Thesis-Farida Yasmin-final

Dependency of Cell Mechanics on
Substrate Topography
Dissertation submitted in partial fulfilment of the requirements
for the degree of “Master of Science” (MSc) of the Program of
Advanced Materials Ulm University
Submitted by
Farida Yasmin
849697
farida.yasmin@uni-ulm.de
Dhaka
June 2016

Head of the Department
Prof Dr. Kay E. Gottschalk
First Supervisor:
Prof Dr. Kay E. Gottschalk
University Ulm
Second Supervisor:
Prof Dr. Rolf Brenner
Universitätsklinikum Ulm

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Contents
Abbreviations …………………………………………………………………………………………………………………………..10
Abstract ……………………………………………………………………………………………………………………………………11
Chapter 1. Introduction............................................................................................................12
Materials Science And Engineering ................................................................................12
1.1.1 Nanomaterials ........................................................................................................12
1.1.2 Biomaterials............................................................................................................13
Importance of Studying Cell Mechanics In Biomaterials................................................14
Chapter 2. Background Information.........................................................................................15
An Introduction To Cell...................................................................................................15
Cell –Surface interaction ................................................................................................17
Theory of Elasticity (Hertz Contact Model) ....................................................................18
Chapter 3. Experimental...........................................................................................................20
Cleanroom Technique ....................................................................................................20
Spin Coating: Deposition By Spinning.............................................................................21
Dip Coating: Preparation of Nanoparticles.....................................................................22
H2 Plasma........................................................................................................................24
Photochemical Deposition .............................................................................................25
Plasma Etching: Reactive Ion Etching (RIE) ....................................................................26
3.6.1 Effects of Oxygen addition .....................................................................................28
3.6.2 Effects of Hydrogen addition..................................................................................28
3.6.3 Effects of CHF3 and Noble gas addition..................................................................28
Electron Microscope.......................................................................................................29
3.7.1 Scanning Electron Microscopy (SEM).....................................................................29
3.7.2 Atomic Force Microscopy (AFM)............................................................................30
Cell Culture Preparation .................................................................................................34
Chapter 4. Results and Discussion............................................................................................35
Fabrication of Nano-Pillars .............................................................................................35
4.1.1 Micellar technique by using block copolymer........................................................35
4.1.2 Photochemical growth of Gold (Au) particle..........................................................37
4.1.3 Reactive Ion Etching (RIE).......................................................................................38
Cell Mechanics on Nanostructure Topography..............................................................41
4.2.1 Indentation depth...................................................................................................41
4.2.2 Measurements........................................................................................................42
Cell-Surface interaction..................................................................................................53
Chapter 5. Conclusion ..............................................................................................................57
Chapter 6. References..............................................................................................................58

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List of Tables
Table 3-1 Preparation of micellar solution.....................................................................................23
Table 3-2 Description of different types of filter............................................................................24
Table 3-3 Preparation of the gold solution for photochemical growth .........................................26
Table 3-4: Settings of AFM .............................................................................................................33
Table 4-1: Experimental data of particle diameter, etching time and average height ..................40
Table 4-2: Elastic modulus with different Samples and Spring Constant.......................................53
List of Figures
Figure 1-1: Correlation of nano and biotechnology [8]..................................................................13
Figure 2-1: Schematic diagram of a Eukaryotic cell [18] ................................................................15
Figure 2-2: Schematic diagram of Fibroblast in ECM [22]..............................................................16
Figure 2-3: Schematic diagram of components of cytoskeleton, a) Microtubules b) Intermediate
filament c) Actin filament [25] .......................................................................................................17
Figure 2-4: Movement of cell by crawling over the surface [28] ...................................................17
Figure 2-5: Schematic representation of a cell possessing different types of forces [30] .............18
Figure 2-6: Schematic diagram of Hertz contact model with a spherical tip (radius R2), loading
force F, Cell radius R1, a is contact radius and δ is indentation depth [34]....................................19
Figure 3-1: Classification of cleanroom [39]...................................................................................20
Figure 3-2: schematic diagram of different steps of spin coating: a) dispensation of photoresist,
b) acceleration, c) spreading of the liquid, d) evaporation [40].....................................................21
Figure 3-3: Schematic diagram of stages of dip-coating process [46]............................................22
Figure 3-4: Preparation of Au micellar solution [50]......................................................................23
Figure 3-5: Structure of PS-b-P2VP blocked copolymer, modified from [51] ................................23
Figure 3-6 Photochemical growth of Au particle [54] ....................................................................25
Figure 3-7: Schematic diagram of optical system of mask aligner Karl SUSS MJB 3 Mask UV 400
[58] .................................................................................................................................................26
Figure 3-8: Schematic representation of the process of etching adopted from [61] ....................27
Figure 3-9: Schematic diagram of Scanning Electron Microscope (SEM) [66] ...............................29
Figure 3-10: Emission of various electrons and electromagnetic waves from the specimen [65] 30
Figure 3-11: Schematic diagram of AFM [69].................................................................................31
Figure 3-12: Schematic diagram of scanning system in AFM, redrawn from [70] .........................31
Figure 3-13: Force spectroscopy mode in AFM [71] ......................................................................32

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Figure 3-14: Calibration of cantilever spring constant, blue line is for cantilever resonance peak
(measured) and red is the Lorentz fit [71] .....................................................................................33
Figure 4-1: Plot of average interparticle distance over withdrawal velocity from experimental
data, from figure it is seen that for 130nm interparticle distance the withdrawal speed is
2.8mm/min.....................................................................................................................................36
Figure 4-2: Interparticle distance changes depending on the concentration of the solution [58] 36
Figure 4-3: HRSEM image of Au nanoparticle after H2 Plasma. A) Grey scale picture and B) after
adjusting threshold, inset hexagonal arrangement (bandpass filter). Scale is 1µm, 30kV. ...........36
Figure 4-4: Plot of Au NPs diameter as a function of exposure time from experimental data......37
Figure 4-5: HRSEM images (at 30kV) of different diameter(average) of Au NPs, a) 9nm, b) 17nm,
c) 30nm, scale is 200nm. ................................................................................................................37
Figure 4-6: a) grey scale image using HRSEM scale 200nm at 30kV; b) after adjusting threshold;
and c) marking for area measurement through imageJ software .................................................38
Figure 4-7: Schematic diagram of a) formation of SiF2 and b) formation of SiF4 [79]...................38
Figure 4-8: Etching rate changes depending on a) DC bias and b) size of the mask [77]...............39
Figure 4-9: Au NPs in 200nm scale, tilted by 30 degree a) average height 75nm and diameter
28nm with Au particle diameter 17nm, b) height 75nm and diameter 49nm with Au particle
diameter 30nm, and c) height 106nm and diameter 49nm with Au particle diameter 30nm ......39
Figure 4-10: AFM pictures (topography) of Samples 2 (130-28-75)...............................................40
Figure 4-11: Height of pillars build by Gwydion, profile 1 corresponds to red line and profile 2
corresponds to yellow line from Figure 4-10 right part. ................................................................41
Figure 4-12: Schematic diagram of indentation test (top) and force-indentation curve (bottom)
[14] .................................................................................................................................................41
Figure 4-13: Sample 1: 130-49-106, Hertzfit indentation depth 100nm, figure A shows the
topography of the cell, C is force-distance curve, B is ‘Young’s modulus error’ vs ‘indentation
depth’ and D is ‘Young’s modulus ‘vs ‘Indentation depth’ curve...................................................42
Figure 4-14: Histogram of Young’s modulus by using Hertz model of 3T3 fibroblast of Sample 1 at
indentation depth 100nm. X-axis is in logarithm, Y-axis is linear scale (calibrated spring constant
0.275 N/m), different colours produced by the addition of one cell with other and this results
come from summation of 10 cells data..........................................................................................43
Figure 4-15: Histogram of Sample 2 at indentation depth 100nm 30 cells measurement,
(calibrated spring constant-0.125N/m), different colours arise by adding one cell with other ....44
Figure 4-16: Histogram of sample 3a at indentation depth 100nm, with spring constant 0.084
N/m, different colours arise from summation of all cells (6cells measurement). .........................45

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Figure 4-17: Histogram of Sample 3b at indentation depth 100nm (5cells measurement) with
calibrated spring constant 0.275 N/m, different colours arise from summation of all cells. ........45
Figure 4-18: Histogram of Sample 1 (top, 10 cells) and Sample 3b (bottom, 5 cells), frequency
scale is different due to the different number of cells measurements, calibrated spring constant
0.275 N/m. Different colours produced by the addition of one cell with other............................46
Figure 4-19: Boxplot for comparing the median of different samples at indentation depth-100nm
........................................................................................................................................................47
Figure 4-20: All samples: Sample 1: 130-49-106, Sample 2: 130-28-75, Sample 3a: 130-49-75 and
Sample 3b:130-49-75 .....................................................................................................................48
Figure 4-21: Boxplot of all samples of Young’s modulus over selected height: 0.4-0.6, 0.9-1.1, 1.4-
1.6 and 1.9-2.1 µm .........................................................................................................................49
Figure 4-22: Young’s modulus VS Height plot of 3 samples: Sample1: 130-49-106, Sample2: 130-
28-75, and Sample3b: 130-49-75 ...................................................................................................49
Figure 4-23: Boxplot of 3 samples of Young’s modulus over selected height, the selected heights
are: 0.4-0.6, 0.9-1.1, 1.4-1.6 and 1.9-2.1 µm .................................................................................50
Figure 4-24: 3D plot of Young’s modulus over height of 3 samples, colour scale bar shows how
frequent the combination of young’s modulus and height was measured, left figures are for the
cell and right figures are for the substrate.....................................................................................52
Figure 4-25: AFM picture (topography) of Sample 1 after background corrections, scale 5µm,
colour scale bar shows the height measured.................................................................................54
Figure 4-26:HRSEM pictures at 5kV, Cells spread over the surface of all types of samples; a)
Elongated triangular shape with formation of microvili, scale 20µm, b) without microvili, scale
30µm c) the formation of lamellipodia and filopodia (yellow arrows), scale 5µm........................55
Figure 4-27: HRSEM picture taken at 5kV, 30 degree tilted, Cell moves attaching the top of the
pillars in all samples (black arrows) a) Sample 2 scale 100nm, b) Sample 1, scale 1µm and c)
Sample 3b 1µm...............................................................................................................................56

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‘Everybody is a genius. But, if you judge a fish by its ability to climb a tree, it will spend its
whole life believing that it is stupid.’
Albert Einstein

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This work is dedicated to my beloved husband who has always inspired me,
supported me and encouraged me. Also to my parents and my little sister
who have always prayed for me and my brother for guiding me.

