge of the PSF and the
statistical characteristic of noise are given, principally the true object function f(x,y) can be
reconstructed. This kind of approach is also known as deconvolution, although deconvolution
can be regarded as subfields of image restoration. However, deconvolution more refers to as
techniques that invert the blurring process in a deterministic way [32].
Formally speaking, deconvolution is an computational approach based on objective criteria to
improve the quality of an image through the knowledge of physical processes that leads to image
formation on the screen (focal plane) [41, 33, 42]. The concept is to reconstruct the degraded
observed image by using a priori knowledge of how the image is degraded during the image
formation and then apply an inverse process (therefore its called de-convolution) to get the
original object [43]. The attempts to find the estimated object from the observed image is also
known as inverse problem.
An inverse problem is the opposite of forward problem. For examples in Newtonian classical
mechanics, where the equation for getting the force applied to an object is the mass multiplied
by its acceleration during its motion (F = ma). In the forward problem, the mass m and the
acceleration a of the object are given. Thus, one can get straightforwardly the force which is
applied to that object that causes it to be in motion. Whereas in inverse problem, one is only
given the force. Then the task is to estimate the mass m and the acceleration a of the object.
Consider another example of the black box of the imaging system that mentioned before. The
forward problem is that the true object is given and are to be imaged by using the black box
which will generate an image according to the parameters of the black box. Now the inverse
problem is that the output image and the parameters of the black box are known, but the input
object needs to be found. This task is obviously more difficult than the forward problem because
there might be no solutions for this.
A convenient way to do the inverse calculation is by Fourier-transforming the imaging equation
so that the integral calculation can be treated with a simple arithmetic calculation (however, this
is a naive approach since it cannot be applied to all frequency range). One of the tasks of Fourier
transform is to simplify the calculation by bringing the calculation into frequency domain in
this case. So, to put them together mathematically, the imaging equation in frequency domain is
28
given by equation 2.7.

G(kx , ky ) = H(kx , ky )F (kx , ky ) + N (kx , ky )
G(kx , ky ) = G0 (kx , ky ) + N (kx , ky )
G0 (kx , ky ) = H(kx , ky )F (kx , ky )
(2.7a)
(2.7b)
(2.7c)
where
G(kx , ky ) = Fourier transform of the recorded image
F (kx , ky ) = Fourier transform of the true object
H(kx , ky ) = Fourier transform of the PSF (Optical Transfer Function / OTF)
G0 (kx , ky ) = Noiseless image
N (kx , ky ) = Fourier transform of the noise
The band-limited problem of the imaging system is described by the OTF H(kx , ky ) which
is zero outside a frequency band domain . The frequency band is therefore called the
bandwidth of the imaging system or the cut-off frequency of the optical system. Thus,
given that the OTF is band-limited, obviously the noiseless image G0 (kx , ky ) is also bandlimited. This property implies that the imaging system is behaving like a low-pass filter,
meaning that it only allows low frequency signal of the object to be transmitted to the image plane.
It turns out that noise is not band-limited in the sense that it exists in all frequency range.
Consequently, although the noiseless image is band-limited, the observed image does not have to
be band-limited because of the addition of noise term according to equation 2.7b.
2.5.1
Non-iterative Image Restoration
The task of deconvolution is to estimate an image Fb to be as close as possible to the true object F .
If the noise is small, no background (only signal of interest comes to the detector) and provided
that the PSF is known, then the observed image can calculated by using equation 2.7a as follows:
G(kx , ky ) = Fb (kx , ky )H(kx , ky )
(2.8)
where
Fb (kx , ky ) = Fourier transform of the estimated object
Equation 2.8 already shows the difficulties in image deconvolution. As explained before
the PSF is basically band-limited and the observed image is generally not. Therefore,
outside the frequency band , there will be inconsistencies happening. The solution of the
equation might not exist or be not unique. This kind of error is often referred as ill-posed problem.
Ill-posed problem can be described in three words: existence, uniqueness and stability. The
stability here means that small perturbation would lead to big errors in solving the inverse
problem. Then, by substituting equation 2.7a into 2.8 and solving for the estimated object Fb ,
29
yields:
Fb (kx , ky ) = F (kx , ky ) +
N (kx , ky )
H(kx , ky )
(2.9)
As an example of how this type of deconvolution works is given in figure 2.14 which is showing a
NIH3T3 cell adhering on a micropatterned substrate with crossbow shape. This image was taken
with wide field fluorescence microscope. The inverse filtering was done under assumptions that
the PSF has Gaussian form with lateral sigma value 0.25 m and without noise. The Gaussian
functions used in this thesis have the form given in equation 2.10.
(x2 + y 2 )
2r2
h(x, y) = e
r2 z 2
2
2
h(r, z) = e 2r e 2z
F W HMr = 2 2 ln 2r
F W HMz = 2 2 ln 2z
(a) Original image
(b) Actin filaments in grayscale
(2.10a)
(2.10b)
(2.10c)
(2.10d)
(c) Actin filaments after inverse filtering
Figure 2.14: Image comparison showing how the image looks like after the inverse filtering
method. The image is showing a NIH3T3 cell adhering on a crossbow micropatterned substrate.
It is obvious that the result of the inverse filtering cannot be used for further quantification
analysis. The result cannot be recognized (2.14c). The main reason of this is because of the
unknown properties of the microscope PSF which results the amplification of noise. As stated
before, the computation of inverse filtering is in the whole range of frequencies. Since the PSF
was generated by assuming that it has Gaussian form, therefore it has a cut-off frequency. Noise
is not band-limited while the PSF is band-limited. The noise can have low values (even zero) in
the high frequency regime, once the inverse filtering is applied, the division by low values would
give greater values (unstable solutions) to the estimated image Fb . Furthermore, the noise term in
30
equation 2.9 would be more dominant in high frequency regime.

The previous discussion makes clear that the band-limited problem leads to serious problems
in image deconvolution. The incapabilities of the imaging system to convey the true object
information at all frequencies (plus the degradation due to noise and background) give no unique
solutions to the imaging equation for estimating the true object. Concretely, this naive approach
cannot reconstruct certain information at certain frequency which in fact does not exist in the
image plane due to the failure of transmitting this information. To be more precise, the imaging
system fails to transmit complete information about the Fourier transform of the object at certain
frequencies. Following the statement taken from this reference [44, 31], A lack of information
cannot be remedied by any mathematical trickery.
In order to solve that ill-posed problem, one must reject the concept of an exact solution of the
imaging equation and look for approximate solutions, i.e. objects which reproduce approximately
the noisy image (observed image). The second approach is to use the knowledge of additional
properties of the unknown object for selecting from the set of approximate solutions those which
are physically meaningful (also known as a priori information) [31].
The Wiener-Helstrom filter
Although this type of filter is not used in this thesis, a brief explanation of this filter would
be a good introduction to understand the problem of deconvolution better. As discussed in
the precious section that the incomplete information transmission of imaging system leads to
band-limited problem. As the result of this incomplete information in the image plane, it is
very difficult to get back the true object form the observed image. Since the direct approach of
deconvolution by inverse filtering is trying to solve the problem in the whole frequency domain,
it turns out that this approach induces ill-posed problem which then yields unsatisfying result.
It turns out that one solution to avoid the ill-posed problem is by introducing a modified filter
WH (kx , ky ) (see equation 2.11) that satisfies the following conditions:
1. At the high frequency regime, the filter should be able to avoid division by zero in order
to prevent noise amplification. This is done by setting the modified filter to zero. Since
in the high frequency regime the signal information is dominated by noise, setting the
modified filter to zero will also prevent restoring information which does not belong to the
true object.
2. At the low frequency regime (regime where the noise component is much smaller than the
object signal), the modified filter should be able to recover the true object signal.
3. At the frequency regime where the noise signal and the object signal are comparable, the
modified filter should be able to make a compromise between complete acceptance and
total suppression of the noise.
Fb (kx , ky ) = WH (kx , ky )G(kx , ky )
= WH (kx , ky )[H(kx , ky )F (kx , ky ) + N (kx , ky )]
(2.11)
31
The Wiener-Helstrom filter has the capabilities to fulfil those requirements. For brevity, the
Wiener-Helstorm filter is defined as:
WH (kx , ky ) =
H (kx , ky )
|H(kx , ky )|2 + N SR(kx , ky )
where
N SR(kx , ky ) = Noise to Signal Ratio
WN (kx , ky )
=
WF (kx , ky )
WN (kx , ky ) = Noise power spectra
= h|N (kx , ky )|2 i
WF (kx , ky ) = Input power spectra
= h|F (kx , ky )|2 i
(2.12)
The Wiener-Helstrom filter is a least square solution of an optimization criteria which minimizes
a cost function that is defined in equation 2.13. As given in equation 2.12, the term NSR enables
the filter to accept low-noise frequency components and reject high noise frequency components.
Figure 2.15 is an illustration of how this deconvolution method works.
Q = h
Z Z
[Fb (x, y) F (x, y)]2 dxdyi
(2.13)
(a) Original image
(b) Actin filaments after applying

Wiener-Helstrom filter with NSR =
15.5437
(c) Actin filaments after applying

Wiener-Helstrom filter with NSR =
100
Figure 2.15: Image comparison showing how the image looks like after Wiener-Helstrom
filtering method. The PSF is assumed to have Gaussian shape with lateral sigma 0.25 m and
distorted by random noise whose NSR is 15.5437 and 100. The image processing was done with
Matlab.
Figure 2.15 clearly shows that the deconvolution works much better compared with the
32
inverse filtering method. The PSF is again assumed to have a Gaussian shape with lateral
sigma 0.25 m. As for comparison, the term NSR was generated with Matlab with values
15.5437 and 100. When the NSR is 15.5437, the deconvolved image subjectively seems to
have more and more detailed structures compared to the original image. But parts of the
image where no cell is adhering seems worse. Moreover, it has a lot of artifacts like the
halos pattern along the boundary of actin filament curvature which makes more difficult
to analyze. When the NSR is set to 100, the background seems better, but it has less
detail structures. The main reason of this variation is because the nature of the PSF of the
microscope and the noise are unknown. They were just given as an assumption, not the true value.
As stated in equation 2.12 that Wiener-Helstrom filter needs the power spectra of the true object
which is not directly provided. Thus, the Wiener-Helstrom filter cannot be precisely implemented
in order to get the estimation of true object. But it has given a lot of improvement compared with
the previous generation of deconvolution.
2.5.2
Statistical Image Restoration
The previous discussion made clear that one needs another approach to reconstruct the true object
from the observed image. The non-iterative approaches still induce ill-posedness, implying
that there might be no solution or no unique or no stable solution of the imaging problem.
Therefore, its necessary to have additional information as a way to reduce the uncertainty of the
approximate solutions. To do this, ones must reformulate the problem by taking into account all
the available information both on the process of image acquisition (noise) and on the object
itself (a priori information, such as non-negativity). The first step in the reformulation of image
deconvolution is to model the noise which is corrupting the data [45].
Before modelling the noise, it is appropriate to introduce the matrix formulation (digitized
version) of equation 2.5 since in digital computing, continuous signal will be converted into
digital signal for further process in a computer. The discretized version of the imaging equation
is given as follows:
y = Hx + b
(2.14)
Where y = {yi }iS is the output or image column vector containing all the pixels in the image,
where i is a multi-index of the image, and S is an appropriate range of i. x = {xj }jR is the
input column vector, where j is a multi-index of the object, and R is the range of j. H is the
discrete form of the PSF, describing the transfer of each pixel in the input to each and every
pixel in the output. Also known as a transfer or imaging matrix. b is background column vector.
The cardinality of S and R doesnt have to have the same dimensions. Moreover, the imaging
33
matrix H has to satisfy the following conditions [46]:

Hi,j 0,
X
Hi,j > 0,
iS
j R,
X
Hi,j > 0,
(2.15)
jR
i S
Equation 2.15 means that for each fixed value of the index or multi-index i or j, there exists at
least one non-zero entry. The matrix H is also assumed to satisfy the normalization conditions:
X
Hi,j = 1, j R,
iS
(2.16)
in case the value of vector b is 0, then it implies that the total number of photons is the same in
the original object and in the image, or the object and the image have the same total flux [46].
In case of Poisson noise, the detected value yi of the image g is the realization of a Poisson
random variable (RV) Yi with the expected value (Hx + b), i.e.
Yi P oisson(Hx + b)i
(2.17)
By assuming statistical independence of the RV Yi (because each pixel in a recorded image is

statistically independent from the others), the probability distribution of Y, for given H, x and b,
is equal to the product of individual probabilities, which is given as follows [47]:
pY (y; x) =
m (Hx+b)i
Y
e
(Hx + b)yi i
i=1
yi !
(2.18)
Equation 2.18 can be used to find an estimate xb of the unknown object corresponding to the
observed image y. Since the probability density pY (y; x) of the data is assumed to be known, and
in this density, the unknown object appears as a set of unknown parameters, then the estimation
of the object reduces to a parameter estimation problem which can be calculated by using the
so-called maximum likelihood (ML) approach by introducing the likelihood function defined by:
LYy (x) = pY (y; x)
(2.19)
Thus, the solution of the estimated object xb is the one which maximizes that likelihood function,
i.e.
xb = arg max
LYy (x)
(2.20)
xRn
Moreover, the likelihood function above is more convenient to be calculated in negative log
form (neglog) since there is a large number of factors. Hence, the maximization problem is
34
transformed into a minimization problem which is given as follows:

xb = arg minn J0 (x; y)
J0 (x; y) =
xR
log LYy (x)
(2.21)
In case of Poisson noise, the function J0 (x; y) is given by the so-called Kullback-Leibler (KL)
divergence (or Cziszr I-divergence), defined by:
DKL (y (1) , y (2) ) =
m
X
(1)
(1)
{yi ln
i=1
yi
(2)
yi
(2)
(1)
+ yi yi }
(2.22)
Hence
J0 (x; y) = DKL (y; Hx + b)
m
X
yi
=
{yi ln
+ (Hx + b)i yi }
(Hx + b)i
i=1
(2.23)
Scaled Gradient Projection (SGP)

A scaled gradient projection method is a class of iterative image restoration that lead to minimize
convex non-linear functions subject to non-negativity constrains and flux conservation constraint
[48]:
min J(x)
sub. to x 0
or
min J(x)
(2.24)
sub. to x 0
N
X
xi = c
i=0
Where J(x) is a Continuous Differentiable Convex Function measuring the difference between
reconstructed and measured data and possibly containing a penalty term expressing additional
information on the solution. The convergency of this method is provided in the same paper [48].
The objective function is the Kullback-Leibler divergence:
J0 (x; y) = DKL (Ax + bg, b)
=
N
X
N
X
i=0 j=0
N
P
Aij xij + b bi bi log(
Aij xij + bg
j=0
bi
(2.25)
))
2.6. Structure Tensor
2.6
35
Structure Tensor
The detection of edges and lines has many application in image processing since it provides a
way to recognize an object in an image. Patterns in an image can be recognized because of the
spatial brightness changes that allows to model the direction of the neighbourhood by applying a
gradient operator that are sensitive to the changes of the brightness. However, it turns out that
determining the orientation by just merely using gray scale value changes would not be robust
against noise [49]. Therefore, another approach needs to be developed that is able to determine
a unique orientation. The structure tensor is a suitable representation that can determine the
orientation of a local neighbourhood. Measuring the orientation of actin stress fibers is done
with the help of the structure tensor. The basic idea is to detect the gradient image and determine
the dominant local orientation of the image gradient.
The structure tensor evaluates the local orientation in a small region of an image [50, 51].
Orientation here means the direction of a vector perpendicular to the lines of constant gray
values. This technique can be used to determine the presence and orientation of edges, the
orientation of texture, and the velocity in image sequences [52].
Firstly, orientation has to be defined since it will conflict with the definition of direction. The
angle of orientation in the structure tensor calculation is defined as the least deviation from the
image gradient [53, 52]. The gradient vector of a structure in an image always points towards the
higher intensity object (to brighter or whiter location in an image). The gradient is represented
as a vector, which can be represented with a magnitude and an angle ranging from 0 to 360
degrees. However, the case is different for orientation. The angle of the orientation is only
defined in the range of 0 to 180 degrees. Assuming that there is a boundary marked by black and
white like shown in figure 2.16. Then the gradient will be pointing towards the white region,
and the orientation is also pointing to the same direction as the gradient. Now if this image is
rotated 180 degree, then the gradient will be pointing towards the opposite direction (downward).
However, the orientation will not change. Because what is important information about the
orientation is that the boundary marking off black and white is still lying in the same direction
(horizontal). Therefore, it is sufficient to limit the range of orientation angle up to 180 degree.
Although the formal definition of the structure tensor orientation demands a parallel situation
with the intensity gradient vector, it can however be converted to be perpendicular with the
gradient vector. This is done for the sake of simplicity in observing the actin stress fibers
orientations together with the nucleus long axis orientation (see Section Experimental Methods
for the discussion). Figure 2.17 demonstrates how the orientation is represented in the structure
tensor calculation used in this thesis.
Let f (r0 ) denotes a two-dimensional image, with spatial dimensions in r0 = (x, y). Assuming
that f is oriented in a region . Since the orientation vector u = (cos(), sin ) should have
the least deviation from f , it means that the dot product between those two vectors is to be
maximized:
max(f (r0 ).u)2 = max(|f (r0 )|2 |u|2 cos2 (f (r0 ), u))
(2.26)
Thus, the dot product will be maximized if the orientation is parallel or anti-parallel with the
36
(a) A Bundary represented as black and white
(b) Rotated image
Figure 2.16: Gradient vector showing the direction from black region to white region.
gradient vector and will be the lowest when they are perpendicular each other. Finding
(denoting the orientation) is done by maximizing the integral of that dot product over the whole
frame of the image (within the local neighbourhood ) [54], meaning:
E(u) = max
kuk=1
= max
kuk=1
= max
kuk=1
= max
kuk=1
(uT f (r0 ))2 d

(uT f (r0 ))(uT f (r0 )T )d
(uT f (r0 ))(f (r0 )T u)d
(2.27)
uT (f (r0 )f (r0 )T )u)d
= max uT [
kuk=1
f (r0 )f (r0 )T d]u
Equation 2.27 will be more convenient if the structure tensor is introduced which is defined as:
J=
f (r0 )f (r0 )T d
Z "
fx20 fx0 fy0

d
fy0 fx0 fy20
(2.28)
Hence, by substituting equation 2.28 to equation 2.27,

E(u) = max uT Ju
kuk=1
(2.29)
In order to introduce local neighbourhood around pixel r, a window function w(r r0 ) is
37
(a) Original image
(b) Structure tensor calculation showing

the filtered structure. The colour shows
the orientation.
(c) Histogram of the orientation angle
Figure 2.17: Orientation definition in structure tensor calculation.
introduced to the structure tensor matrix, yielding

J=
w(r r0 )f (r0 )f (r0 )T d
Z "
hfx20 iw hfx0 fy0 iw

d
hfy0 fx0 iw hfy20 iw
(2.30)
where
hg, hiw =
w(r r0 )g(r0 )h(r0 )d
In Practice, computing the structure tensor can be done by applying a gradient operator like the
Sobel operator for computing the gradient and then apply a smoothing filter (like Gauss filter)
for windowing computation. If the smoothing operator is denoted by B and the gradient operator
38
with respect to coordinate p and q is denoted as Dp and Dq respectively, then

Jpq = B(Dp .Dq )
(2.31)
where the dot sign (.) denotes the pixel-wise multiplication.

It turns out that the tensor J is a symmetric, positive semi-definit matrix, and therefore there will
exist two orthogonal eigenvectors with non-negative eigenvalues where the one with the largest
eigenvalues 1 maximizes equation 2.29 and also acts as the local orientation [55, 50].
Having found the components of structure tensor matrix, then the local orientation, coherency,
and energy for each pixel respectively can be calculated easily by using equation 2.32 [51, 56].
2Jxy
Jyy Jxx
2
(Jyy Jxx )(Jyy Jxx ) + 4Jxy
C =
(Jxx + Jyy )
E = T race(J) = Jxx + Jyy
tan 2 =
(2.32a)
(2.32b)
(2.32c)
Where
C = Coherency
= Orientation angle
E = Energy
Coherency is in the range between 0 and 1, with 1 indicating highly oriented structures, and 0
indicating isotropic area. High energy means high values of the gradient, indicating an abrupt
changes in brightness. Therefore, pixels with higher energy values indicates the structure is
prominent in those pixels.
Eigen values have a role in giving information regarding the classification of orientation:
1. 1 = 2 0
This situation indicates that the region is homogeneous, no preferred orientation occurs.
2. 1 > 0, 2 0
This situation indicates that the region has single orientation.
3. 1 > 0, 2 > 0
This situation indicates that the region has multiple orientation.
39
40
Chapter 3
Materials and Methods
The experimental methods which are discussed here include the stamp fabrication, sample
preparation, data acquisition, and image processing.
3.1
Stamp Fabrication
The master stamp for 2D substrates is produced by using two-photon lithography process (Direct
Laser Writing - DLW). It involves the absorption of two photons at once in the focal spot of
femtosecond laser in the photosensitive liquid material. Once the energy of the absorbed photons
exceeds a specific threshold of the photoinitiator molecules inside the photoresist, a highly
localized chemical polymerization event would occur within the focus of the laser beam [57].
The design of the pattern in one master stamp consists of 10 10 fields. The dimension of one
field is 300 300 m corresponds to the stage size defined by the DLW machine. And within
one field, there are 4 same patterns which is shown in figure 3.1. The design is generated with
Matlab in which the design consists of coordinates showing the tracks for the piezo stage to write
the patterns.
The writing process is done by polymerizing the photoresist along a line track defined by the
movement of the piezo stage. In writing the stamp, the piezo stage moves line per line, this
means that the piezo stage moves from left to right horizontally, then shifts vertically for a
defined distance (in this case, the distance between lines is 0.25 m) and redo the line writing.
As the piezo stage is in horizontal motion, the power can be turned on or off defined by the
user. The on state is indicated by blue lines and off state is by red lines in figure 3.1. Since the
polymerization occurs during the on state, this will make the tracks depicted by blue lines to
be embossed while the tracks depicted by red lines will show caved patterns. The depth of the
patterns is about half the size of the laser beam focus (about 500 nanometer).
There are 5 different pattern designs in one stamp, so each pattern has 20 fields drawn on it. The
writing process for 1 master stamp may take up to 70 hours. After the writing process is done,
the sample is taken out from the machine and go to the developing process. The developing
procedure is as follows:
1. Incubate the sample in a mixture solution of isopropanol and MIBK (Methylisobutylketon)
with ratio 1:1 (by volume) and wait for 10 min.
41
42
Chapter 3. Materials and Methods
Figure 3.1: Design of pattern on stamp in 1 field.