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DECLARATION
I hereby declare that I wrote the present dissertation with the topic: Dependency of Cell
Mechanics on Substrate Topography independently and used no other aids than those cited. In
each individual case, I have clearly identified the source of the passages that are taken word for
word or paraphrased from other works. I also hereby declare that I have carried out my
scientific work according to the principles of good scientific practice in accordance with the
current “Satzung der Universität Ulm zur Sicherung guter wissenschaftlicher Praxis“[Rules of the
University of Ulm for Assuring Good Scientific Practice].
…………………………..
Farida Yasmin
Ulm, 10.06.2016

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ACKNOWLEDGEMENT
I’m obliged to my first supervisor Prof. Dr. Kay E. Gottschalk to give me this opportunity to work
in a wide field of Biomaterials & Biophysics, also I show my gratitude to Prof. Dr. Rolf Brenner to
be my second supervisor. My deepest respect to my Ex-supervisor Dr. Alfred Plettl, I’m grateful
to Dr. Axel Seidenstüker to train me well in Cleanroom Technique, Scanning Electron Microscopy
and for being nice to me. I show courtesy to Fabian Endele, Tanja and Anja for helping me during
my work. I’m thankful to Patrick Paul to introduce me to Atomic Force Microscopy. I mostly
grateful to Nicole Sieber for helping me a lot with measurements. I want to thank Ulla Nolte to
do the cell culture for me. I want to thank my husband Dr. Mohammad Abbas Uddin, my friend
Sean Harvey and Dr. Nabiul Hassan, and Patrick Paul for being my proof reader.

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ABBREVIATIONS
AFM Atomic Force Microscopy
Au NPs Gold nanoparticles
ECM Extracellular matrix
HRSEM High Resolution Scanning Electron Microscopy
PMMA Poly methyl methacrylate
P2VP Poly(2-vinylpyridine)
PS-b-P2VP
BCMT
Polystyrene-block-Poly-2-vinylpyridin
Block Copolymer Micellar Technique
RIE
Silicon
F
Ar
He
H
HF
CF4
CHF3
E
3T3
Sccm
Reactive Ion Etching
Si
Fluorine
Argon
Helium
Hydrogen
Hydrogenflouride
Tetrafluoromethane
Trifluoromethane
Elastic constant
3-day transfer, inoculum 3 x 105
cells
Standard Cubic centimetres

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ABSTRACT
Mechanical property of materials behave differently when they are subjected to another
materials with different topography, particularly for biological cells. Cytoskeleton and nucleus,
components of biological cells, varies in their mechanical properties such as cell stiffness and
rigidity when another material is applied. Also, depending on the topography cell stiffness
changes. In this regard, the nature of interaction between soft materials like cell to hard
materials like Silicon is worth exploratory. Silicon has been used as a biomaterials for long
however, very few researches were carried out to measure the mechanical properties of
fibroblast cells on nanostructure of Silicon. Therefore, this study investigated the mechanical
property of 3T3 fibroblast (mouse embryonic dermal cell) on Silicon nanostructure surface and
corresponding interaction between them. In this work Silicon nano-pillars were fabricated from
n-type Silicon wafer with different heights and diameters.
Characterization of the fabricated nano-pillar surface was carried out with HRSEM (High
Resolution Scanning Electron Microscopy), and the elasticity of cell on substrate was measured
by Atomic Force Microscopy (AFM). It was observed that elasticity of substrate was increased
from 0.13MPa to 1MPa when the diameters and heights of nano-pillar were increased. On the
other hand, elasticity of 3T3 fibroblast varies with diameter and height of the Silicon substrate of
which the lowest diameter and height of pillars have highest cell stiffness. It was also found that
pillars with same diameter but with different heights have similar elasticity for cells and
substrates which suggests that different pillar heights do not have significant effect on elasticity.
Increasing the cell thickness the Young’s modulus decreased, indicates that leading edge have
higher stiffness than other part of the cell. Cells were well spread and firmly attached on the
Silicon nanostructure and movement of cell was taken place on the top of the pillars.

Chapter 1: Introduction Page 12 of 63
Chapter 1. INTRODUCTION
This chapter discusses the methods for fabrication of nanostructure substrates and the
relationship between the nanotechnology and biotechnology. In addition, the importance of
studying cell mechanics are explained.
MATERIALS SCIENCE AND ENGINEERING
Materials Science refers to the investigation of the relationship between the structure and
property of materials whereas materials engineering focused on the design and synthesis of the
new products or materials. Structure and property are the most important in materials science
and engineering, including processing and performance that influences the structure and
property of materials [1]. Nanomaterials exhibits some special chemical, mechanical, optical,
magnetic and electronic properties on surface due to the size of the material on micro to nano
scale. The optical property of nanomaterials varies a lot from the bulk material due to the
differences in the refractive index which measures the electromagnetic radiation. Similarly
mechanical properties such as strength and elastic modulus of bulk materials will be different for
nanomaterials [2].
1.1.1 Nanomaterials
Nanomaterial is regarded as the materials with a size range of 1-100 nm. Nanotechnology refers
to the process and synthesis techniques and characterisation of nanomaterials (crystalline and
amorphous) in detail. The synthesis or fabrication of nanomaterials is important due to its small
size and the mass with high surface area [3].
There are two conventional methods for fabrication, bottom-up and top-down. In top-down
approach, the bulk material are chopped, layer by layer, to a small material [4]. Consequently,
the waste is high but the method is fast and has very good control on particles shape and
spacing. Photo-lithography, Reactive Ion Etching (RIE), ball milling etc. are considered as the top-
down process [5]. On the other hand, the bottom-up process is similar to making a building by
placing bricks one by one. Nanostructure manufacturing or synthesizing is carried out by
assembling atoms or molecules. This process can be carried out from homogeneous nucleation
from liquid and vapour or heterogeneous nucleation on substrate, which is able to give properly
ordered nanostructure by means of building block. Wet chemistry routes e.g. precipitation,
reduction, sol-gel process, chemically and topologically pattern surface, organic block copolymer
are the most common types of bottom-up process to fabricate nanostructure [3].

1.1.2 Biomaterials
Biomaterial science mostly focuses on the interaction of materials in the biological environment
although the study is surrounded by physics, chemistry, biology, engineering and medicine.
‘Biomaterials’ as defined by DF Williams in 1987, ‘is a nonviable material used in a medical
device, intended to interact with biological system ’[6] . The material that should be used as
biomaterial should have biocompatibility and can perform specific tasks. The materials should
not be toxic, should be mechanically stable, should not make corrosion or degradation in vivo
and should be non-carcinogenic [6]. Nanomaterials can be used as biomaterials which can help
to develop new device such as diagnostic sensor or drug delivery system with precise dosage [7].
Fig 1-1 shows the relationship between the nanotechnology and biology and application of
them. The dashed line indicates that there is a possibility to make bionanodevice and
bionanosystem.
Figure 1-1: Correlation of nano and biotechnology [8]
Presently several materials including polymers have been used as biomaterials, for example,
polyurethane used in heart valve, teflon in vascular graft, hydrogel as contact lenses,
hydroxyapatite in healing bones, titanium alloy and some ceramics i.e. alumina used in dentistry,
polyethylene in hip prosthesis etc. [3].
Silicon (Si) as a material has wide range of applications from the kitchen to computer chips to
the human body. The use of Si as a biomaterial has been going on for decades due to
biocompatibility and biodurability and other chemical properties such as low surface tension,
ans hydrophobicity. Si provides one of the best biodurability, however, Si elastomers have lower
tensile strength or tear resistance than other elastomers. Si elastomers have been used in blood
coagulation prevention since 1940 because of its hydrophobicity. Some other important

applications of Si are in orthopaedics (hand and foot implants) kidney dialysis, blood oxygenator,
aesthetic implants such as breast implant etc. [6] [9].
IMPORTANCE OF STUDYING CELL MECHANICS IN BIOMATERIALS
In the biomaterials industry it is crucial to study the cell-materials mechanics due to interaction
of cells with different materials, such as in implanted devices where cells interact with materials
at nanoscale. The topography of material has significant influence on cell response [10] for
example, when the substrate has nano island on the surface, the cell morphology and focal
adhesion will be significantly different from the flat surface [11]. Furthermore, depending on the
materials that cells are interacting, cell response will vary. Cells will interact with metals
differently than polymer due to the high stiffness and stability and ordered atomic structure of
metals [12]. Cell mechanics has a great effect on cell proliferation, migration and differentiation.
It is also necessary to investigate the mechanical response of the cells to an external force such
as any chemical and physical signals. Heart is beating by expansion and contraction so blood is
pumped out to the body consequently, that creates a mechanical stress to the cells [13].
Additionally, analysing nanomechanics is getting important in cancer cell research. The variation
in elasticity in normal cell and metastatic cell can be measured in nanomechanics by using
Atomic Force Microscopy (AFM) [14]. There are other ways for quantitative analysis of
mechanics of cells such as Micropipette aspiration (MA), Magnetic Twisting Cytometry (MTC),
Optical and Magnetic tweezers. However, there are limitations using these techniques, for
example, in AFM the results rely on the spring constant of the tip of the cantilever and the
interaction between the tip and the cell surface so, there is a chance that the mechanical
property might be misinterpreted [15] [16].
In this study, fabrication of nano-pillars of Silicon was done using bottom up and top down
methods to investigate the mechanical properties of cells more specifically; elasticity of 3T3
fibroblast using AFM.

Chapter 2: Background Information Page 15 of 63
Chapter 2. BACKGROUND INFORMATION
This chapter includes detailed information of cell structure, functions and mechanics of the cells-
surface interaction. For the statistics the Hertz model has been used therefore, the theory of
elasticity and Hertz Model are also explained.
AN INTRODUCTION TO CELL
Cells are living organisms and the basic unit of life, and therefore perform and control many
body functions. There are two main types of cells: Eukaryotic and Prokaryotic. Animal cells
belong to Eukaryotic type. The component of a Eukaryotic cell are Nucleus, Cytoplasm,
Cytoskeleton, Golgi apparatus, Endoplasmic reticulum etc. [17]. Figure 2-1 shows the
components of a Eukaryotic cell.
Figure 2-1: Schematic diagram of a Eukaryotic cell [18]
The cell is surrounded by an Extracellular matrix (ECM), which is a non -cellular component with
strong biochemical and biomechanical behaviour and are responsible for tissue morphogenesis.
Two main classes of ECM are proteoglycans (PGs) and fibrous protein which are collagen,
fibronectin, elastin and laminins. These have different shapes and sizes with structural and
adhesive functions [19] [20] . The components of ECM are responsible for organizing the
orientation of the matrix such as cytoskeleton, which is situated inside of the cell and it’s
orientation can be controlled by the matrix situated outside the cell [20].

Fibroblast are situated in the loose connective tissues in ECM (Figure 2-2) and plays an important
role by proliferating, migrating and producing the collagen matrix whenever a tissue is injured
which helps damaged tissue to be repaired. However, the skin fibroblast cells are different from
others and they show different plasticity in the same cell culture [21].
Figure 2-2: Schematic diagram of Fibroblast in ECM [22]
Every eukaryotic cell possess an internal skeleton called the cytoskeleton. There are three main
components of cytoskeleton: actin filaments, microtubules and intermediate filaments. These
components are associated with proteins [23] and give the cell mechanical stability, shape and
capability to move from one place to another [17]. They form a network that inhibits any
deformation but when any force is applied form outside they can reorganize and maintain the
intracellular arrangement. The order of stiffness between three components are, microtubule>
actin filament> intermediate filament. Actin filaments are highly organised by proteins, and
possess isotropic, bundled and branched networks which are responsible for cell to cell
communication. Actin filaments and microtubules are associated with polarized subunit of
polymer, but intermediate filaments are not polarized and not able to assist with movement of
the cell [24]. Figure 2-3 shows the structure of the three components where, microtubules is the
biggest in diameter compare to others.

Figure 2-3: Schematic diagram of components of cytoskeleton, a) Microtubules b) Intermediate filament c)
Actin filament [25]
The link between ECM and cells is maintained by Integrin, a cell adhesion molecule, and a
receptor protein. Integrin usually has two subunit of transmembrane glycoprotein called α and
β, which are non-covalently associated [26].
CELL –SURFACE INTERACTION
Fibroblast moves over the surface by crawling smoothly in cell culture and when it moves, it
makes elongated triangular formation. One of the triangle sides forms lamellipodia, but the
other two sides try to move backward or even remain motionless [27]. Movement involves three
steps, the leading edge extend and attach to the substratum then backside of the cell is pulled
forward, figure 2-4 [28].
Figure 2-4: Movement of cell by crawling over the surface [28]

Living cells feel different types of forces such as shear stress, compression and stretching and
hence have contractility which has an impact on cell functions. Wound healing, migration and
cytokinesis are regulated by the cell’s contractility in which the interaction mechanism between
the proteins of actin filament are responsible [29]. In figure 2-5 shows how the living cell binds
to the surface through integrin and can feel some forces.
Figure 2-5: Schematic representation of a cell possessing different types of forces [30]
When living cells are subjected to a substrate, they interact through transmembrane receptors
like integrin, a component of ECM on the substrate. Integrin forms a complex in the intracellular
side through focal adhesion and as a result ECM connects to the actin cytoskeleton or stress
fibres. Cells are also able to sense the substrate rigidity and therefore, adopt their structure and
can respond to forces as little as 5 pN [30].
THEORY OF ELASTICITY (HERTZ CONTACT MODEL)
Elasticity is defined as the regaining of the original formation when an applied forces is removed,
as the applied forces causes deformation of the structure. All materials possess elastic property
to a certain point [31]. Elasticity is described by Hooke’s law in terms of stress (𝛔) and strain (ε)
where, stress is an externally applied force per unit area and strain is the amount of deformation
caused due to force [1] [32].
According to the Hook’s law stress is proportional to strain in the form:
𝛔 = Eε (2.3.1)
E is a constant and it is called elastic modulus and dependent on the materials [32]. However,
elasticity of soft materials such as biological materials is determined by Hertz Contact Model
which depicts the elastic deformation of two homogeneous bodies contacting each other. This
model is widely used to measure the elastic property in a time scale by collecting force-distance
curve [33]. This model considers some assumptions such as:

 The tip and cell material properties are homogeneous and isotropic;
 The geometry of the communication between the tip and cell is axisymmetric, steady and
smooth;
 The contact between two bodies is adhesionless and frictionless [34].
In this work spherical tip is used and the force (F) acting on the cell is described by
𝐹 =
4
3
𝐸∗
𝑅
1
2 𝛿
3
2 (2.3.2)
Here, E* is the effective Young’s modulus and R is the combined curvature of the tip (R1) and cell
(R2) and δ is the indentation depth. It also presumed that the cell is flat so its radius goes to
infinity. Since the tip is harder than the cell, the Young’s modulus of the tip is also considered to
be infinity [33]. For the force-distance curve and for fitting the curve the equation (2.3.1) will be
applied.
Figure 2-6: Schematic diagram of Hertz contact model with a spherical tip (radius R2), loading force F, Cell
radius R1, a is contact radius and δ is indentation depth [34]
The effective Young’s modulus is express as [33]
𝐸∗
=
𝐸
(1−𝜐2)
(2.3.3)
Where, 𝞾 is the poisson’s ratio and for Hertz model the assumed value for biological sample is
0.5 [35].
Combining two equation (2.3.1 and 2.3.2), the expression of the Young’s modulus is [33]
𝐸 =
3
4
𝐹(1 − υ2)𝑅−1/2
𝛿−3/2
(2.3.4)
It should mention that for the spherical indenter, the Hertz model will match satisfactorily if the
indentation depth is smaller than the curvature of the tip [36]. Although, the Hertz model is
lacking in giving absolute measurements it is still valuable to differentiate mechanical properties
between living cells and substrates.

Chapter 3: Experimental Page 20 of 63
Chapter 3. EXPERIMENTAL
This chapter discusses the materials and methods used in this study. This includes spin-Coating,
Dip-Coating, H2 Plasma, Photochemical growth and Reactive Ion Etching (RIE) for fabrication of
the nanostructure surface. Along with, the principle of the High Resolution Scanning Electron
Microscope (HRSEM) and Atomic Force Microscopy (AFM) explained here. An introduction to
Cleanroom technique also given. Cell culture preparation and calibration of AFM cantilever have
been explained.
CLEANROOM TECHNIQUE
Cleanroom is an environment clean from pollutants such as dust and chemical vapour.
Cleanroom technique controls temperature, humidity, pressure and electrostatic charge which
prevents contamination and maintain particulates i.e. oil, grease, hair etc. Cleanroom is required
to manufacture the healthcare products, pharmaceutical products as well as in industry research
on small particles [37].
The classification of cleanroom express as a number such as ‘class 100’, ‘class 1000’ and so on.
Classification depends upon the cleanliness of air in terms of particle diameter and density. For
instance, ‘Class 100’ means in each cubic foot there are less than 100 particles which are larger
than 0.5 microns and it is equal to ISO 5 cleanroom [38]. In this study fabrication was carried out
in cleanroom class 100.
Figure 3-1: Classification of cleanroom [39]

SPIN COATING: DEPOSITION BY SPINNING
Spin coating is a widely used technique to prepare a thin film on substrate in nanotechnology
because it is easy and fast and can be uniformly deposed on the substrate [40]. The precondition
for spin coating technique is the layer-building material should be viscous to float on the surface
and soluble in an evaporating solvent with an acceptable evaporating rate. it is also necessary
that the solvent has higher evaporating rate to get a homogeneous and failure free layer [5].
Coating the substrate should be on a turntable and the liquid should be placed on the middle of
the substrate. The rotation will start with a lower angular velocity which will be then rapidly
increased. The liquid solution then spread towards the substrate edge and consequently, a
uniform thin film will be formed [41] [42] [43]. Figure 3-2 shows different steps of Spin-Coating.
Figure 3-2: schematic diagram of different steps of spin coating: a) dispensation of photoresist, b)
acceleration, c) spreading of the liquid, d) evaporation [40]
The viscosity and density of the liquid photoresist and the thickness and the rotation time are
main factors in spin coating. Higher spin speed and longer spin time form thinner film. The
interaction between solution and air is not as strong compared to the interaction between the
substrate and the solution. The thickness of the layer depends on the viscosity and
concentration of the fluid. When the solvent evaporates, the concentration of the fluid increase
hence the viscosity is increased [44] [42].
In this study, Si (100) n-type (phosphorus) (CrysTec, Germany) wafer was used as substrate with
a diameter of 3 inch and thickness of 380 µm. A thick positive photoresist, PMMA (poly methyl
methacrylate) which is a commercial e-beam/deep UV photoresist supplied by ‘Allresist’ has
been used on Si wafer to coat the whole surface by using ‘Convac 1001S’. This coating helped to
adhere broken particles which was generated during the cutting of the wafer by diamond tip.