2. Dry the sample carefully with N2 gas so that the patterns are not damaged.
3. Having the sample cleaned, put the sample into the plasma chamber. When the pressure
bar reaches at 0.3 mbar, apply the plasma for 1 min.
4. Take out the sample from the plasma chamber and put it into glass petri dish filled with
Trichloro(octadecyl) silane in toluene (1 mM). Wait for 10 min.
5. Rinse the sample with isopropanol and dry it again with N2 gas carefully.
6. Embed the sample on a microscope slide and the master stamp is ready to be a negative
replica (template) for making the replicate stamp.
Once the master is produced, it will become the template for making a replicate stamp. The
replicate stamp is made of silicone, PDMS (Polydimethylsiloxane) that resembles the cavity
patterns on the master stamp. The cavity patterns on the master stamp will make relief patterns on
the PDMS stamp after PDMS casting and peeling off, i.e. the replicate stamp will have inverted
patterns of the master stamp. To mold a stamp from the template, the steps are as follows:
1. Prepare the PDMS with ratio 10:1, ten parts of base (liquid PDMS) and one part curing
agent. Then mix it well to make sure that all base and the curing agent are mixed
homogeneously. During the mixing, it will incorporate a lot of air that traps gases inside
the solution.
2. Wait for about 30 minutes for degassing to make the gases inside are gone.
3. Dispense a tiny amount of mixture (a single drop would be sufficient) onto the template.
4. Put a glass stripe onto the drop and add some weight (about 0.5 g) on the glass stripe.
5. For curing process, put them into the incubator at 60 and wait for about 2 hours.
3.2. 2D Environments
43
6. The next step is to peal off the stamp from the template.
7. The finalizing step is to cut the boundary of the thin PDMS layer to get a homogeneous
thickness. The best cutting line is in the form of octagon where the patterns are in the
middle of it.
3.2
3.2.1
2D Environments
Sample Preparations for 2D Substrates
The PDMS stamp is then used for functionalizing the gold substrate area, i.e. to print a variety of
molecules in submicrometer resolution patterns on gold substrates where the cells will attach
and spread on that location. The flexible PDMS stamp was coated with hydrophobic thiol
(Octadecylmercaptone/ODM, Aldrich - fibronectin protein binds to these molecules) and was
dried gently with an inert gas, N2 gas. Then it was brought into tight contact with the surface of
a cover glass containing a thin layer of deposited gold (with a thin titanium adhesion layer). This
step is very critical in transferring the patterns onto the gold substrates due to the low aspect
ratio making the stamp easily deformed as mention in theoretical section. This is the reason why
the stripe glass is used as the backbone of the PDMS stamp.
The time applying the stamp and removing it again is about 10-20 seconds. The hydrophobic
alkanethiol molecules are transferred only to regions of the glass surface that contact the
raised regions of the stamp and thus the patterns will correspond to the shape of the raised
regions. When transferred to the gold surfaces, these molecules self-assemble into a molecular
monolayer or self-assembled monolayer (SAM) that is limited to the regions of the islands
created on the original master. Having removed the stamp, the gold substrate is rinsed with pure
ethanol and dry it again with N2 gas. The next step, a solution containing non-adhesive thiol
was added to the patterned substrate to passivate the remaining regions so that cells can only
adhere on the functionalized regions. Hydroxyl-terminated Hexa(ethylene glycol) alkanethiol
HS(CH2 )11 (OCH2 CH2 )6 OH, referred to as EG-6OH solution is used for the passivation
with the incubation time 10-15 minutes. The non-adhesive thiol self-assembles between the
hydrophobic SAM-covered islands, thus forming a continuous SAM over the entire substrate.
Figure 3.2 is a schematic drawing of the stamping procedures.
After incubation, the substrate is again cleaned with pure ethanol and dried carefully with N2
gas. Now the gold substrate is ready to be coated with the FN protein. The concentration of
the protein is 1:100 (by volume in which the protein stock solution is 1 mg/mol), 1 parts of
protein and 100 parts of buffer solution PBS (Phosphate buffered saline). The incubation time is
1 hour in incubator at 37C. The FN adsorbed only on the hydrophobic surfaces in the defined
islands, while the inrvening PEG-covered barrier regions remained uncoated (Figure 3.3) and
hence nonadhesive.
After the FN incubation, NIH3T3 fibroblasts were plated on the FN-coated micropatterned
substrates in DMEM (Dulbeccos Modified Eagle Medium). The cells preferentially attached
and spread on the location where the surface was functionalized. Before fixation and staining,
44
(a) Preapartion of a PDMS stamp using replica molding.
(b) Pattern transfer by microcontact printing ( CP).
Figure 3.2: A schematic outline of patterning by preparation of a PDMS stamp using replica
molding, and pattern transfer by microcontact printing ( CP). [58]
the cells were incubated for 2.5-3 hours in the incubator at 37C.
3.2.2
Cell Fixation and Staining
After incubation, the cells were fixed in 100 l of 4% paraformaldehyde (PFA) in PBS for 10
min. Then the washing process is done 3 times with 0.1% Triton X-100 in PBS for 5-10 min in
each washing step. The cell staining procedure is done in 2 steps:
1. Primary antibody staining:
(a) Anti-fibronectin polyclonal (rabbit) with concentration 1:400 in BSA (Bovine Serum
Albumin).
(b) Anti-Paxilin monoclonal (mouse) with concentration 1:500 in BSA.
2. Incubate for 1 hour and wash with 0.1% Triton X-100 in PBS 3 times for 5-10 min in each
washing step.
3. Secondary antibody staining:
(a) Cell nucleus staining with DAPI (4,6-diamidino-2-phenylindole) with concentration
1:1000 in BSA.
(b) Actin filaments staining with Phalloidin Alexa 488 with concentration 1:200 in BSA.
(c) Fibronectin staining with anti-rabbit Cy3 with concentration 1:500 in BSA.
(a) 60 V-shape. The length is 30

m, the width is 3.5 m.
(d) L-shape. The length is 30

m, the width is 3.5 m.
45
(b) Equilateral triangle. The

length is 30 m, the width is
3.5 m.
(e) Cross bow. The length is

45 m, the width is 3.5 m and
the radius is 18 m.
(c) Perpendiculat triangle. The

length is 30 m, the width is
3.5 m.
(f) Arrow. The length is 45 m,

the width is 3.5 m and the
length of the head is 25 m.
Figure 3.3: Fibronectin pattern on the functionalized surface
(d) Paxilin staining with anti-mouse Alexa 647 with concentration 1:200 in BSA.
washing step.
BSA is used for reducing the unspecific. After fixation and staining are done, the samples are
embedded on a microscope slide by using mowiol solution.
3.2.3
Data Acquisition
An Axio Observer equipped with Apotome module microscope is used for data acquisition of
the samples. For getting high quality images, a 63x oil objective lens (NA = 1.4) was used. The
illumination time was set automatically by the microscope system.
3.2.4
Image Processing for 2D Cases
Matlab is mostly used for doing image processing and analyzing. In 2D cases, the analyzes
include cell body and nucleus area measurement, nucleus orientation, nucleus elongation and
stress fiber orientation.
46
Cell Body and Nuclei Area Measurement

The actin channel is considered to represent the cell body and therefore this channel is used
for the measurement of cell body area. The cell nucleus area is measured in DAPI channel.
The image processing steps for measuring the area are depicted in figure 3.4. First, canny edge
detection is performed to detect the boundary of the cell. Then image dilation is performed with
1-3 pixel size diamond structure element to connect the unconnected pixels. Having connected
all the pixels along the boundary, filling holes can be performed in order to make a binary image
of the cell body. The cell area is represented by the total number of white pixels contained in
the binary image multiplied with the pixel area resolution. The same method is also applied for
measuring the cell nucleus area.
Figure 3.4: Steps in measuring cell body area with Matlab.
The significancy test or student-t test is performed on the following pair test: (angularbar;eq.triangle),(angular-bar;perp. triangle),(angular-bar;L-Shape),(eq.triangle,perp.triangle),
(eq.trianlge;L-Shape),(perp.triangle,L-Shape),(arrow;cross-bow).
Cell Nucleus and Actin Stress Fiber Orientation Measurement

The cell nucleus orientation measurement is done by fitting the ellipse and the angle defined by
major ellipse axis and x axis is the orientation angle of the nucleus, whereas the distribution of
actin stress fiber is measured by using structure tensor algorithm. For both nucleus and actin
stress fiber, the orientation is defined within 0 and 180 degree.
The performance of the structure tensor was first evaluated with synthetic random fiber network
by comparing the measured orientation distribution with the expected values.
47
Evaluation of Structure Tensor

A similar way like evaluating the deconvolution in the previous section, evaluating the structure
tensor will also be done in 2 ways, first by evaluating the synthetic random fiber network, and
afterwards by evaluating the real data.
A synthetic image in the form of random fiber network was used for evaluating the performance
of the structure tensor. The results are shown in figure 3.5.
(a) Original image
(c) Relative coherency of the orientation distribution
(b) HSV mode, showing the fiber network

orientation calculated by the structure tensor
(d) Rose plot showing the evaluation of the sructure tensor
Figure 3.5: The orientation of the synthetic random fiber network calculated by the structure
tensor.
Comparison of the orientation distribution between the true data and the one calculated by the
structure tensor is presented in rose plot as given in figure 3.5d. From that rose plot, one can
observe that the orientation distribution calculated by the structure tensor has a good correlation
with the true data. The difference in the number of data taken into the calculation causes the
inexpediency in the amplitude of the rose plot. The number of true data was 95, indicating that
there was 95 bars in figure 3.5a, while the number of data for the calculation of the structure
tensor was about 25.000. The number of data processed by the structure tensor is much more
48
than the true data because of the local neighbourhood calculation which is dependent upon the
size of the smoothing window function.
For real data measurements, cell on the arrow island was used as an example. The results are
given in figure 3.6. Notice that the orientation can also be represented in a rose plot diagram as
displayed in figure 3.6d after the angles are converted to be perpendicular to the gradient vector,
i.e. the orientation now lies along the iso-gray intensities values.
(a) Original image
(c) Relative cohorency of the actin stress fibers
(b) HSV mode, displaying the color code for the