At first, PPMA was spread on the wafer at low speed (rpm 500) for 10 seconds. Later the speed
was increased to 2000 rpm and ran for 40 seconds in order to dry the solution. The samples
were cut into the size of 10x5 mm2
to be analysed in High Resonance Scanning Electron
Microscope (HRSEM). After cutting, the substrates were cleaned in ultrasonic bath with acetone
and isopropanol for 12 minutes to remove the photoresist, and immediately dried in nitrogen
flow to avoid the film coating of acetone and isopropanol.
DIP COATING: PREPARATION OF NANOPARTICLES
Dip coating method is an easy and faster method for the preparation of the thin film from a
chemical solution. There are several ways to dip-coat such as drain Coating, angle-dependent dip
coating and classical dip coat from different types of solution. Solution could be inorganic
precursor or metallo-organic precursor and can be prepared by hydrolysis or condensation or
self-organization. Self-assembly principle, a spontaneous organization due to non-polar
interactions i.e. H-bond, Van Der Waals force, London force, is one of the best methods for
nano-structuring and thin film formation. The chemical solution contains amphiphilic molecules
that are composed of a hydrophilic and a hydrophobic part [45]. This process is usually based on
three separate steps as shown in figure 3-3.
Figure 3-3: Schematic diagram of stages of dip-coating process [46]
i) Immersion and dwell time: The samples are dipped into the precursor with a
constant velocity and defined time. Time is needed for the interaction between the substrate
and the coating solution to complete the wetting.
ii) Deposition and drainage: The substrate is then pulled out with a constant velocity.
When the substrate is pulling out and upwards there is a flux and excessive solution is drained

out. This specific velocity is important for determining the distance between the particles on the
substrate surface.
iii) Evaporation: The evaporation from the fluid is occurred and hence the thin film
deposition is made [45].
In this work, PS-b-P2VP (Polystyrene-b- poly-2- vinylpyridine) was used which is a diblock
copolymer and supplied by ‘Polymer Source, Inc’. This is a better approach for making
homogenous nanostructure array after which the copolymer can be removed completely. The
polymer is used with an appropriate non-polar solvent, toluene, to get the deposition of the
nanoparticles nearly ordered array on flat surface [47]. Poly(2-vinylpyridine)(P2VP) is hydrophilic
and forms the core while Polystyrene (PS) is hydrophobic and forms the outer shell. PS-b-P2V
polymer is dissolved in toluene, therefore spherical reverse micelles formed in the solution. With
the addition of salt of gold (HAuCl4) in the solution and stirring, gold migrates into the core of
the micelles, figure 3-4. Micelles are loaded with the same amount of metal salt at the
equilibrium [47] [48] [49].
Figure 3-4: Preparation of Au micellar solution [50]
PS(1800)-b-P2VP(770) was mixed with toluene and kept for one week under magnetic stirring.
Gold (lll) Chloride hydrate salt from ‘Sigma-Aldrich’ was then added and kept for another week
under magnetic stirring to help the polymer to be self-assembled. The structure of the PS-b-
P2VP and the preparation of micellar solution are given below.
Figure 3-5: Structure of PS-b-P2VP blocked copolymer, modified from [51]
Table 3-1 Preparation of micellar solution
Item Identity Quantity
Polymer(PS-b-P2VP) 1800-770 100 mg
Solvent Toluene 20 mL
Salt HAuCl4.H2O 50 mg

Before using the solution, filtration was done to avoid any potential contamination or pollution.
For filtration, three different types of filter media was used with different pore size, Table 3-2.
Table 3-2 Description of different types of filter
Filter Type (can be used in) Pore size
CHROMAFIL Xtra PTFE Non-polar media 0.45 µm
CHROMAFIL GF Highly contaminated media 1.0 µm
Millex FG Hydrophobic Flouropore 0.2 µm
After every dip coating the substrate was checked in the High Resolution Scanning Electron
Microscope (HRSEM) (Hitachi S5200 at 30 kV) to measure the distance as it varies with the
solution. Depending on the distance between the particles the withdrawal velocity was different.
The different velocity were 2.8, 3.6, 4.2 and 4.8mm/min. The interparticle distance was
calculated using ImageJ software.
H2 PLASMA
Plasma is increasingly used in semiconductor technology for removing carbon contamination,
native oxide layer, and also used in etching process [52]. There are different types of plasma i.e.
Oxygen, Hydrogen and Argon plasma which reacts with the deposited molecule on the surface,
break them down and convert into volatile compound. H2 plasma is gaseous and electrically
neutral which contains electron, ions, neutral atoms and molecules. Hydrogen has very small
molecular weight and energy therefore, sputtering is not possible but removing organic polymer
from the surface is possible [53].
When hydrogen plasma generated, it creates chemically active species and ions i.e. H* and H+
with low kinetic energy. The mechanisms are following [52]:
H2 + e → H + H+
+e (3.4.1)
H + e → H* + e (3.4.2)
H+
+ H → H* + H+
(3.4.3)
H*, H+
, H and e are hydrogen radical, hydrogen ion, hydrogen atom and electron respectively.
These all have strong role to remove the chemicals and oxide layer from the surface [53]. In this
study, ‘TePla 100-E’ was used to remove the polymer completely from the surface of the
substrate. To use this device, first vacuum was created in the chamber with pressure less than
0.05 mbar for plasma ignition. Then H2 gas was allowed to the quartz chamber for 15 min at 0.25

mbar for pre-treatment. Finally, H2 plasma was carried out for 90 min to remove the polymer.
The power was 160 W, the frequency was 2.46 GHz and the pressure was 0.8 mbar.
PHOTOCHEMICAL DEPOSITION
After dip-coating and then H2 Plasma treatment, the size of the gold naoparticles on the
substrate are quasi-hexagonally ordered and the size is small (average size is 9nm).Therefore, to
make a bigger size of gold particles, photochemical deposition process was used. In this process,
a solution was prepared by mixing gold salt (HAuCl4.H2O) with Phtalatester and irradiated under
UV light. The advantage of using Phtalatester is that it does not evaporate in UV light and also
absorbs in low spectral range. Exposure time is important due to the size of the growth of the
particles [54]. Figure 3-6 shows the effects during exposure.
Figure 3-6 Photochemical growth of Au particle [54]
The principle of this process is that when UV is exposed to the gold complex, gold salt absorb the
UV light and Cl-
ion from the gold salt solution is oxidised by absorbing the energy and the
reduction of gold particles take place. Here is the reaction,
2AuIII
Cl-
4
ℎ𝜐
→ 2 AuII
Cl-
3 +Cl2 (3.5.1)
2AuII
Cl-
3 → AuI
Cl-
2 + AuCl-
4 (3.5.2)
2AuI
Cl-
2 → Au0
+ AuCl-
3 +Cl-
(3.5.3)
From the reaction it can be seen that this process involves two steps – first, the Auis gradually
reduced to Au atom and followed by agglomerate with existing gold particles on the substrate to
make small metal cluster[55] [56].
After preparing the solution the substrate has been exposed to UV light by seeding machine
from ‘Karl SUSS MJB 3 Mask UV 400’, West Germany. Figure 3-7 shows the schematic diagram of
this seeding machine. This machine has an Hg (Mercury) short-arc lamp surrounded by the
ellipsoidal mirror and maximum power (350 W) of the light can be used at two wavelengths:

365nm and 390nm. When the radiation was discharged by turning on the light, the radiation is
collected by the ellipsoidal mirror and then focused to the cold light mirror. Only short
wavelength light is reflected to Fly’s eye lens and the condenser lens adjust the light intensity.
There is a filter to block undesirable wavelength, but in this work no filter was used. By using the
lens plates it is possible to remove diffraction effect during the experiment. With the help of
surface mirror and front lens, the beam exposes to the substrate vertically [57].
Figure 3-7: Schematic diagram of optical system of mask aligner Karl SUSS MJB 3 Mask UV 400 [58]
After putting the substrate on the chuck, 20µl of the solution has been poured on each substrate
using a pipette. Exposure time was 1.5 minutes for 17nm diameter and 3.5 minutes for 30nm
diameter of the particles. Then the substrates were cleaned with acetone and isopropanol for
12min and dried in nitrogen flow. But there is a chance that the organic molecule could be
present on the substrate so H2 plasma was carried out again for 15min. The preparation of gold
salt solution is given in Table 3-3.
Table 3-3 Preparation of the gold solution for photochemical growth
Phtalatester Gold Salt (HAuCl4.H2O)
Density 1059 g/l Molar mass 339.79 g/mol
Weight 2056.99 mg Weight 3.3 mg
Volume 1.95 ml Concentration 0.005 molarity
Plasma Etching: Reactive Ion Etching (RIE)
Etching is the process of removal of materials from a substrate. There are two main division of
etching: wet chemical and physical dry etching. In wet chemical, materials are removed by using
liquid chemicals or etchants. Specific patterns are protected by masks, otherwise the whole
surface will be etched away by liquid chemicals. In wet chemical etching there are three basic
steps are followed [59]:

 The liquid etchant diffused to the structure;
 A redox reaction occurs between the liquid etchant and the materials to be removed;
 By-products diffused from the surface
On the other hand, the physical dry etching process is carried out in gaseous phase by high
kinetic energy such as particle bombardment, chemical reaction or combination of both
followed by evaporation. In general, when the high kinetic energy ion, electron or proton in
touch the substrate knocks out the atoms from the surface. There are three different types of
dry etching: Chemical Plasma Etching (PE), Reactive Ion Etching (RIE) and Ion Beam Etching (IBE).
RIE gives high etch rate and high selectivity due to combination of physical sputtering and
chemical activity [59][60].
RIE is plasma assisted etching which involves the generation of glow discharge of a feed gas, i.e.
CF4 for Si etching, by which high kinetic energy particles, neutral atoms, electrons, radicals and
positive/negatives ions are produced. Since the substrate is placed on the coupled electrodes
and obtains a negative charge so the positive ions are attracted to the substrate and diffused to
the surface to start etching [61]. Figure 3-8 shows the schematic diagram of this process. This
process is associated with several steps, such as formation of ions, radicals, diffusion,
adsorption, chemical reaction desorption and pumping out the reacted product.
Figure 3-8: Schematic representation of the process of etching adopted from [61]
Bombardment makes free radicals as active sites which then adsorb and react with the
substrate. For example, Fluorine bombardment in Si wafer etching, reaction between F atom
and Si produces SiF4, which is volatile. To desorb the SiF4, a high vapour pressure is needed at
substrate temperature. Reacted products mostly go back to the plasma region therefore, it is

necessary to pump out afterwards otherwise, there is a chance to dissociate with others and
resorption will take place [61].
In this work, ‘Oxford Plasmalab 80Plus ICP65’ has been used for the RIE and the etching process
occurs vertically therefore, anisotropic etching profile can be reached. The gases produced are
CF4,CHF3, O2, Ar, hence, the influence of these gases will be explained here.
Using CF4 gas gives the following mechanism [62]:
CF4 → F* + CF3 (3.6.1)
CF4 + e → CF3
+
+ F* (3.6.2)
Si + 4F* → SiF4 ↑ (3.6.3)
3.6.1 Effects of Oxygen addition
The mixture of different gases have different influence on etching rate. For instance, addition of
O2 (< 5%) to CF4 plasma increases the density of F atom and consequently rate of etching as seen
in following reaction. However, addition of O2 over 15% decrease the density of fluorine [63].
CF4 → F* + CF3 (3.6.4)
CF4 + e → CF3
+
+ F* +2e (3.6.5)
O2 → O* + O (3.6.6)
O2 + CF*
x → CO2 + COF2 (3.6.7)
CF4 + O → COF2 (3.6.8)
3.6.2 Effects of Hydrogen addition
The addition of H2 on CF4 plasma reduces the density of F atom thus etching rate due to the
formation of HF. H2 also reacts with CF3 radical and produce polymeric precursor which forms a
layer of CxFy on the surface. At high concentration of H2 (> 30%) etching will stop due to the
polymerization on the surface [60].
3.6.3 Effects of CHF3 and Noble gas addition
Addition of CHF3 in CF4 plasma does not make any significant changes because CF4/CHF3 is very
similar to CF4/H2 system, however, CHF3/HF has lower internal energy than CF4/H2 system.
CHF3/HF can be produced by mixing CF4 with H2 [60].
Noble gases, mostly Argon (Ar) and Helium (He) are added to stabilize the plasma. Addition of Ar
can make ion bombardment on the surface consequently, it increases the anisotropic etching.
Helium is used in order to cool the substrate from the front or back side [60].

Finally, the Au NPs were removed by Loguls solution and then rinsed with Millipore water.
Loguls solution was prepared by adding 4gram of Potassium Iodide from ‘Prolabo’, 1gram of
Iodine from ‘Merck’ in 150ml deionised water.
ELECTRON MICROSCOPE
To study the structure of the feature in nanometer range, Scanning Electron Microscope (SEM)
and Atomic Force Microscope (AFM) have been used. Furthermore, using electron microscope, it
is possible to see the single atom and soft materials like biological cells in a very low voltage.
3.7.1 Scanning Electron Microscopy (SEM)
SEM gives the information about the topography, morphology, composition and crystallographic
structure of the feature. A voltage is applied to the electron gun to heat up the filament
(cathode) which then emits thermo-electrons after it reaches a defined temperature [64]. The
filament is usually made of tungsten which is about 0.1mm and is heated up to about 2800K. The
thermo-electrons known as electron beam, are then forced to go to anode by applying positive
voltage (1-30kV) to the anode [65], figure 3-9.
Figure 3-9: Schematic diagram of Scanning Electron Microscope (SEM) [66]
When the high energy electrons enter to the specimen they scatter and lose their energy. Some
of them are absorbed in the specimen, and some of them are emitted from the specimen as
secondary electrons, back scattered electrons, Auger electrons, figure 3-10 [65]. Secondary
electrons are produced from inelastic collision with the atom of the specimen, and with an
energy less than 50eV provides information about the topography of the specimen. The

backscattered electrons produced from elastic collision are higher energy greater than 50eV
[67]. These electrons come from a deeper region of the specimen and are sensitive to
composition, therefore provides information about the atomic number [65]. In this work, Hitachi
S5200 High Resolution Scanning Electron Microscopy (HRSEM) was used with a cold emission
gun and tungsten cathode.
Figure 3-10: Emission of various electrons and electromagnetic waves from the specimen [65]
3.7.2 Atomic Force Microscopy (AFM)
AFM is one of the best modern techniques in biomaterials and nanomaterials field and has a
great contribution in cell study. The working principle is simple comparing to electron
microscopes; AFM detects the forces acting between the AFM tip (attached to a very flexible
cantilever) and the surface of the substrates [68]. Figure 3-11 shows schematic diagram of an
AFM setup where a laser light is focused on the back side of the tip, which is reflected and then
detected by the photodiode. To form an image, cantilever comes close to the surface of the
sample and scans line-by-line. By doing so it feels deflection due to the tip-sample interaction.
This deflection is detected by the photodiode detector [69].

Figure 3-11: Schematic diagram of AFM [69]
AFM possesses a piezoelectric scanner that moves over the surface of the sample. During image
acquisition, the scanner moves fast along the horizontal line and slow along the vertical line and
takes data points. The space between the data points is called ‘step size’ and for this experiment
64 data points were taken. Once it finished scanning across the horizontal line it comes back to
its perpendicular position and start scanning the second line and continues, figure 3-12 [70].
Figure 3-12: Schematic diagram of scanning system in AFM, redrawn from [70]
In this work force spectroscopy mode was used in AFM which assists to measure force at a
specific point. Here, the tip and cantilever move up and down to the surface. When the distance
between the tip and surface of the sample is big, no deflection is recorded (Force=0) [68]. When
tip-sample distance decrease (approaching red colour in Figure 3-13), at some point tip jumps
into contact to the surface (attractive force) and this effect called ‘snap-in’. When the cantilever
contacts with surface it applies some forces, from here it is possible to investigate Young’s

modulus or stiffness of the surface. After that at some point cantilever feels repulsive force and
retracts (blue colour in figure 3-13) from the surface but for a while tip tries to keep in contact
because of adhesion [71].
Figure 3-13: Force spectroscopy mode in AFM [71]
For this experiment, AFM NanoWizard 3 (JPK instrument, Berlin, Germany) was used and there is
an optical microscope (Zeiss Axiovert 200) fitted to see the cells. The cantilever is made of
Silicone (B500-CONTAuD-5), coated with gold. The tip is spherical and high density diamond-like
carbon with 500 nm ±10% in diameter. The nominal spring constant is 0.2 N/m, but actual spring
constant derived from the calibration data.
Before running the experiment calibration of the cantilever was carried out at its resonance
frequency by thermal noise method which is commonly used and highly automated [72]. The
spring constant was determined in two steps. First, from the slope of the force curve which
shows the sensitivity of the cantilever and second, resonance frequency from the spectrum [34].
JPK software was used in contact mode on a hard surface i.e. glass in liquid, in this case DMEM
medium so, that there will be no indentation of the surface for the calibration. Figure 3-14
shows the spectrum of the fluctuations of the cantilever as a function of frequency, and from
thermal noise data the value of the spring constant was calculated to be 0.147N/m [71].

Figure 3-14: Calibration of cantilever spring constant, blue line is for cantilever resonance peak
(measured) and red is the Lorentz fit [71]
For imaging, quantitative imaging (QI) mode was used, which is developed for AFM by JPK
instrument, which works while not applying lateral force therefore, it helps to control the
vertical force at every pixel. This mode makes AFM imaging easy and faster by controlling tip-
sample force at each point of the image. There are other advantages of using this mode, such as,
no need to adjust the set point or gain during scanning and it also gives the information about
elasticity, adhesion and dissipation [73].
The settings of the AFM are given below:
Table 3-4: Settings of AFM
Setpoint 4nN
Z length 3500nm
Extend time 100ms
Extend speed 35µm/s
Retract time 35ms
Retract speed 100µm/s
Fast 30µm
Slow 30µm
X-Offset 0µm
Y-Offset 0µm
Grid angle 0 degree
Pixels 64*64
Pixel ratio 1:1

Extend sample rate 100kHz
Retract sample rate 100kHz
Add. retract 50nm
Motion time 5ms
Acceleration 1.5ms
Time for image 9.73min
CELL CULTURE PREPARATION
In this work 3T3 fibroblast was used which is an embryonic mice skin cell supplied by Medicine
Department in University Ulm. First, the substrate was sterilised with ethanol and dried at room
temperature. Second, fibronectin coating was made on the Si substrate to adhere fibroblast on
the surface. For fibronectin coating, 5% solution was poured on the substrate for 2 hours. After
preparing the cells, they were placed into the incubator. The environment of the incubator is 37°
C, 98% humidity and 5% CO2 and kept for 24 hours. Cell culture was done by Ulla Nolte in
Experimental Physics department, University Of Ulm. The petri dishes uses for cell culture are
from ‘TPP’ made in Switzerland. These Si substrates can be reused for cell culturing once the
experiment is done.

Chapter 4: Results and Discussion Page 35 of 63
Chapter 4. RESULTS AND DISCUSSION
FABRICATION OF NANO-PILLARS
One of the part of this thesis work is to fabricate well-ordered nano-pillars, and this involved
several steps. The results of these steps are given here.
4.1.1 Micellar technique by using block copolymer
Micellar technique is comparatively easier way to make thin film on a substrate. The distance
between particles can be determined by pulling out the substrate with a constant velocity, figure
4-1 or changing the concentration of the solution, figure 4-2. In this work, the substrate was
pulled out with a constant velocity to determine the distance of particles.
During dip-coating, the substrate was withdrawn vertically with a constant velocity U. The model
for dip-coating given by Landau and Levich is:
h = 0.946 * √
𝜎
𝜌𝑔
Ca2/3
(4.1)
Here, h is the thickness of the wetting film on the substrate after removal from the micellar
solution, Ca is the capillary number, is surface tension and 𝜌 density of the solvent.
Now, equation (4.1) will be valid when the capillary number, 𝐶𝑎 = µ
𝑈
σ
, where µ is the dynamic
viscosity [74]. The maximum thickness will be proportional to U2/3
. After evaporation of the
solvent, the thickness is proportional to the deposited micelles and the areal density of the
deposited micelle, monolayer film is also proportional to U2/3
[75]. Therefore, the interparticle
distance is proportional to U-1/3
[58]. From Figure 4-1 it is observed that when the velocity
increases the distance between the particles decreases and vice versa. In addition, with constant
interparticle distance the pulling out velocity varies depending on the types of substrate. For
example, if the distance was kept at 100nm, the velocity for Si was 4.8mm/min while for SiO2 the
velocity was 10mm/min.