orientation
(d) A rose plot showing the orientation of the stress fibers
Figure 3.6: The actin stress fibers of cell adhering on the arrow island evaluated by the structure
tensor.
3.3. 2.5D Environments
3.3
3.3.1
49
2.5D Environments
Sample Preparations for 2.5D Substrates
The 2.5D microscaffold structures on cover slips were also built with DLW machine where the
writing process may take 1 day for 10x10 fields. The fields consisted of microscaffolds in the
shape like U letter (30 80) m and V letter whose length is 80 m and with angle variation: 30,
45, 60, and 90. The height was set to 20 micron. The writing process was done in 2 steps. The
first step was to polymerise the ormocomp (a member of ORMOCER (ORganically MOdified
CERamics family, a protein-binding photoresist containing Igracure 369 as a photoinitiator
[57]) resist according to the design (see figure 3.7a). The radius of the bars was 2 m and the
radius of the Ormo pillar was 4.5 m. The second step was to coat the supporting pillar of the
structure with a photoresist composed of monomer PEG-DA (poly-ethylene glycol diacrylate),
pentaerythritol tetraacrylate (PETA, Sigma Aldrich) and Irgacure 369 (Ciba) as the photoinitiator
[59] (see figure 3.7b). The radius of the PEG pillar was 7.5 m. The pillar coating process was
done after washing the polymerised ormocomp structure from the first writing step with ethanol.
Once the writing was done, then the sample was rinsed with a mixture of ethanol and MIBK ,
then dried carefully with N2 gas. The schematic of the structures are given in figure 3.7.
(a) Ormo structures. The radius of the Ormo pillar was 4.5
m, the height was 20 m and the length was 80 m.
(b) PEG pillar structures. The radius of the PEG pillar was
.5 m and the height was 20 m.
Figure 3.7: Two steps of writing: Ormo writing and PEG pillar coating. The pictures are
without scale.
50
The incubation time for fibronectin was 30 minutes with the same concentration as in the 2D
case. Afterwards, the sample was washed with PBS and then NIH3T3 cells were seeded on the
structure and were incubated in DMEM medium for 2.5-3 hours in incubator at 37C.
3.3.2
Cell Fixation and Staining
After incubation, the cell were fixed in 100 l of 4% paraformaldehyde (PFA) for 10 min. The
washing process was done 3 times with 0.1 % Triton X-100 in PBS for 10 min in each washing
step. The next step was to stain the cell in 2 steps:
1. Primary antibody staining:
(a) Anti-fibronectin polyclonal (rabbit) with concentration 1:400 in BSA.
(b) Anti-Paxilin monoclonal (mouse) with concentration 1:500 in BSA.
washing step.
3. Secondary antibody staining:
(a) Cell nucleus staining with DAPI with concentration 1:1000 in BSA.
(b) Actin filaments staining with phalloidin Alexa 568 with concentration 1:100 in BSA.
(c) Fibronectin staining with anti-rabbit Alexa 647 with concentration 1:200 in BSA.
(d) Paxilin staining with anti-mouse Alexa 488 with concentration 1:200 in BSA.
washing step.
3.3.3
Data Acquisition for 2.5D Cases
The acquisition was done by using Confocal Laser Scanning Microscope (LSM510, Zeiss). For
the best resolution, a 63x oil objective lens (NA = 1.4) was used. The general procedure was
done according to this protocol [60].
3.3.4
Image Processing for 2.5D Cases
The image processing and analysis that were involved in the 2.5D case are cell surface area
and volume measurements which include cell body and nucleus. Since images taken with
confocal microscope has poor SNR, the images are first deconvolved with SGP algorithm for
fast processing and good results [61] to increase the accuracy of the geometric measurements.
Before the deconvolution algorithm is applied to the real data, it will be evaluated with synthetic
3D images like cube and sphere and also with 2D synthetic random fiber network.
The synthetic objects are convolved with 3D PSF Gaussian (2D Gaussian PSF for synthetic
random fiber) and then added with Poisson noise to mimic the imaging process in the confocal
microscopy. The performance of the SGP algorithm is viewed in terms of how the results
correlate with the true data, the number of iteration. Least square error (LSE) with the original
data and discrepancy error (error between after and previous iteration).
51
Evaluation of Deconvolution
Evaluation of deconvolution performance is done by analyzing the synthetic images (random
fiber network, cubes and spheres) image generated with Matlab and compared with the real data
which were taken from the lab.
Synthetic Image Analysis
The synthetic image for 2D is generated with Matlab. Its a kind of fiber network whose
orientation, length, and location are in random. The original image is then can be assumed to
be the true object. This true object then will blurred (convolved) with normalised Gaussian
PSF (sigma radius 0.25 m) as a representation of passing through a microscope system. Then
for representing detector recording, the noiseless image will be added with Poisson noise. The
original image, blurred image, and blurred-noisy image are given in figure 3.8.
(a) Synthetic fiber network in grayscale
(b) Blurred synthetic fiber network with Gaussian PSF
(c) Blurred and noisy synthetic fiber network. The

noise is Poisson distributed
Figure 3.8: Synthetic random fiber network
The SGP deconvolution matlab codes has several optional input arguments. However, 4 different
optional input arguments that were used in this thesis: INITIALIZATION, STOPCRITERION,
BG, and TOL. Other input arguments are set as default. INITIALIZATION input argument
is the choice for starting point and it has 5 options:
52
1. 0
The starting point is initialized with all zero starting point
2. 1
The starting point is random
3. 2
The starting point is initialized with the same value of the matrix input image gn
4. 3
The starting point is initialized with: ones(size(gn))*sum(gn(:) - bg) / numel(gn)
5. x0
The starting point is initialized by user (double array data type)
The value of BG is defined by estimating the background of the matrix input image. The
background estimation was done by using imopen command and take the average of all pixels.
TOL values are between 101 and 102 for both actin and nucleus deconvolution. There are 4
kinds of stopping criteria which are used in the SGP deconvolution program:
1. iter > M AXIT .
Meaning that the iterations will be stopped when the iteration reaches the maximum
iteration that is set.
2. ||xk xk1 || <= tol ||xk || OR iter > M AXIT .
Meaning that the iterations will be stopped when the reconstruction error is reaching the
minimum value denoted by the tolerance value. The reconstruction error is calculated by
taking the absolute value of the difference of the next and previous iteration.
3. |KLk KLk1 | <= tol |KLk | OR iter > M AXIT
Meaning that the iterations will be stopped when the KL divergence is reaching the least
value.
4. (2/N ) KLk <= tol OR iter > M AXIT
Meaning that the iterations will be stopped when the discrepancy error is reaching the least
value.
Different variation of input arguments would yield different result. For Actin cytoskeleton and
nucleus deconvolution, stopping criteria number 2 or 3 is often used. The INITIALIZATION
value of 2 or 3 is often used for actin cytoskeleton and 0 for nucleus. The evaluation of the
deconvolution is mainly subjectively evaluated how the blurriness is reduced in all stack of the
image and whether or not there are vanishing structures. Deconvolving nucleus is easier than
deconvolving actin cytoskeleton.
The deconvolution of object in figure 3.8 is given in figure 3.9 with difference value of tolerance.
As shown in figure 3.9 that the tolerance value at 104 seems give the best looking among
others. When the tolerance is at 106 , some structure seems vanishing. However, in practice,
tolerance value of 101 is often used because of the poor signal contained in actin cytoskeleton.
53
(a) Original image
(b) Tolerance 103 . The number of iterations is 25
(c) Tolerance 104 . The number of iterations is 46
(d) Tolerance 105 . The number of iterations is 75
(e) Tolerance 106 . The number of iterations is 170
(f) Tolerance 105 . The number of iterations is 14
Figure 3.9: Deconvolution of synthetic fiber network with difference tolerance.
Therefore to prevent vanishing structures, the tolerance values will be set between 101 to 102
for deconvolving the real data, depending on subjective criteria in evaluating the vanishing
structures. The LSE and discrepancy values are given in figure 3.10.
Both LSE and discrepancy error of the deconvolution for every tolerance values give the same
results at the same iterations. This can be observed from the overlapping graph in figure 3.10.
This is obvious because the value of tolerance is just used for stopping the iteration, not changing
the LSE or the discrepancy error at every iterations. By looking at the LSE graph in figure 3.10,
54
the lowest value of LSE is reached at iteration 14. And the result of the deconvolution at 14th
iteration with tolerance 105 is given in figure 3.9f.
Its obvious that although the LSE is the lowest at 14th iteration, the results of the deconvolution
seems worse. LSE method seems does not always a good parameter for evaluating the results.
Evaluation of Minkowski Algorithm for Geometric Measurements
The geometric measurements are done with the help of Matlab codes from [62]. The performance
of Minkowski algorithm for measuring the geometric measurements like volume and surface area
is performed in synthetic cube and sphere which are blurred and noisy. The measurements will
also be compared after the cube and the sphere are deconvolved to see if the deconvolution would
increase the accuracy. The size of the cube is 5 micron, and the radius of the sphere is 2 micron.
Then this cube is convolved with 3D Gaussian PSF whose radial FWHM is 0.394 m and its
axial FWHM is 0.626 m. These values were taken from the average of the PSF measurements
for LSM510 in the lab. After convolution, Poisson noise will be added to the objects. The voxel
sizes in x, y, z are assumed to be 0.081 m, 0.081 m, and 0.31 m respectively which mimic
the voxel sizes of the real data. The original cube, blurred cube, blurred-noisy cube, and the
deconvolved cube are given in figure 3.11. While figure 3.12 corresponds to the sphere.
Due to the blurring process and noise effect, the lateral and axial resolution will be degraded.
Table 3.1 demonstrates the comparison tests to assess the significance of these degradation as
well as to demonstrate the performance of the Minkowski algorithm in computing the volume
and the surface area.
Table 3.1: Comparison tests for assessing the significance of blurring and noise degradation as
well as demonstrating the performance of the Minkowski algorithm
Original
Blurry
Blurry + noisy
Deconvolved
Starting
slice number
42
35
35
41
Ending slice
number
58
63
63
60
Original
45
55
Blurry
Blurry + noisy
Deconvolved
43
43
44
57
57
58
Shape
Condition
Cube
Sphere
Volume
Surface Area
128.66 (theory: 125)

236.12
260.60
148.47
33.62 (theory:
33.51)
47.99
45.02
37.74
128.10 (theory: 150)

208.83
267.81
139.89
50.63 (theory:
50.27)
72.63
74.00
55.22
As demonstrated in table 3.1 that the blurring and noising process cause the starting and the
ending slice number to be different from the original objects. But after deconvolution, they seem
closer to the original one.
The calculation of volume and surface area are strongly dependent on how good the segmentation
is, because the Minkowski code need the 3D image input in binary format. And there is some
errors between the expected value and the measured value. The errors would be worse in case of
55
shapes with sharp edges (or corners, like cube). But for the shapes which do not have too many
edges like spheres, the errors would be decreased as given in table 3.1 where the volume and
the surface area of the original image, the measured values are pretty close to the theoretical
values. These results show that the deconvolution is able to increase the accuracy of geometric
measurements.
Cell Nucleus and Cell Body Volume Measurement in 2.5D Cases
The methods for estimating the volume and the surface area of the cells adhering on 2.5D
microscaffolds are as follows:
1. Create a mask from the maximum intensity projection of the 3D stack image. This is done
with the help of canny operator in the Matlab Image Processing Toolbox. In particular
for nucleus, the size of the mask is increased for several pixel in order to ensure that the
nucleus periphery is conserved during the pixelwise multiplication which will be done in
the next step.
2. Perform pixelwise multiplication to all slices of the image by this mask.
3. Estimate a global thresholding by using Otsu method (in this thesis, the Otsu is applied to
the MIP) and employ this value to all slices. If the thresholding resulted from Otsu method
is zero, then the thresholding value is set manually to 0.0039 which is very often a good
number in most cases.
4. Perform a median filtering (the size that was used in this thesis is [10 10]) to all slices in
order to reduce the speckles resulted from the thresholding.
5. The 3D binary image (see figure 3.13f) as the result of the filtering is then used to estimate
the volume and the surface area. The calculation is directly done by using the Matlab
routine taken from [62]. The Matlab file is freely distributed in Matlab Exchange File
website by the author.
These steps are also applied to measure the cell body volume, including the deconvolved 3D
images.
Actin Distribution at the Bottom Side and the Top Side of the Nucleus
In 2.5D environments, the actin filaments cover the whole surface of the nucleus to support its
position in the cell. Since the function is for supporting, it is intuitive to estimate and to compare
the distribution of the filaments at the bottom and the top side of the nucleus. The procedure is
as follows:
1. Perform the MIP of the DAPI channel and crop the nucleus (see figure 3.14b).
2. Perform the edge detection with canny operator to detect the boundary of the nucleus. This
process will create a binary image of the nucleus (see figure 3.14b).
3. Find the centroid and estimate the radius of the nucleus binary image.
56
4. Create a bigger mask by increasing the estimated radius by 2-3 m depending on how
close the nucleus to the structure. Avoid creating the mask that includes the structure so
that the actin filaments that binds to the structure not to be included in the calculation (see
figure 3.14d.
5. Perform the thresholding by using the Otsu method to the MIP of 3D stack in actin channel
and apply this value to all slices.
6. Perform a pixelwise multiplication with the big mask to all slices of the 3D stack images
that have been thresholded.
7. Assess the density of the actin filaments at the bottom and the top side of the nucleus. The
bottom side is defined as the slices from the middle section of the nucleus goes to the first
slice. The top side is defined from the middle section goes to the last slice. And the middle
section is defined by estimating the middle slice number of the deconvolved nucleus since
the deconvolved one provides a clear segmentation at the top and the bottom sides.
Cell Nucleus and Actin Stress Fiber Orientation Measurement