Figure 4-1: Plot of average interparticle distance over withdrawal velocity from experimental data, from
figure it is seen that for 130nm interparticle distance the withdrawal speed is 2.8mm/min
Figure 4-2: Interparticle distance changes depending on the concentration of the solution [58]
After dip-coating, polymer was removed from the surface through H2 plasma so that the
distance between the Au particles can be measured. The Au NPs are arranged hexagonally
which also indicated the particles were self-assembled on the substrate. Figure 4-3A shows the
hexagonal distribution of particles taken by HRSEM at 30kV while figure 4-3B shows the
threshold image using ImageJ software 1.46r version by applying bandpass filter. The inset of
figure 4-3B shows the hexagonal arrangement of Au particles. The average distance between the
particles was measured at 130nm.
Figure 4-3: HRSEM image of Au nanoparticle after H2 Plasma. A) Grey scale picture and B) after adjusting
threshold, inset hexagonal arrangement (bandpass filter). Scale is 1µm, 30kV.

4.1.2 Photochemical growth of Gold (Au) particle
Nanoparticles have size dependent properties, therefore, the precise fabrication is important.
Photochemical growth is one of the easiest and fastest method to seed particle [76]. It is
important to mention that depending on the number of monomer of the block copolymer, the
size of the gold particles may vary. It has been shown that by using PS(325)-b-P2VP(75), the size
of the Au NPs was 2.9±0.4nm while using PS(1350)-b-P2VP(400), the size of Au NPs was
7.9±1.2nm [47]. In this work PS(1800)-b-P2VP(770) was used as block copolymer and the
average diameter of the gold particle was found to be 9nm. The photochemical growth process
was conducted with exposure time 1.5 minutes and 3.5 minutes and found that the average
diameter of the Au NPs are 17nm and 30nm respectively. Figure 4-4 shows the relation between
diameter and exposure time.
Figure 4-4: Plot of Au NPs diameter as a function of exposure time from experimental data.
Figure 4-5 shows that particles were getting bigger after seeding. In general, particles up to
30nm are monodisperse and longer reaction time will cause the dislocation or disorder of the
particles [76]. After dip-coating the particles were found to be spherical but when the size was
increased the particles were getting less and less spherical. The reason could be that particles
were contaminated with air while transporting the substrate to the cleaning box or the humidity
was not ideal.
Figure 4-5: HRSEM images (at 30kV) of different diameter(average) of Au NPs, a) 9nm, b) 17nm, c) 30nm,
scale is 200nm.
a) b)
a)
c)
a)

Less spherical particles can be made spherical by annealing at 720°C for 1 hour afterwards [77].
The diameter of Au NPs were determined by converting the picture to its threshold then
analysed the particle area of each particle in the software as seen in figure 4-6.
Figure 4-6: a) grey scale image using HRSEM scale 200nm at 30kV; b) after adjusting threshold; and c)
marking for area measurement through imageJ software
4.1.3 Reactive Ion Etching (RIE)
CF4 gas is used to etch to fabricate well-ordered nano-pillar array using Au NPs as mask in
Reactive Ion Etching (RIE). Although CF4 gas yields higher etching rate but it also removes Au
nanoparticles. Therefore, a fluorocarbon layer close to the Au NPs is necessary to reduce under
etching [78]. During the experiment, the mixture of CF4-CHF3 gases with flow rate 2sccm:20sccm
(Standard Cubic centimetres) and low pressure 1mTorr was used. The temperature was
maintained at 25°C by liquid nitrogen. The DC bias was maintained at 96V by changing plasma
power between 56 and 63W, and the system was operated by PC 2000 software.
In CF4 based Si etching, F radicals adsorb on the surface and react to produce SiFx layer on the
surface. Two F atoms form SiF2 on the upper level and are removed. But when more F atoms are
available, it forms SiF4 and desorbs [79]. Figure 4-7 shows the formation of SiF2 andSiF4.
Figure 4-7: Schematic diagram of a) formation of SiF2 and b) formation of SiF4 [79]
Depending on etching time the height of the pillars varies and therefore etching rate can be
measured by plotting height vs etching time. During etching time the rate and the shape of the
a) b)
a)
c)
b)
a)
a) b)

pillar depends on DC bias. In the previous study Si etching rate was found to be 4nm/min with
30W power [78] while in this study the etching rate was 5.20nm/min with 56 to 63 W. Using high
power makes the applied voltage high and this makes ion bombardment faster hence, increases
etching rate. In addition, the aspect ratio (width over height) was experimentally found to be
0.52. A study showed that increasing power also causes decreasing selectivity [80]. Selectivity is
defined by the ratio of etching rate of two different materials. During RIE, erosion of gold atoms
occurs due to the sputtering. Therefore, the selectivity of these two materials was calculated to
be around 5. Another study [77] showed that depending on the size of the mask etching rate
varies, figure 4-8 but in this work it was not so obvious.
Figure 4-8: Etching rate changes depending on a) DC bias and b) size of the mask [77]
Figure 4-9: Au NPs in 200nm scale, tilted by 30 degree a) average height 75nm and diameter 28nm with
Au particle diameter 17nm, b) height 75nm and diameter 49nm with Au particle diameter 30nm, and c)
height 106nm and diameter 49nm with Au particle diameter 30nm
The diameter of the pillars was measured on top and on full width high maxima. For 17nm
diameter of Au NPs, the average diameter of the pillars on the top was 28nm and at FWHM
calculated to be 34nm. For 30nm Au NPs the diameter on the top was 49nm and at FWHM was
52nm. Since cells are interacting with pillars only on the top therefore, the top diameter was
counted for the measurement.
Table 4-1 shows the heights of the pillars with corresponding Au NPs diameter. It is observed
that for 17nm and 27nm diameter of Au NPs, the average pillar heights are a bit larger. It can be
explained that the DC bias was a bit higher (98 V) and therefore the rate was higher.
a)
))
b) c)

Table 4-1: Experimental data of particle diameter, etching time and average height
Average Diameter (nm) Etching time (min) Average height of nano-pillars (nm)
15 13 67
15 10 47
17 13 75
27 12 66
27 20 116
30 22 106
Figure 4-10 shows the AFM pictures using tapping mode. Some areas on the surface shows no
pillars due to scratches on the substrate surface during handling. The etching rate without any
mask was higher than with mask therefore, the pillars height adjacent to the scratch are higher
than the normal height of the pillars. Figure 4-10 (right) showed how the height was measured
by Gwydion software by making cross section over the scratch (yellow and red line). From Figure
4-11 it is seen that the height adjacent to the scratch was around 400-500nm and the scratch
area is rough.
Figure 4-10: AFM pictures (topography) of Samples 2 (130-28-75)

Figure 4-11: Height of pillars build by Gwydion, profile 1 corresponds to red line and profile 2 corresponds
to yellow line from Figure 4-10 right part.
CELL MECHANICS ON NANOSTRUCTURE TOPOGRAPHY
This section discusses indentation depth, evaluation of the measurements, and comparison of
stiffness of cells and between the substrates. Cell-surface interaction are also discusses here.
4.2.1 Indentation depth
Hertz model is used for the measurements and there are two conditions for indenter. If these
two are met then Young’s modulus can be calculated from Hertz model. The conditions are that
the indenter will not deform and there will be no added collaboration between sample and
indenter. Figure 4-12 (top) shows the schematic diagram of indentation test, where the
cantilever is moved down to the sample by distance z, called ‘height measured’. But the
cantilever is bending in the opposite direction (x), indenting the sample by δ. The indentation
can be estimated by deducting the deflection of the cantilever from height measured. The
bottom figure is the deflection of the cantilever [14].
Figure 4-12: Schematic diagram of indentation test (top) and force-indentation curve (bottom) [14]

Finding the right indentation depth is important, otherwise measurement will vary. Although,
due to the properties of living cells such as elasticity, viscosity and adhesion, the elucidation of
the experimental data would be difficult. Figure 4-13 was drawn using Python and a program
named ‘hertzfit’ written by Christian Bühler [81]. Using this program it is possible to see
topography of one cell with Force-Distance curve. Figure 4-13C shows the contact point in the
force-distance curve (red point). If the hertzfit fits this point nicely then measurement of the
indentation depth can be done from figure 4-13B and 4-13D. Figure 4-13A shows the topography
of the cell, 4-13C is force-distance curve, 4-13B is ‘Young’s modulus error’ vs ‘indentation depth’
and 4-13D is ‘Young’s modulus ‘vs ‘Indentation depth’ curve. To get the acceptable indentation
depth, several force-distance curve with different fitting and different cells were investigated. In
figure 4-13 an indentation depth 100nm was chosen because at this point Young’s modulus start
to be steady. Once the indentation depth is chosen, the evaluation of all cells was carried out by
an extension of the program called ‘Hertzfolder’.
It is important to mention that if the indenter is on the nucleus the indentation depth will be
higher than if the indenter is far from the nucleus and importantly, small indentation depth (5-
10% height of the cell) is acceptable in Hertz model [14].
Figure 4-13: Sample 1: 130-49-106, Hertzfit indentation depth 100nm, figure A shows the topography of
the cell, C is force-distance curve, B is ‘Young’s modulus error’ vs ‘indentation depth’ and D is ‘Young’s
modulus ‘vs ‘Indentation depth’ curve
4.2.2 Measurements
Evaluation of data was done by MATLAB, scripts were written by Dr. Tobias Paust, Jonas Pfeil
and Fabian Port, University Ulm. For this work some changes have been made in the scripts. The
axis of the Young’s modulus was presented as the logarithm for a better view. The set point was
C D