Likewise in the 2D case, the actin stress fiber orientation distribution is calculated with the help
of the structure tensor. But in the 2.5D case, maximum intensity projection (MIP) is performed
to the image stacks to approximate the actin filaments distribution of the whole cell. Afterwards,
the structure tensor is applied to the MIP.
57
(a) LSE with the corresponding tolerance 103
(b) LSE with the corresponding tolerance 104
Figure 3.10: LSE and discrepancy error of synthetic fiber network deconvolution with difference
tolerance.
58
(a) Original cube. Scale bar 1.5 m
(b) Blurred cube. Scale bar 2 m
(c) Blurred and noisy cube. Scale bar 2 m
(d) Deconvolved cube. Scale bar 1 m. The number

of iterations is 59
Figure 3.11: Synthetic cube.
59
(a) Original sphere. Scale bar 2 m
(b) Blurred sphere. Scale bar 2 m
(c) Blurred and noisy sphere. Scale bar 1 m
(d) Deconvolved sphere. Scale bar 1 m. The number

of iterations is 47
Figure 3.12: Synthetic sphere.
60
(a)
(b)
(c)
(d)
(e)
(f)
Figure 3.13: Steps in measuring the nucleus volume (a-b-c-d-e-f). (a) Perform the MIP on the
stack image. (b) Create a mask with the help of canny operator to detect the boundary of the
nucleus. Notice that the size of the mask is increased for conserving the periphery of the nucleus.
(c) Perform the pixelwise multiplication on the stacks with the mask. This figure shows the slice
number 17 out of 40 slices. (d) Threshold the MIP by using the Otsu method and apply the
threshold value to all slices. This figure shows the slice number 17 out of 40 slices. (e) Filter all
slices by using a 10 10 median filter to reduce the speckles. This figure shows the slice number
17 out of 40 slices. (f) Isosurface rendering of the filtered stacks. This figure is without scale.
61
(a)
(b)
(c)
(e)
(d)
(f)
Figure 3.14: Steps in measuring the nucleus volume (a-b-c-d-e-f). (a) Perform MIP on the stack
image. (b) Crop the MIP image in the region of interest (nucleus) and perform the segmentation.
Then estimate the centroid and the radius of the segmented image. (c) Create a bigger mask
by increasing the radius of the nucleus binary image. The radius can be increased by 2-3 m
depending on how close the nucleus to the structure. Avoid creating the mask that include the
structure. (d) Perform the pixelwise multiplication with this big mask to all slices of the 3D
stack in the actin channel. This figure shows the slice number 13 out of 40 slices. (e) After
masking the slices, perform the Otsu thresholding to these masked slices of the 3D image. And
then perform the pixelwise multiplication again by using the thresholded masked slices to the
corresponding slice number This figure shows the slice number 13 out of 40 slices. (f) Estimate
the actin density at the bottom and the top side of the nucleus.
62
63
64
Chapter 4
Results and Data Analysis
The analysis of the results is mainly divided into 4 parts, analysis of the nucleus and the stress
fibers orientation, the nucleus and the cell body area, the nucleus elongation, and the nucleus and
the cell body volume. In particular, the analysis will also include the data taken from the master
thesis of Mona Jaggy [63].
Fixated and stained cells adhering on shorter patterned substrates are given in figure 4.1 and cells
adhering on longer patterned substrates are displayed in figure 4.2.
In general, the features of cells adhering on 2D patterns, either on the normal or the bigger one,
they are relatively the same, i.e. there are a lot of ventral stress fibers formed on the basal plane
of the cells. Particularly, the stress fibers seem more prominent upon nonadhesive edges than on
adhesive edges. By looking at the paxilin channel, one can immediately observe that the focal
adhesion are mostly mature at the edges on the patterns which are the anchor points of the stress
fibers. These results are in accordance with Thrys experiments in one of his paper [2].
4.1
Cell Nucleus and Stress Fiber Orientation
The structure tensor is applied to quantify the stress fiber orientation distribution of cells adhering
on 2D patterned substrates and in 2.5D environments. Concerning the orientation, as outlined in
the theoretical section, the orientation is defined as the smallest deviation of the image gradient
vector. Hence it can be parallel or antiparallel with the gradient vector. However, for the sake
of simplicity when comparing the orientation of actin stress fibers with the orientation of cell
nucleus, the stress fiber orientation is rotated 90. The rotation can be anticlockwise or clockwise
in such a way that the range of the orientation is within 180. This rotation indicates that the
orientation now is pointing perpendicular with the gradient vector or lying along the line of
iso-gray intensity values. Figure 4.3 shows the actin stress fiber orientation distribution when it is
parallel with the gradient vector while figure 4.4 shows the distribution when it is perpendicular
with the gradient vector and combined with the cell nucleus orientation. Another thing which is
important to mention regarding the structure tensor feature used in this thesis is that it does not
measure the strength (intensity) of the stress fibers; only the coherency of the orientation. Thus,
the dominant coherency does not indicate the strength of any stress fibers.
65
66
Chapter 4. Results and Data Analysis
(a) 60 V-shape
(b) 60 V-shape
(merged)
(c) Equilateral Triangle
(d) Equilateral Triangle (merged)
(e) Perpendicular Triangle
(f) Perpendicular
Triangle (merged)
(g) L-Shape
(h)
L-Shape
(merged)
(i) Arrow
(j) Arrow (merged)
(k) Cross Bow
(l) Cross
(merged)
Bow
Figure 4.1: NIH3T3 cells adhering on various normal patterned 2D substrates. Green: actin
filaments, magenta: fibronectin, blue: nucleus, red: paxilin. The scale bar is 10 m.
4.1. Cell Nucleus and Stress Fiber Orientation
67
(a) V-shape 50 m, 30
(b) V-shape 50 m,
30 (merged)
(c) V-shape 50 m, 45
(d) V-shape 50 m,
45 (merged)
(e) V-shape 50 m, 60
(g) V-shape 60 m, 30
(i) V-shape 60 m, 45
(k) V-shape 60m, 60
(m) L-Shape 60 m
(f) V-shape 50 m,
60 (merged)
(h) V-shape 60 m,
30 (merged)
(j) V-shape 60 m,
45 (merged)
(l) V-shape 60m,

60 (merged)
(n) L-Shape 60 m
(merged)
Figure 4.2: NIH3T3 cells adhering on various bigger size of 2D patterned substrates. Green:
actin filaments, magenta: fibronectin, blue: nucleus, red: paxilin. The scale bar is 10 m.
68
(o) U-Shape 60 m
(q) U-Shape 80 m
(p) U-Shape 60
m (merged)
(r) U-Shape 80
m (merged)
Figure 4.2: (Continued) NIH3T3 cells adhering on various bigger size of 2D patterned substrates.
Green: actin filaments, magenta: fibronectin, blue: nucleus, red: paxilin. The scale bar is 10 m.
Figure 4.4 displays the orientation of the nucleus together with the orientation of the actin stress
fibers when cells adherent on shorter size of micropatterned substrates. Generally, the orientation
of the nucleus was in accordance with the dominant orientation of the ventral actin stress fibers.
This suggests that the nucleus orientation is influenced by the dominant orientation of the actin
stress fibers. This behaviour was observed in cells adherent on L-shapes, triangles, V-shapes and
crossbows. However, for cells adherent on crossbow patterns, the nucleus long axis does not
orientate exactly in a similar fashion as the stress fibers.
For cells adherent on L-shapes, triangles and V-shapes, a lot of ventral stress fibers were
assembled along straight edges of the shapes and crossing over the non-adhesive region. Cells
adherent on the crossbow patterns displayed contractile stress fibers (peripheral bundles)
over non-adhesive region and a polymerizing meshwork within ruffles at the adhesive border
(4.1k,4.1l). This is in accordance with what has been studied in [11, 64, 65] that concave
fibronectin patterns like crossbow can prevent the development of conspicuous stress fibers and
induce a structural asymmetry in the plasma membrane which then leads to polarization to the
curved zone. Additionally, these stress fibers are said to enable cells to translate the geometry of
their microenvironment into an internal organization of the cytoskeleton that allows them to
bridge distant adhesion site, and to ensure a mechanical balance and continuity within tissues [10].
Cells adherent on L-shape (30 m) islands also show different dominant stress fiber orientation
compared with the 60 V-shapes (30 m) (see figure 4.4a and 4.4b) although both structure
69
(a) 60 V-Shape
(b) Equilateral Triangle
(c) Perpendicular Triangle
Figure 4.3: Relative coherency of the stress fiber orientation on various patterned 2D substrates.
70
(d) L-Shape
(e) Arrow
(f) Cross Bow
Figure 4.3: (Continued) Relative coherency of the stress fiber orientation on various patterned
2D substrates.
71
(a) 60 V-Shape
(b) L-Shape
Figure 4.4: Cell nucleus and stress fiber orientation on various patterned 2D substrates. The
rose plots are normalized to the maximum orientation value.
72
(c) Equilateral Triangle
(d) Perpendicular Triangle
Figure 4.4: (Continued) Cell nucleus and stress fiber orientation on various patterned 2D
substrates. The rose plots are normalized to the maximum orientation value.
73
(e) Arrow
(f) Cross Bow
Figure 4.4: (Continued) Cell nucleus and stress fiber orientation on various patterned 2D
substrates. The rose plots are normalized to the maximum orientation value.
74
are open patterns. To associate the cell division orientation with the stress fiber and nucleus
orientation, Thry in his paper [66] showed that the division axis of cells adherent on L-shape
has pronounced orientation along the hypotenuse of the pattern. Likewise, figure 4.4b shows
that the long axis of the nuclei points dominantly to the same direction along the hypotenuse.
Moreover, the actin stress fibers have a strong coherency in the same orientation as nucleus.
Collecting these results, it seems that the actin stress fibers on L-shapes contribute to the
elongation of the cell nucleus in such a way that they orientate to the dominant orientation of the
actin stress fibers.
By comparing figure 4.1a and figure 4.4a for cells adhering on the 60 V-shapes (30 m), one
might think that the data represented by the structure tensor does not correlate with the direct
observed image (figure 4.1a shows the strong bundle stress fiber is oriented along the hypotenuse
while figure 4.4a shows that the dominant stress fiber orientation is at 60). It should be pointed
out that what is directly seen in the image does not have to be always the same as represented
by the structure tensor calculation. As outlined before that the structure tensor used here does
not evaluate the strength (intensity) of the stress fiber but it evaluates the coherency of the
local orientations and sum the coherency values that are in the same orientation. Although the
actin stress fibers have the dominant coherency at the orientation of 60 (or along one side
of the bars) as given in figure 4.4a, the intensity of a bundle of stress fibers that connects the
edges of non-adhesive region is still the strongest as can be easily observed with bare eyes
in figure 4.3a. The distribution looks like this because the cell also makes several bundles
of actin stress fibers pointing from one edge to the side of the adhesive bar (see figure 4.4a).
And some of these bundles are in the same orientation as the adhesive side of the bar. Hence
during the calculation of the structure tensor, it will increase the coherency at the direction of 60.
Cells adherent on the equilateral triangle responded to distribute their stress fibers homogeneously
on the adhesive region. Therefore, by counting in the symmetry of the structure, their nucleus
orientation oriented to the apices of the structure. However, when cells adherent on the
perpendicular triangle, they responded to distribute their stress fibers dominantly along the
hypotenuse of the adhesive region and the stress fiber orientation on this shape is very similar
with the L-shape. Correspondingly, their nucleus oriented along this dominant stress fiber
orientation.
Taking a closer look at the actin stress fiber distribution calculated by the structure tensor, cells
adherent on the arrow and on the crossbow patterns surprisingly have different actin stress fiber
orientation (figure 4.4e, 4.3e, 4.4f and 4.3f) although the distance between edges in the head part
of the arrow and the crossbow were made the same and they showed the same nucleus long axis
orientation. In some cases of cells adherent on the arrow islands, they displayed some variation
of stress fiber distribution which look like in figure 4.5a and 4.5b. This might contribute to
the difference of the coherency of the actin stress fiber orientation calculated by the structure
tensor. It seems that the geometry of the head of the island is the reason to this difference. The
arrow head has straight shapes, while the crossbow head has curved shapes. As demonstrated in
[65] that cells responded to form lamellipodia than stress fibers on curved islands. Therefore
the stress fibers on the straight edges tend to have more variation in the orientation than on the
curved shapes.
75
(a)
(b)
Figure 4.5: Its quite often to observe cells adhering on the arrow islands to have bundles of
actin stress fibers displayed in these figures. The bundles do not always anchor from one edge to
another edge of the non-adhesive islands, but sometimes from one edge of the arrow leg to some
points on the arrow head which give more variation of orientations. Eventually, this variation
will be affecting the relative coherency computed by the structure tensor. Green: actin filaments,
magenta: fibronectin, blue: nucleus, red: paxilin. The scale bar is 10 m.
When cells adhere on other V-shapes that are longer and have variation in angles, the results are
given in figure 4.6 and 4.9. It should be pointed out that the data shown in figure 4.6 has been
combined according to the angles of the patterns. For example, the 30 V-shape has 2 sizes: 50
m and 60 m. The data of those two different sizes but in the same angles are combined into
one dataset.
Cells adherent on longer size of V-shapes whose angles are 30 and 45 responded to orientate
their long axis nucleus according to the dominant stress fiber orientation. Direct observation to
the images show that this behaviour was caused by the direct contact of the stress fibers along
the adhesive bars with the periphery of the nucleus. As the result, the nucleus was physically
orientated by the actin stress fibers to the direction lying between the 2 adhesive bars. Whereas
for angle 60 and L-shape, they responded to orientate their nucleus along the hypotenuse of the
patterns. However, when cells adherent on the shorter size of 60 V-shapes (30 m), the long
axis nucleus orientation showed to be in accordance with the dominant stress fiber orientation
(see figure 4.4a). Seemingly, because the size of the shorter islands was comparable with the
nucleus, the actin stress fibers were dense enough near the nucleus so that they could directly
orientate the nucleus. When the size of the islands were enlarged, the actin stress fibers were not
dense enough near the nucleus so that they do not directly orientate the nucleus. This argument
is based on the observation that the mean nucleus area is smaller on the shorter V-shapes than
on the longer V-shapes (see section Cell Nucleus and Cell Body Area Measurements). The
nucleus indeed still has a certain orientation which was governed by the stress fibers orientation
but it became less pronounced.
76
(a) 30 V-shape (longer dimension)
(b) 45 V-shape (longer dimension)
Figure 4.6: Relative coherency of the stress fiber orientation on enlarged 2D patterned substrates.
The rose plots are normalized to the maximum orientation value.
77
(c) 60 V-shape (longer dimension)
(d) L-Shape (longer dimension)
Figure 4.6: (Continued) Relative coherency of the stress fiber orientation on enlarged 2D
patterned substrates. The rose plots are normalized to the maximum orientation value.
78
(e) U-Shape (longer dimension)
Figure 4.6: (Continued) Relative coherency of the stress fiber orientation on enlarged 2D
patterned substrates. The rose plots are normalized to the maximum orientation value.
Studying cells adhering on 2D islands which induce the same cell shape (like 60 V-shape vs
equilateral triangle; L-shape vs perpendicular triangle) reveals an important fact that cell shape
does not determine the actin organization [67]. The study of cells adherent on 2D V-shapes
shows that if there is no direct contact between the actin stress fibers with the periphery of the
nucleus, then the long axis nucleus orientation dominantly points along the hypotenuse of the
patterns.
For cells spreading in 2.5D 60 V-shape (30 m), 2.5D L-shape (30 m) and 2.5D triangles
(equilateral and perpendicular whose side length is 30m), they responded to orientate the long
axis nucleus as shown in figure 4.7. In these 2.5D environments, the long axis nucleus orientation
on V-shapes, L-shapes and perpendicular triangles was oriented along the hypotenuse of the
structures. Particularly for 60 V-shape (30 m) in 2D and 2.5D environments, they did not have
similar long axis nucleus orientation: in 2D, cells responded to orientate the nucleus orientation
along the adhesive bar while in 2.5D the orientation was along the hypotenuse.
Figure 4.8 shows the image rendering of NIH3T3 cells adhering and spreading over various
microscaffold structures whose length is 80 m.
For cells adherent on 2.5D structure with the length of 80 m, the cell nucleus and the stress
fiber orientations are given in figure 4.9 and 4.10.
The most distinguished features of actin stress fiber orientations between 2D and 2.5D is that in
79
Figure 4.7: Nucleus orientation. A. Microscaffold structures. A. Nucleus orientation definition.