4nN. For the experiment, it was tried to collect the data far from the nucleus which is only
cytoskeleton and substrate. Three different types of samples of Silicon has been used to
investigate the elasticity. Samples differ by their diameter and height. Samples are named by the
distance-diameter-height, all are in nm range. For example, Sample 1: 130-49-106, Sample 2:
130-28-75, Sample 3a: 130-49-75 and Sample 3b: 130-49-75.
4.2.2.1 Sample 1: 130-49-106
Figure 4-14 shows the histogram of the substrate with 3T3 fibroblast, which indicates that
elasticity distribution of the cell has a wide range of 102
Pa to 104
Pa. In contrast, for the substrate
the distribution is not wide. For this sample, the most frequent Young’s modulus for cells is, Ecell
~ 3.7kPa and for the substrate, ESubstrate ~ 1MPa. Different colours were produced by addition of
one cell with another with a total of 10 cells. The reason for wide range of elasticity value for
cells could be due to the different component of cytoskeleton. As stated in Section 2.1, the
stiffest components of the cytoskeleton is the microtubules so the value of Young’s modulus
more than 10kPa may represent the microtubules. Young’s modulus is between 1 and 10kPa
represent actin filaments. And the lowest value, less than 1kPa is for intermediate filaments. To
find out the contribution of these three components to the mechanics, immunofluorescence
imaging can be performed by using confocal laser scanning microscope.
Figure 4-14: Histogram of Young’s modulus by using Hertz model of 3T3 fibroblast of Sample 1 at
indentation depth 100nm. X-axis is in logarithm, Y-axis is linear scale (calibrated spring constant 0.275
N/m), different colours produced by the addition of one cell with other and this results come from
summation of 10 cells data.
A study [82] showed that 3T3 fibroblast has an elasticity range from 4-100kPa and authors
suggested that elasticity of cells comes from large contribution of actin filament rather than
other components. Other components such as intermediate filament also contribute but
microtubules do not have significant contribution to stiffness. Therefore, it can be said that in

these experiments most frequent elasticity belongs to actin filaments and the value more than
104
Pa correspond to microtubules.
4.2.2.2 Sample 2: 130-28-75
Figure 4-15 shows the histogram of Sample 2, in this case the indentation depth was 100nm (30
cells measurements).
Figure 4-15: Histogram of Sample 2 at indentation depth 100nm 30 cells measurement, (calibrated spring
constant-0.125N/m), different colours arise by adding one cell with other
In contrast with Sample 1 figure 4-14, the Young’s modulus of this substrate is less, which is
ESubstrate ~ 0.5MPa. From Figure 4-15, the distribution of the cell is wide between 102
and
5*104
Pa. The most frequent elastic modulus of the cells measured in this case is Ecell~ 10kPa. It
was observed that decreasing the pillar diameter and height resulted in decreasing stiffness for
the substrate.
A study showed that the cell stiffness changes with the substrate stiffness and on a rigid surface
the cell spread well and the stiffness of the cell increased. Authors worked with human
mesenchymal stem cells (hMSCs) and found that with increasing substrate stiffness of 1-30kPa,
cell stiffness increased 1-7kPa [83]. In contrast figure 4-16 showed that elasticity of substrate
was decreased but the cell elasticity increased compared to the Sample 1. In Sample 2 Pillar
height and diameter are lower than Sample 1 and it also showed decreasing elasticity of the
substrate but increasing cell elasticity.
A study [84] mentioned that the Rotsch et al. (1999) investigated elastic modulus of 3T3
fibroblast and found that while the cortical stiffness for stable edge was 12kPa but for the
leading edge the stiffness was 3-4kPa. Comparing to the results in this study it can be reasonably
claimed that the stiffest part of the cell belongs to the stable edge of the cell due to the stress
fibres. Mahaffy et al. [84] investigated Young modulus by applying two different models, well-

adhered and non-adhered regions for 3T3 fibroblast, for the former one elasticity was
1.6±0.2kPa and for later, elasticity was 0.6±0.1kPa. Both are smaller than this work.
4.2.2.3 Sample 3a: 130-49-75
From figure 4-16 it’s observed that for Sample 3a, the most frequent elastic modulus for cell is
ECell~1kPa and for the substrate ESubstrate ~1.3*105
Pa. The calibrated spring constant was
0.084N/m therefore, it can be said that with a soft cantilever the elasticity will be less.
Figure 4-16: Histogram of sample 3a at indentation depth 100nm, with spring constant 0.084 N/m,
different colours arise from summation of all cells (6cells measurement).
4.2.2.4 Sample 3b: 130-49-75
In contrast with Sample 3a, Sample 3b (figure 4-17) has a stiffer spring constant (0.276N/m).
Therefore, the elasticity is higher than Sample 3a. Here, the most frequent elasticity calculated
for cell was ECell~4.3kPa and for the substrate was ESubstarte~1MPa.
Figure 4-17: Histogram of Sample 3b at indentation depth 100nm (5cells measurement) with calibrated
spring constant 0.275 N/m, different colours arise from summation of all cells.
Since Sample 1 and Sample 3b have same diameter with different height of the pillars, the
results were compared to see the contribution of height on elasticity.

Figure 4-18: Histogram of Sample 1 (top, 10 cells) and Sample 3b (bottom, 5 cells), frequency scale is
different due to the different number of cells measurements, calibrated spring constant 0.275 N/m.
Different colours produced by the addition of one cell with other.
From figure 4-18 it can be observed that sample 1 and sample 3b (with same k and diameter of
the pillars) do not have significant difference in elasticity even though they have different height.
Therefore, it can be suggested that different height of the pillars do not have a significant effect
on the elasticity. The most frequent elasticity for the substrate were same but for cells, Young’s
modulus was Ecell~3.7kPa for Sample 1 and was Ecell~4.3kPa for Sample 3b.
To make comparison of Young’s modulus among the samples, boxplots were created, figure 4-19
showing the median at indentation depth 100nm. The corresponding median shows upper part
of the box. The highest median corresponds to Sample 3b, 6.02kPa and the lowest is 1.2kPa for
Sample 3a. The highest maximal belongs to Sample 2. Since the cell number are not same for all
samples eventually, the comparative results may not be fully representatives of the samples and
may need further study. Codan et al. [85], measured elasticity of living 3T3 fibroblast on glass
and found the Young’s modulus median is 5.2kPa. Codan et. al., used squared pyramidal tip
therefore the model is different from this study. Depending on the model, the elasticity values
will differ from each other.

Figure 4-19: Boxplot for comparing the median of different samples at indentation depth-100nm
Figure 4-20, Young’s modulus was plotted over height for the samples. It demontrates the
Young’s modulus of corresponding contact point through MATLAB Program. From here it is seen
that the Young’s modulus is decreasing with increasing height of the cell except Sample 3a,
which shows very different intervals compared to other samples. The highest Young’s modulus
was measured at near to zero micron. It should be noted that there could be 5-10% error in
measurement of height due to glueing the substrate onto the petri dishes. In the region of 2.5 to
3.5µm, the elasticity decreased for Sample 3a, but increased for other 3 samples and showed
very rough intervals. It can be explained that there are some area where cells overlaped on each
other and this height is coming from this overlaping. However, over 3.5µm height, the nucleus
could be the responsible for this height and shows very different elasticity under force. It was
mentioned before that the Hertz model is only valid at low indentation depth, but when the
indenter is on the nucleus area the indentation will be large. This may also explain this uneven
elasticity. The change of Young’s modulus with height also indicates that cells are
heterogeneous.
Median:
3843.8 Pa 4987.7 Pa 1193.3 Pa 6015.6 Pa

Figure 4-20: All samples: Sample 1: 130-49-106, Sample 2: 130-28-75, Sample 3a: 130-49-75 and Sample
3b:130-49-75
Solon et. al. [86] showed that the stiffness of 3T3 fibroblast differ depending on the distal
(17kPa) and proximal (5kPa) regions and higher parts of the cell are soft and homogeneous. In
similar to this study, authors found that the lower part of the cell is the stiffest and the
contribution of the substrate stiffness on cell stiffness. But authors found that over 700 nm of
height of the cell the Young’s modulus remain constant which is dissimilar with this thesis work.
It was suggested that when the cell thickness is more than 700 nm (same as this work), the
deformation of the cell distribute only into the cell body but were not transmitted to the
substrate.
Since Sample 3a shows rough intervals, to see details of Young’s modulus over height, boxplot of
selected heights such as 0.4-0.6 µm, 0.9-1.1 µm, 1.4-1.6 µm and 1.9-2.1 µm is presented in
figure 4-21. It is observed that the highest median correspond to Sample 3a in 0.4-0.6 µm but in
other parts it’s different. From here it is clear that something is wrong with Sample 3a. The
problem could be related to calibrated data.

Figure 4-21: Boxplot of all samples of Young’s modulus over selected height: 0.4-0.6, 0.9-1.1, 1.4-1.6 and
1.9-2.1 µm
From figure 4-21 it is seen that when the cell thickness is low the elasticity is high. Therefore, the
0.4-0.6 µm height the Young’s modulus is higher than others. But Park et. al. [87], showed
different result with 3T3 fibroblast, authors showed that elasticity increase with increasing cell
thickness (570nm-4700nm). The leading edge has lower elastic constant than the cell body
which is dissimilar to the current work.
Sample 3a was omitted from Young’s modulus over height plot and corresponding boxplot,
presented in Figure 4-22 and Figure 4-23 respectively.
Figure 4-22: Young’s modulus VS Height plot of 3 samples: Sample1: 130-49-106, Sample2: 130-28-75, and
Sample3b: 130-49-75
Rotsch et. al., [88] worked with 3T3 fibroblast and showed that the dynamics is different for
active edge and stable edge. The leading edge height profile was rather flat between 0.4-0.6 µm