B. Cell control. C. Nucleus orientation on L-shape. D. Nucleus orientation on perpendicular
microscaffold. D. Nucleus orientation on 60 V-shape. F. Nucleus orientation on equilateral
triangle. Magenta: fibronectin. Blue: DAPI staining. [63].
2.5D, the orientation was dominantly along the adhesive bars of the structures. And the strong
actin stress fibers that connect the non-adhesive edges was not observed as occurred in the 2D
environments. The stress fibers in 2.5D were more distributed on the adhesive regions than on
non-adhesive regions. Apparently, cells adherent in 2.5D environments behave in such way
because they need to make a stronger bound to the structure for the purpose of supporting the
position of the cell.
For cells adherent in 2.5D L-shapes (80 m) and in 2.5D V-shapes (80 m) with angles of
45 and 60, they responded to orientate their long axis nucleus along the hypotenuse of the
structure. This nucleus orientation does not correspond to their dominant stress fiber orientation.
However, the nucleus orientation in 2.5D 30 V-shape was coherent with their dominant stress
80
(a) 2.5D V-shape 30
(b) 2.5D V-shape 45
(c) 2.5D V-shape 60
(d) 2.5D L-Shape
(e) 2.5D U-Shape
Figure 4.8: NIH3T3 cells adhere and spread over various 2.5D microscaffold. Green: actin
filaments, red: paxilin, magenta: fibronectin, blue: nucleus. The scale bar is 10 m.
(a) 2.5D V-shape 30, 80 m
(b) 2.5D V-shape 45, 80 m
(c) 2.5D V-shape 60, 80 m
Figure 4.9: Relative coherency of the stress fiber orientation on 2.5D structure.
81
82
(d) 2.5D L-Shape, 80 m
(e) 2.5D U-Shape, (30 80) m
Figure 4.9: (Continued) Relative coherency of the stress fiber orientation on 2.5D structure.
83
(a) 2.5D V-shape 30, 80 m
(b) 2.5D V-shape 45, 80 m
Figure 4.10: Cell nucleus and stress fiber orientation on various shapes of 2.5D microscaffold.
The rose plots are normalized to the maximum orientation value.
84
(c) 2.5D V-shape 60, 80 m
(d) 2.5D L-Shape, 80 m
Figure 4.10: (Continued) Cell nucleus and stress fiber orientation on various shapes of 2.5D
microscaffold. The rose plots are normalized to the maximum orientation value.
85
(e) 2.5D U-Shape, (30 80)m
Figure 4.10: (Continued) Cell nucleus and stress fiber orientation on various shapes of 2.5D
microscaffold. The rose plots are normalized to the maximum orientation value.
86
fiber orientation because based on direct observation to the image (figure 4.8a), the shape of the
structures are close enough to the size of the nucleus resulting a direct contact of the stress fibers
along the adhesive bars with the nucleus periphery. Thus the nucleus orientation on 2.5D 30
V-shape is oriented directly by these actin stress fibers. For cells adherent on 2.5D U-shapes, the
nucleus orientation is coherent with the dominant stress fiber orientation (see figure 4.8e).
Correlation between Cell Division and Stress Fiber Orientation on Arrow and Crossbow
shape
The difference of the actin stress fiber distribution on the arrow shape and the crossbow shape
might help to understand the reason of why the spindle orientations of cells are different (as
demonstrated in [64],see figure 4.11). When cells enter the mitosis, stress fibers disassemble
and most proteins disengage from focal adhesions resulting in small anchoring sites [68, 2].
Then they round up and the cell membrane over the non-adhesive edges retracts rapidly while
the anchoring sites resist the cell rounding by forming retraction fibers that originate from their
adhesive contacts with ECM [10]. It turns out that in cultured cells, cortactin is distributed
along retraction fibers and ezrin (ERM-Ezrin/Radixin/Moesin family proteins that connect lipid
membranes to the actin cytoskeleton [69]) accumulates at the proximal end of the retraction
fibers, forming cortical landmarks at the contact with the cell body [2]. Eventually, these cortical
landmarks serves as a map of the projection of the interphase adhesion pattern on the mitotic cell
that guides spindle orientation [2, 66].
It was demonstrated that during development, actomyosin contractility participates in the spatial
restriction of polarity cues and helps in determining the orientation of the spindle [70, 2]. The
direction of this actomyosin contractility is obviously related with the stress fiber orientation
that can be estimated by the structure tensor. Therefore, by knowing the stress fiber orientation,
one can estimate on which sides the cortical cues would be dominantly distributed in the cell body.
The stress fiber orientations of cells adhering on the arrow shape are closer to the y axis than that
on the crossbow shape, some cortical cues would be dominantly located close to the vertical
axis (y axis). Thus, there will be torques that orientates the mitotic spindles approaches to
the y axis. For cells on the crossbow shape, since the majority of the stress fiber orientations
are along x axis, therefore the cortical cues would be dominantly distributed along x-axis,
resulting forces that orientates the mitotic spindles along the x axis as well. Therefore, the
cell division orientation occurs as shown in figure 4.11. However, one remark from both
patterns, although cells show a similar fashion of cell body and the nucleus long axis orientation,
they show different ways of division. So, this situation does not obey the Hertwig rule
(trends of the spindle orientation which is to be aligned with the long axis of the cell [71, 14, 15]).
4.2
Cell Nucleus and Cell Body Area Measurements
Figure 4.12a, 4.12b and 4.12c show the results of the area measurements when cells adhering on
the 2D shorter patterned substrates. The figures show the area of the nucleus, the cell body and
the ratio of both respectively.
4.2. Cell Nucleus and Cell Body Area Measurements
87
(a) Mitotic cells on arrow-shaped (left) and on crossbow-shaped (right)

fibronectin micropatterns (red). Retraction fibres (green, second row), spindle
poles (red) and the chromosomes (blue) were labelled. Sequences of images
acquired in time-lapse 10 phase contrast microscopy show examples of cell
division on both patterns. Time is given in minutes. Scale bar, 10m.
(b) Pattern geometry used for the calculation of w(): the pattern
outline along which retraction fibres are anchored (dark orange) and
the corresponding density of force generators (blue dots) are indicated.
The experimentally observed spindle orientation is displayed as angular
histograms (light blue).
(c) Nucleus and stress fiber orientation on arrow