compared to the stable edge (2-3 µm) and the leading edge is softer than the stable edge. The
result contrasted with this study, increasing thickness of the cell, elasticity decreased and elastic
modulus was highest at the flat part, figure 4-22. Haga et. al., [82] discovered that nucleus has
10 times lower elasticity than its surrounding area. It is considered that over 3.5 µm height is
attributed the nucleus and it also shows lower elasticity than its surrounding area.
Figure 4-23: Boxplot of 3 samples of Young’s modulus over selected height, the selected heights are: 0.4-
0.6, 0.9-1.1, 1.4-1.6 and 1.9-2.1 µm
From figure 4-23 it is seen that highest Young’s modulus corresponds to Sample 2 in all region. It
indicates that cells are stiffest on Sample 2 which has lowest diameter and height of the pillar.
On the other hand, Sample 1 and 3b have significant different elasticity with increasing cell
thickness. When thickness of the cell is low (0.4-0.6 and 0.9-1.1 µm), Sample 3b has higher
elasticity than Sample 1 but in thicker area (1.4-1.6 and 1.9-2.1 µm) Sample 1 has higher
elasticity than Sample 3b, indicates that cells stiffness is slightly dependent on pillars height with
increasing thickness of the cell.
It is calculated that in ~1µm2
area the average number of pillars in all samples remained same
but the average top area of the pillars that the cells were interacting with was different. In
Sample 2 it was 666nm2
but for Sample 1 and 3b the average area on the top of the pillars is
almost 3 times larger at 2078nm2
. Therefore, Sample 1 and 3b should give the stability to the
cells better than Sample 2, but here it shows different. It should be noted that other samples
showed an indentation depth of 150nm as well but Sample 2 showed only 100nm indentation
depth in ‘hertzfit’, this implies that cells are stable on Sample 2. The reason might be related to
focal adhesion. Ghibaudo et. al., [89] showed that fibroblast shows strong dependency of
adhesion on spacing between the pillars. But in this study, the spacing between the pillars are
the same. But yet, Kuo et al., [90] showed that focal adhesion is dependent on size of the pillars

and the cell-line. Authors worked with different cell lines such as CHO, MDCK and C2C12, and
observed their interaction with different size of the pillars, 200nm and 700nm. Authors
discovered that focal adhesions decreased on small pillars and mentioned that Chien et al.
showed that when cells have small focal adhesion they exert stronger force because small focal
complex become matured to large focal adhesion and thus exert contractile force on the
substrate. This exerted force on the substrate cause the pillars to bend. Moreover, Yim et. al.,
[91] showed s similar result. Therefore, it can be said that because of the small focal adhesion
form on small size of the pillars, Sample 2 possesses large force hence, highest stiffness. Biggs et.
al. [92] mentioned that when height of the nano-feature is small the focal adhesion increase. As
it pointed out before (2.2 section), cells form focal adhesion through integrin and in 3T3
fibroblast there are three different types of integrin: α5β1, α5β3, clone of α5β1/α5β3. They recruit
focal adhesion molecules and form strong focal adhesion with fibronectin which is a component
of ECM, in Sample 2 compared to other Samples.
To find out the statistical significance of these results, p value was determined pairwise. The null
hypothesis, h values are 1 and 0. When h=0, the test failed to reject the null hypothesis at the
5% significant level and vice versa when h=1. Here are the P and h value for 0.4-0.6 µm height
are:
S1-S2: P = 4.81*10-5
(h=1), S1-S3b: P = 0.009 (h=1), S2-S3b: P = 0.125 (h=0);
From these value it is seen that only the S2-S3b null hypothesis is significant.
On the other hand for 1.9- 2.1 µm height the P and h values are:
S1-S2: P = 0.023 (h=0), S2-S3b: P = 0.002 (h=1) and S1-S3b: P = 0.055 (h=0);
From these it is observed that S2-S3b pair statistic is not significant but other two, S1-S2, S1-S3b
are significant.
In 0.9-1.1 and 1.4-1.6 µm height none of them are significant.
S1-S2: P = 1.02*10-4
(h=1), S1-S3b: P = 0.02 (h=1), S2-S3b: P = 0.015 (h=1); (0.9-1.1µm);
S1-S2: P = 0.007 (h=1), S1- S3b: P = 0.014 (h=1), S2-S3b: P = 0.003 (h=1); (1.4-1.6µm).
Individual measuerements of Young’s modulus over height of the cells (left) and the substrates
(right) for 3 samples represented in figure 4-24. All of them show decrease of elasticity with
increasing height for cells however, for the substrates, it is steady.

Figure 4-24: 3D plot of Young’s modulus over height of 3 samples, colour scale bar shows how frequent
the combination of young’s modulus and height was measured, left figures are for the cell and right
figures are for the substrate.
The most frequent elastic modulus for cells were for Sample 1 : 5-3kPa in 0.5- 0.9 µm height, for
Sample 2: 10-7kPa in 0-1 µm height and for Sample 3b: 10-6kPa between 0.6 and 0.8 µm height.
It can be seen that lowest part of the cell was the stiffest part and it corresponded to edge of the
cell. From figure 4-24 it is noticed that cell heights were different for most frequent elasticity,
which might be due to glueing the substrates onto the petri dishes.
Ning et. el., [93] did similar work with the specification: distance between the pillars was 700nm,
diameter of pillars was 200nm and height was 300nm. The stiffness for 3T3 fibroblast at an
indentation depth of 300nm was found on flat surface was 2.4kPa and on nano-pillars was
1.5kPa. The stiffness was found higher in Nano-channels (distance 555-diameter 150- height
140nm) is 2.25kPa. In contrast, in this study the cells were stiffer on the pillars than on the flat
surface, Table 4-2. Therefore, it can be said that when the substrates have more groove the
Sample 3b (5 cells measurements)
Sample 2 (30 cells measurements)
Sample 1 (10 cells measurements)

elasticity of the cell will increase. Additionally, a study [16] stated that the elasticity of 3T3
fibroblast is 140±30 dyne/cm2
(14±3Pa) which is much smaller than this study.
Table 4-2: Elastic modulus with different Samples and Spring Constant
Sample type Calibrated Spring
constant (k) mN/m
Elasticity of
cell (kPa)
Elasticity of
substrate (MPa)
Sample 1 (130-52-106) 276.1 3.7 (10 cells) 1
Sample 2 (130-34-75) 124.6 10 (30 cells) 0.5
Sample 3a (130-52-75) 83.94 1 (6 cells) 0.13
Sample 3b (130-52-75) 276.1 4.3 (5 cells) 1
For Hertz model, it assumed that cells are homogeneous and this model tells about the static
Young’s modulus but not the dynamic young’s modulus. Furthermore, cells are heterogeneous
so the evaluation of Young’s modulus using Hertz model may give an error. Also, cells have
viscoelastic property but the Hertz model neglects this.
It is mentioned before that AFM measurements rely on the spring constant (k) therefore, it is
very important to do the calibration of the cantilever carefully otherwise, the results will be
incorrect. Some of experimental error might have interrupted the calibration, thus in current
study there are different values for the spring constant. The probable reasons are:
 Cantilever can be contaminated, thus increasing spring constant (k) value;
 The liquid medium might have different density therefore k value will change;
 The laser focused on the cantilever might be in different position that’s changes the
sensitivity;
 For thermal noise analysis, temperature is one of the parameters, which can also change
k value etc.
CELL-SURFACE INTERACTION
Figure 4-25 shows the AFM picture of Sample 1, built by JPK software. With AFM It is possible to
see topography but not the pillars because the samples cannot be tilted. Moreover, the principle
of AFM is different from electron microscopy. The principle is already explained in experimental
part so it will not be repeated here. Therefore, to see cell-surface interaction, HRSEM (Hitachi
5200) was used. The samples for HRSEM were prepared by the Electron Microscopy
Department, assisted with Professor Paul Walther.

Figure 4-25: AFM picture (topography) of Sample 1 after background corrections, scale 5µm, colour scale
bar shows the height measured
In section 2.2 it is stated that fibroblast moves smoothly and makes elongated triangle,
lamellipodia form on one side and extend forward. When lamellipodia moves it makes focal
adhesion to the substrate but it also detached when it reaches its proximal position. Apparently,
in figure 4-25, it is seen that cells are attached to the surface and spread very well regardless of
the height or the diameter of the pillar. Cells are very flat on the surface. Generally, when cells
are loosely attached to the surface they look spherical [94]. In this case cells were not spherical

so cells are firmly attached. It was also observed that when cell moves it only attaches to the top
of the pillars as shown in black arrows, figure 4-27.
Figure 4-26:HRSEM pictures at 5kV, Cells spread over the surface of all types of samples; a) Elongated
triangular shape with formation of microvili, scale 20µm, b) without microvili, scale 30µm c) the formation
of lamellipodia and filopodia (yellow arrows), scale 5µm
a) b)
c)

Figure 4-27: HRSEM picture taken at 5kV, 30 degree tilted, Cell moves attaching the top of the pillars in all
samples (black arrows) a) Sample 2 scale 100nm, b) Sample 1, scale 1µm and c) Sample 3b 1µm.
a)
a) b)
c)

Chapter 5: Conclusion Page 57 of 63
Chapter 5. CONCLUSION
In this study the elasticity of 3T3 fibroblast was investigated on Silicon nanostructures.
Nanostructure was prepared by both conventional methods: bottom up and top down
techniques. Dip coating process, which is a bottom up technique, used to make thin film with
constant withdrawal speed using block copolymer micellar gold solution and subsequent H2
plasma gives the deposition and interparticle distance of 130nm of the Au NPs. From here
hexagonally ordered gold nanoparticles were created with 9nm average diameter of Au NPs.
From controlled photochemical growth it was able to make Au NPs bigger in size to 17nm and
30nm with exposure time 1.5 and 3.5 minutes respectively. After conducting RIE which is top
down technique, the cylindrical like and hexagonally ordered nanopillars were produced with
different heights (75nm and 106nm) and diameters (28nm and 49nm) with aspect ratio, width
over height 0.52. This study showed that stiffness of cells are higher when diameter and height
of the pillars are small. The most frequent elastic modulus belongs to actin filaments. It was
observed that with increasing thickness of the cell the Young’s modulus decreased and different
heights of the pillars do not have significant influence on elasticity for the substrate and cells. It
is discovered that the cell was spread over the surface regardless of pillar heights or diameters.
Depending on the shape of the tip and calibration data of cantilever, the measurement will be
changed so it is necessary to use one spring constant to evaluate all data. Furthermore,
depending upon the model applied for the measurement the results will vary. Hertz model was
used to evaluate the data and this model is based on some assumptions. An evaluation can be
carried out with Finite Element Analysis (FEA) and results can be compared. Additionally, it can
be compared with cancerous fibroblast cell or even treated cells with inhibitor. Finally, since
living cells are interacting with nano-sized pillars and cells exert focal adhesion, there will be
pulling of the pillars therefore, the pillars might tilted and deformation of the pillars can be
determined.

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