shaped micropatterns
(d) Nucleus and stress fiber orientation on

crossbow shaped micropatterns.
Figure 4.11: The stress fiber orientation revealed by the structure tensor helps to understand
why the spindle orientation of cell on arrow and crossbow shape is different although they have
the same cell elongation [11].
88
ECM determines the cell shape. Comparing the 2D open patterns (like V-shape and L-Shape)
with the 2D closed patterns (like triangles), it seems that cells have more freedom to make bigger
basal cross-sectional area when they adhere on the open patterns. This is shown by the lower
mean value of nucleus area that the closed patterns possess than that is possessed by the open
ones. The cell body area are smaller when they adhere on the 60 V-shapes and the equilateral
triangles than when they adhere on the perpendicular shapes. This is obvious because from
geometrical point of view, the area of V-shapes and equilateral triangle is smaller by a factor of
sin 60 compared with the perpendicular patterns (L-Shape and perpendicular triangle, in this
case). For patterns like arrow and cross-bow, the cells relatively have the same cell body area
because the dimension of the arrow and the crossbow were made similar.
In contrast to the cell body, the nucleus area is similar between the open and the closed patterns.
However, the significancy test showed that there is 1 star of significant level between 60 V-shape
and the equilateral triangle although cells have similar cell shape in both patterns. This revealed
that the nucleus were more compressed on closed patterns than on open patterns. The actin stress
fibers might be the reason of this. As shown in figure 4.3b and 4.1c that the coherency of actin
stress fibers is strong in the circumference of the equilateral triangle suggesting stress fibers are
crowded on the periphery of the nucleus. Furthermore, due to the small size of the patterns that
is comparable with the cell nucleus size, these actin stress fibers could give more compression
on the nucleus resulting smaller basal cross-sectional nucleus area. This is in accord with the
experiments done in [72] in which the author showed the role of actin cytoskeleton in deforming
the nucleus shape.
The nucleus area on L-shape is smaller than on perpendicular triangle although they
have similar cell body area. The reason for this is because of the membrane tension (actin
curvature) between the edges of the L-shape over the non-adhesive region that pushes the nucleus.
For the arrow and the crossbow, the nucleus area increased compared to the open and closed
patterns. This seems obvious given that the cell body is increased as the adhesive area increases,
suggesting the changes in the osmotic equilibrium that increases the hydrostatic pressure inside
the cells. Upon cell swelling, cells release ions through activation of K + channels and/or
anion channels, KCL cotransport, or parallel activation of K + /H + exchange and CL /HCO3
exchange [73]. The nucleus volume has to increase to balance the increase of the cell body
so that the karyoplasmic ratio can be kept constant. It turns out that the nucleus in this case
increased more radially than axially.
As the size of the cell body increases by increasing the size of the islands, the mean area of the
nucleus is a little bit increasing, although not significant as shown in figure 4.13. This study
revealed at least 2 points, that it is easier for cells to enlarge their cell body area than their nucleus
and by increasing the cell body area, the compression to the nucleus decreases.
To provide a clarity comparison with Jaggys data ([63]) in the same shape but in 2.5D, the
maximum projection of nucleus area measurements data in her master thesis is given in figure
4.14.
89
(a) Box-plot showing cell nucleus area on various shapes of 2D micropatterned substrates.
(b) Box-plot showing cell body area on various shapes of 2D micropatterned substrates.
Figure 4.12: Box-plots showing the area of the cell nucleus, the cell body, and the ratio of both
on various shapes of 2D micorpatterned substrates. The bottom and top of the box are always
the first and third quartiles. The band inside the box is always the second quartile (median). The
whiskers extend to the most extreme data points in which the default is 1.5 IQR (Interquartile
Range). The green dots are the plot of the raw data.
90
(c) Box-plot showing the ratio of the cell-nucleus to the cell-body area ratio of cells on various shape on various
shapes of 2D micropatterned substrates.
Figure 4.12: (Continued) Box-plots showing the area of the cell nucleus, the cell body, and the
ratio of both on various shapes of 2D micorpatterned substrates. The bottom and top of the box
are always the first and third quartiles. The band inside the box is always the second quartile
(median). The whiskers extend to the most extreme data points in which the default is 1.5 IQR
(Interquartile Range). The green dots are the plot of the raw data.
91
(a) Cell nucleus area comparison in 2D.
(b) Cell body area comparison in 2D
Figure 4.13: Cell nucleus and cell body area comparison as the size of the islands are enlarged.
The enlarged island of 30 V-shape has dimension 50 m and 60 m. The enlarged 45 V-shape
has dimension 60 m. The enlarged L-shape has size 60 m.
92
Figure 4.14: A. Cells on substrates. B. Nucleus MIP area. C. Nucleus circularity. D. Nucleus
elongation. Magenta: fibronectin. Blue: DAPI staining. [63].
Comparing the nucleus area when cells adhere on the same shape but in different environments
(between 2D and 2.5D), the main different resides between the V-shape and equilateral triangle.
In 2.5D environments, the mean nucleus area on the open patterns (V-shape) has smaller values
than on the closed ones, whereas in 2D cases, it is in converse. The mean value in 2.5D was
about 140 m2 . In 2D it was about 115 m2 . For other shapes, they look quite similar. In 2D
cases, cell nucleus area on equilateral triangle shape is the smallest among other shapes. In
most 2.5D cases, as explained previously that the stress fiber orientation is prominent along the
direction of the bars of the structure. This indicates that the stress fiber distribution in 2.5D
equilateral triangle is more like a web to support the nucleus, so the compression acts axially
rather than radially as occurs in 2D equilateral triangle.
Figure 4.15a, 4.15b, 4.15c show the MIP measurements of the cell nucleus, cell body, and the
ratio of both respectively in 2.5D V-shapes with longer dimension and variation in angles.
To give a clear picture of nucleus area comparison in 2D (both for shorter and longer dimension
patterns) and in 2.5D, the quantitative data are given in figure 4.16. The data shows that in
longer 2D, the nucleus has bigger area than in shorter 2D and 2.5D. This indicates that in shorter
patterns and 2.5D, the actin cytoskeleton is denser near the nucleus so that it can compress the
nucleus radially. In contrast to longer 2D, the actin cytoskelton is not as dense as in shorter 2D
93
(a) Box-plot showing cell nucleus area in various shapes of 2.5D microscaffold.
(b) Box-plot showing cell body area in various shapes of 2.5D microscaffold.
Figure 4.15: The MIP area of the cell nucleus, cell body, and the ratio of both in various shapes
of 2.5D microscaffold.
94
(c) Box-plot showing the ratio of the cell-nucleus to the cell-body area ratio in various shapes of 2.5D microscaffold.
Figure 4.15: (Continued) The MIP area of the cell nucleus, cell body, and the ratio of both in
various shapes of 2.5D microscaffold.
4.3. Cell Nucleus Elongation
95
which gives more space the nucleus to expand radially.

It is also observed that the cell nucleus area are also relatively conserved as the cell body size
increases and put into 2.5D environment (see figure 4.16 and 4.17). It seems that the actin
cytoskeleton network in 2.5D environments radially limits the space of the nucleus, suggesting
that in 2.5D cases, the nucleus is also compressed radially like in the 2D shorter dimension.
Particularly for 2.5D environment, the effect of this actin cytoskeleton distribution near the
nucleus induces an indentation on the nucleus periphery as will be discussed in section Nucleus
Indentation.
4.3
Cell Nucleus Elongation
Figure 4.18 shows the results of cell nucleus elongation measurements when cells adhere on
shorter 2D patterns. There is no significant changes in elongation except for cells adhering on
arrow patterns.
Figure 4.19 shows the results of cell nucleus elongation measurements when the cells adhere and
spread on 2.5D microscaffold.
By comparing the mean values of the nucleus elongation with Jaggys data, the smallest
elongation is given in the case of equilateral triangle. Her data also shows that the most
elongated nucleus is when cells spread on L-Shape. Likewise, the nucleus was more elongated
when the size of 2.5D V-shape is varied in angles and dimensions as shown in figure 4.19. Having
compared these values, the nucleus is more elongated in 2.5D than in 2D. To support this idea,
the nucleus elongation is given in more detail as displayed in figure 4.20. So, it is clear that the
nucleus is more elongated in 2.5D environments.
4.4
Cell Volume and Surface Area
For the volume and area measurements, some results are given in table 4.1.
Table 4.1: 3D Geometric Measurements of the Cells Spreading on 2.5D structures
Structure
V-Shape, 30
V-Shape, 45
V-Shape, 60
L-Shape
U-Shape
Channel
Actin
Nuc
Actin
Nuc
Actin
Nuc
Actin
Nuc
Actin
Nuc
Volume (3 )
Raw
Decon
3432.02 2327.14
1227.82 604.74
2849.93 1876.56
744.51
571.10
1763.62 1209.45
624.87
558.27
3525.08 1984.94
449.86
357.72
4879.25 3554.98
1393.60 1204.00
Surface Area (2 )
Raw
Decon
5009.12 4375.62
626.54
398.93
3672.98 3599.43
503.39
553.08
3842.71 3163.64
402.52
385.95
3902.66 3880.97
348.03
339.71
7570.48 7091.17
846.66
801.60
Karyoplasmic Ratio (N/C) Surf/Vol (1/m)

Raw Decon
Raw Decon
1.46 1.88
0.36 0.26
0.51 0.66
1.29 1.92
0.26 0.30
0.68 0.97
2.18 2.62
0.35 0.46
0.64 0.69
1.11 1.96
0.13 0.18
0.77 0.95
1.55 1.99
0.28 0.34
0.61 0.66
96
(a) The nucleus area in shorter dimension 2D vs longer dimension 2D
(b) The nucleus area in shorter dimension 2D vs 2.5D
Figure 4.16: The comparison of nucleus area when cells adhere on shorter patterns, longer
patterns, and in 2.5D environments.
4.4. Cell Volume and Surface Area
97
(c) The nucleus area in longer dimension 2D vs 2.5D
Figure 4.16: (Continued) The comparison of nucleus area when cells adhere on shorter patterns,
longer patterns, and in 2.5D environments.
98
(a) Cell body area comparison in 2D shorter micropatterned substrates and 2.5D
(b) Cell body area comparison in 2D longer micropatterned substrates and 2.5D
Figure 4.17: Cell body area comparison as the size of the islands increases and put in the 2.5D
environment. The 2.5D shape has a length of 80 m.
4.5. The Actin Cytoskeleton Supports the Cell Nucleus in 2.5D Environment
99
Figure 4.18: Box-plot showing cell nucleus elongation on various shape of 2D patterns.
It is quite difficult to extract information from the data in table 4.1 because it has statistical
problem in the sense that it is still lack of number of data. In each structure, the number data
was only one to be processed. In spite of this problem, one remark that is quite significant is
the L-shape. As outlined previously that in the 2.5D L-shape, the nucleus is the most elongated.
This is even more confirmed by looking at its surface to volume ratio which is high, because 2
objects of the same volume, however, the more elongated the object, the larger the surface area
to volume ratio. Furthermore its karyolasmic ratio is the smallest among other shapes.
Changes in nuclear shape or volume would alter the properties of the pore complex of the
nuclear membrane which regulates the transport of nucleic acids, proteins, and ions between the
cytoplasm and nucleus [74, 75]. Furthermore, it was demonstrated that cells which are more
flattened would have greater functional size of nuclear pores and therefore the rate of transport
of nucleoplasmin-coated gold particles into the nucleus was also greater [74, 6]
4.5
The Actin Cytoskeleton Supports the Cell Nucleus in

2.5D Environment
In 2.5D environment, the nucleus is supported by the actin cytoskeleton so that it can stays
at a certain height from the ground. This situation is depicted in figure 4.21a and 4.21b. To
quantitatively asses this circumstance, the actin cytoskeleton density was measured at the bottom
and at the top side of the nucleus (see the Materials and Methods for processing this problem).
The bottom side is defined as the slices from the middle section of the nucleus goes to the first
100
Figure 4.19:
Box-plot showing cell nucleus elongation on various shapes of 2.5D
microscaffolds.
slice. The top side is defined from the middle section goes to the last slice. And the middle
section is defined by estimating the middle slice number of the deconvolved nucleus since the
deconvolved one provides a clear segmentation at the top and the bottom sides. The quantification
results are given in figure 4.21c.
The total density at the bottom side of the nucleus was greater than that at the top side. At the
bottom side, the integration gave a value of 0.605, while at the top side it gave 0.395. These
values seem reasonable given that the actin cytoskeleton at the bottom side has to support the
weight of the nucleus and the actin cap. However, there might be another possibility that if
the tensions (surface and line tension) of the actin cytoskeleton at the bottom side are strong
enough to counter the weight of the nucleus and the actin cap, then the density does not have to
be greater at the bottom side than at the top side. However, this needs further investigations.
4.6
Nucleus Indentation
It was also observed an indentation happening on the periphery of the nucleus (see figure 4.22).
To investigate further this indentation, the nucleus area in every slices of the images are calculated
and the results are displayed in figure 4.23.
4.6. Nucleus Indentation
101
(a) The nucleus elongation comparison when cells adherent on shorter dimension of 2D and 2.5D
(b) The nucleus elongation comparison when cells adherent on longer dimension of 2D and 2.5D
Figure 4.20: The actin cytoskeleton supports the nucleus from the bottom side. The 2.5D
U-shape has a dimension of (30 80) m.
102
(a) The actin cytoskeleton and the nucleus viewed

from the top side
(b) The actin cytoskeleton and the nucleus viewed

from the bottom side
(c) The density of the actin cytoskeleton at every slices
Figure 4.21: The actin cytoskeleton supports the nucleus from the bottom side. The 2.5D
U-shape has a dimension of 30 80 m.
103
(a) Before deconvolution
(b) After deconvolution
Figure 4.22: Indentation occuring on the perihery of the nucleus of the cell spreading on 2.5D
U-Shape. The structure has the length of 30 80 m.
104
(a) Nucleus indentation of cells spreading on 2.5D 30 V-(b) Nucleus indentation of cells spreading on 2.5D 45 VShape
Shape
(c) Nucleus indentation of cells spreading on 2.5D 60 V-(d) Nucleus indentation of cells spreading on 2.5D L-Shape
Shape
(e) Nucleus indentation of cells spreading on 2.5D U-Shape
Figure 4.23: Cell nucleus area in every slices showing an indentation happening around the
middle section of the nucleus.
105
It turns out that this indentation might be the reason of why the nucleus are more elongated
in 2.5D environment than in 2D environments (see figure 4.20b). The tension of the actin
cytoskeleton near the periphery of the nucleus is that strong in such a way that it can induce an
indentation. This indentation also proves that the compression of the nucleus in 2.5D environment
is the reason of why the MIP nucleus area in 2.5D environments is comparable with the one on
shorter patterns. It means that the compression of the actin cytoskeleton also contributes to the
shrinkage of the nucleus area (or MIP area).
106
107
108
Chapter 5
Discussion and Outlook
This study has shown that the actin cytoskeleton has roles in affecting the nucleus geometry.
However, there are some remarks that need further investigations.
Concerning the experiment of cells adherent on longer size of micropatterned substrates, these
experiments might give clues about the mechanism of cell size and shape control which are an
important attribute of cells to regulate their growth. Cells that are too small or too large would
lead to the asymmetrical cell division due to miss-positioning of the centrosomes (see figure 5.1)
[1].
Concerning the nucleus indentation, it also still needs further investigations by giving more
datasets. Furthermore, the way to observe nuclear plasma indentation needs to be improved by
using, for example, dyes that binds specifically to the nuclear membrane.
3D geometrical measurements of cells, for example, karyoplasmic ratio and surface to volume
ratio, are important to have further investigations because cell shape and cell size are related with
the volume regulatory mechanism. Furthermore, it was demonstrated that the actin cytoskeleton
has been linked to the activity of ion channels in the cell membrane, hence it plays a part in
secretion and volume regulation [76]. Additionally, nuclear deformation like the indentation is
quite interesting events if this indeed happens because nucleus also showed undulation when
cells are in a hyper-osmotic condition (see figure 5.2). Hyper-osmotic stress decreased nuclear
size and increased nuclear lacunarity, indicating that while the nucleus was getting smaller, the
pores and channels interdigitating the chromatin had expanded [77].
Physical connection that links the actin cytoskeleton to the nucleus might induces conformation
of macromolecules inside the nucleus during the activities of the actin cytoskeleton. One way to
assess this is by measuring the chromatin condensation parameter [78] to see if the defined ECM
contribute to the chromatin condensation. The level of chromatin condensation is a measure of
the silencing/activation of chromosomal territories and therefore impacts on gene expression [78].
In addition, assessing the chromatin condensation can also be measured by deconvolving the 3D
stack of nucleus image with using different input parameter of SGP deconvolution algorithm.
An example of this deconvolution is given in figure 5.3.
The difference of the actin filament networks density between the bottom and top of the nucleus
109
110
Chapter 5. Discussion and Outlook
(a) Left: Normal cell division. Middle: Asymmetrical cell division because
centrosomes do not reach the cell centre. Right: Asymmetrical cell division
because centrosome position is unstable.
(b) Left: Normal chromosome segregation. Middle: Insufficient spindle

elongation because the cell size is too large causes astral microtubules do not
reach the cell cortex. Right: Insufficient chromosome segregation because of
the limited space.
Figure 5.1: Possible scenarios of centrosome centering [1].
in 2.5D environment can be further investigated by flipping the cover glass sample after cells
fully spread over the structure to change the gravity direction. This is done to check if the
quantification of the actin filament density shows the same behaviour (we observed in a single
measurement a higher actin density at the bottom than at the top of the nucleus). It is also point
111
Figure 5.2: Schematic drawing showing changes in nuclear morphology with osmotic stress
[77].
of interest to check the actin density in 2D environment at the top and bottom side of nucleus
and then compare the behaviour with the 2.5D cases.
The last remark is about the number of datasets that are still lacking. The experiments which are
lacking of datasets are the longer size of 2D micropatterned substrates and 2.5D microscafolds.
It would give a more convincing quantification when the number of data is sufficient.
112
(a) Volume rendering of the nucleus after deconvolution, showing the

chromatin structure.
(b) Surface rendering of the nucleus after deconvolution, showing the

chromatin structure.
Figure 5.3: Different input parameter of SGP deconvolution algorithm may show the chromatin
structures.
113
114
Chapter 6
Summary
This study demonstrates that the actin cytoskeleton and the actin stress fibres are links that
connects the cell environment to the nucleus geometry (the elongation, long axis orientation,
area, volume, and surface area). By collecting the results of the nucleus and actin stress fiber
orientation, the study of cells adherent on micropatterned V-shape substrates and 2.5D V-shape
microscaffolds gives indication that the nucleus orientation seems to be defined by the dominant
stress fiber orientation.
The design of the ECM defines the cell shape. The cell shape is characterized by the actin
cytoskeleton distribution in the whole cell and the actin stress fibers that define the cell body
structure and the nucleus structure. The actin cytoskeleton and the stress fiber orientation
near the nucleus contribute in deforming the nucleus leading to the changes in the elongation
and cross-sectional area. Cells on the enlarged V-shape structures(in angle and dimension
in comparison to the size of the nucleus, e.g. 60 and 50 m length) have their long axis in
parallel with the hypotenuse of the patterns, giving a clue that the nucleus elongation will guide
the mitotic spindle to rule the cell division orientation. Thus, this makes clear that the actin
cytoskeleton and the actin stress fibers play a role in determining the cell division.
However, the study of adherent cells on the arrow and the crossbow shapes also reveal important
results that seem to contradict with the Hertwig rule stating that cells tend to divide along the
cell long axis. The study on the arrow and the crossbow show that cell long axis orientation
is not the main actor for determining the cell division, but the spatial location of the adhesive
region leading to the specific distribution of cortical cues. Furthermore, the distribution of
cortical cues can be predicted by looking at the dominant orientation of stress fibers calculated
by the structure tensor.
Coming to the studies in 2.5D environments brings new perspectives to the roles of the actin
cytoskeleton. It turns out that the nucleus has to be supported in such environments. And this
task is done by the actin cytoskeleton network. Moreover, the actin distribution near the nucleus
in 2.5D environment seems to compress the nucleus in such a way that the nucleus is more
elongated in this environment than in 2D environments. Furthermore, the cross-sectional nucleus
area is reduced and induces an indentation near the periphery.
115
116
Chapter 6. Summary
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Acknowledgment
The author would like to thank people who were involved in this work:
1. Prof. Martin Bastmeyer as the head of the Institute of Zoology, KIT who has given a great
opportunity to conduct this Master Thesis.
2. Dr. Franco Weth as the main supervisor for his helpful suggestions and stimulating ideas.
3. Benjamin Richter and Tatjana Autenrieth as the direct supervisors who are mainly involved
in this thesis. Thanks for their great patience and care in giving training, sharing their
knowledge, and stimulating discussion.
4. The member of the Cell Group in the institute: Andrea Scheiwe, Hao Chen, Michael
Bachman, Mona Jaggy, Stephanie Frank for giving their valuable time in sharing their lab
experiences and fruitful discussion.
5. Marco Linke from the group of Ulrich Schwarz at the University of Heidelberg for his
great advices in image processing tasks and critical reading of the manuscripts.
6. Riccardo Zanella from the Department of Matemathics and Computer Sciences at the
University of Ferrara, Italy for his helpful comments in performing Scaled Gradient
Projection (SGP) deconvolution.
7. David Legland from INRA - Institut National de la Recherche Agronomique, French for
his helpful comments in measuring the volume of 3D objects by using the computation of
Minkowski measures.
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T HE I NFLUENCES OF THE ACTIN

C YTOSKELETON ON THE P ROPERTIES OF

Examiner: Prof. Dr. Martin Bastmeyer

Declaration of Original Work

Karlsruhe, den 30.04.2014.

Materials and Methods

Results and Data Analysis

Discussion and Outlook

Figure 1.1: In situ vs in vitro cell microenvironment [10].

Actin Stress Fibers

Chapter 2. Theoretical Background

(a) Models of stress fibers structure and contractility [18]

(b) Model of stress fibers formation [18]

(c) Types of stress fibers in cultured animal cells [19]

Figure 2.1: Actin stress fibers structures

2.2. Cell-Matrix Adhesion Contacts

Cell-Matrix Adhesion Contacts

Chapter 2. Theoretical Background

2.2. Cell-Matrix Adhesion Contacts

(a) Formation of focal complexes

(b) Formation of focal adhesions (or focal

(c) Fibrillar adhesions are pulled out from

Chapter 2. Theoretical Background

Figure 2.5: Schematic diagram of adhesion assembly stages [20].

2.4. Image Formation

2. Buckling with high aspect ratio plates

(a) Stamp with rectangular cross section

(c) Lateral Collapse

(d) Roof Collapse

Figure 2.6: Micropattern stamp and its failure modes [30]

Chapter 2. Theoretical Background

2. Degradation because of the process of image recording.

Degradation Due to the Blurring Process

2.4. Image Formation

Chapter 2. Theoretical Background

Figure 2.8: Expansion of a unit-step function using Fourier series [33].

2.4. Image Formation

(a) Original image without frequency components

(c) High-pass filtered image

(b) Low-pass filtered image

(d) Band-pass filtered image

Figure 2.10: A generalized model of an imaging system [35].

Chapter 2. Theoretical Background

Figure 2.11: Diffraction limited according to Abbe theory [35].

2.4. Image Formation

f (x0 , y 0 )h(x, y; x0 , y 0 )dx0 dy 0

Chapter 2. Theoretical Background

f (x0 , y 0 )h(x x0 , y y 0 )dx0 dy 0

Degradation Due to Noise

f (x0 , y 0 )h(x, y; x0 , y 0 )dx0 dy 0 + n(x0 , y 0 )

2.5. Image Restoration

Chapter 2. Theoretical Background

given by equation 2.7.

Non-iterative Image Restoration

2.5. Image Restoration

(a) Original image

(b) Actin filaments in grayscale

(c) Actin filaments after inverse filtering

Chapter 2. Theoretical Background

equation 2.9 would be more dominant in high frequency regime.

2.5. Image Restoration

[Fb (x, y) F (x, y)]2 dxdyi

(a) Original image