RADIATION-INDUCED BYSTANDER EFFECT

CHUNG WING SHUN VINCENT

A0110760W

A THESIS SUBMITTED

FOR THE BACHELOR DEGREE OF SCIENCE (HONS) IN PHYSICS

DEPARTMENT OF PHYSICS

NATIONAL UNIVERSITY OF SINGAPORE

2017

I

Abstract

When cells are exposed to ionizing radiation, DNA damage will occur in the cell. This

DNA damage can be visualised under the fluorescent microscope using

immunofluorescence staining technique. Studies have shown that non-irradiated

cells in the vicinity of those irradiated cells had displayed radiation effects as well [1].

This phenomenon is known as the radiation-induced bystander effect. In this work,

we developed a method of accurately detecting the radiation-induced bystander

effect on cells using a thin CR39 solid state nuclear track detector. A bulk etching

procedure was developed in this project to thin down CR39 below 20 m thickness

using 1.0M KOH/ethanol at 50C. After exposing to radiation, latent tracks formed on

the CR39 were etched using the optimum track etching conditions of 6.25M NaOH,

75C and 4 hours of etching to investigate track properties such as track area,

density of tracks and aspect ratio of minor to major axis of the tracks. Two

approaches were used to demonstrate the bystander effect in cells. The first

approach was a proof-of-principle experiment and the second approach was a more

detailed investigation in which thin CR39 was used to differentiate irradiated cells

from the non-irradiated cells. The experimental results showed that the cells exhibit

potential bystander effect.

III

Table of Contents

Chapter 1: Introduction ............................................................................................... 1

1.1. Motivation ........................................................................................................ 1

1.2. Alpha Particle Radiation .................................................................................. 2

1.3. Radiation Effects on Cells ................................................................................ 4

1.4. Principle of Track Detector CR39 .................................................................... 5

Chapter 2: Experiment Methodology .......................................................................... 9

2.1. Optimisation of Track Etching Conditions ........................................................ 9

2.2. Track Angle Variation Experiment ................................................................. 11

2.3. Thin CR39 Preparation .................................................................................. 14

2.4. Bystander Effect Experiment ......................................................................... 16

2.4.1. Preparation of Custom Cell Dish and Thin CR39 .................................... 17

2.4.2. Cell Irradiation and Dose Calculation ...................................................... 18

2.4.3. Fixing of Cells and Pre-staining Cell Imaging .......................................... 19

2.4.4. Immunofluorescence Staining Procedure................................................ 20

2.4.5. Etching of CR39 and Cell Imaging .......................................................... 20

2.4.6. Image Matching Method for Bystander Effect ......................................... 21

Chapter 3: Results and Discussion .......................................................................... 23

3.1. Optimisation of Track Etching Conditions ...................................................... 23

3.2. Track Angle Variation Experiment ................................................................. 26

3.3. Thin CR39 Preparation .................................................................................. 33

3.4. Bystander Effect Experiment ......................................................................... 37

Chapter 4: Conclusion .............................................................................................. 45

Chapter 5: Future Work ............................................................................................ 47

Chapter 6: Acknowledgement .................................................................................. 49

Chapter 7: Bibliography ............................................................................................ 51

IV

List of Figures

Figure 1: Schematic diagram of etched track for normal angle of incidence alpha

particle (left) and oblique angle of incidence alpha particle (right) [10] ....................... 6

Figure 2: Variation in etch cone structure [11] ............................................................ 6

Figure 3: Schematic diagram of source holder (left) and actual experiment source

holder with Kapton orange film (right)....................................................................... 10

Figure 4: Schematic diagram for track etching experiment setup (side view) ........... 10

Figure 5: Schematic diagram of collimator (left) and actual experiment collimator

(right) ........................................................................................................................ 13

Figure 6: Schematic diagram for track angle variation experiment setup ................. 13

Figure 7: Masked CR39 a) before etching and b) after etching ................................ 15

Figure 8: Bystander effect experiment setup ............................................................ 16

Figure 9: Schematic diagram of the custom cell dish ............................................... 17

Figure 10: Thin CR39 with a cut for reference purpose ............................................ 17

Figure 11: Images of tracks after etching duration of a) 2 hours b) 3 hours c) 4 hours

d) 5 hours and e) 6 hours ......................................................................................... 24

Figure 12: Graph of track area against the duration of etching. The optimum etching

time of 4 hours produced an average track area of 898 m2. ................................. 25

Figure 13: Simulated probability distribution of overlapped tracks using circular track

area of 89m2 and track density of 145 tracks/mm2 ................................................. 25

Figure 14: Images of track formation on CR39 at different angles of incidence a) 10

b) 20 c) 30 d) 40 e) 50 f) 60 .............................................................................. 27

Figure 15: Graph of aspect ratio of tracks’ minor to major axis against angle of

incidence .................................................................................................................. 28

Figure 16: Graph of track area against angle of incidence ....................................... 28

Figure 17: Graph of track density against angle of incidence ................................... 29

V

Figure 18: Output energy distribution from SRIM after passing through 10.0 mm of

air gap at normal incidence ...................................................................................... 31

Figure 19: CR39 track area distribution for normal angle of incidence ..................... 32

Figure 20: SRIM output energies of alpha particles at different angles of incidence 33

Figure 21: SRIM output energies of alpha particles after passing through varying

CR39 thickness ........................................................................................................ 34

Figure 22: Bulk etching rates for NaOH/ethanol (blue) and KOH/ethanol (red) at 50C

with concentration varied from 0.5M to 2.5M. Repeated experiments were carried out

for KOH/ethanol 1.0M and 1.5M (black). .................................................................. 35

Figure 23: -H2AX fluorescent images for a) positive control and b) negative control.

Red bright spots denote regions of DNA damage. ................................................... 38

Figure 24: -H2AX fluorescent images for a) exposure region b) shielded region c)

middle region ............................................................................................................ 39

Figure 25: Overlaying of pre-staining bright field image onto CR39 along the

reference line. .......................................................................................................... 41

Figure 26: DAPI image superimposed on CR39 track image for bystander effect

experiment. The red boxes denote cell nuclei which were hit by an alpha particle. . 41

Figure 27: Cropped image from Point X, Y and Z in Figure 26. White cross denotes

the exact hit location of the alpha particle on the cell. .............................................. 42

Figure 28: -H2AX fluorescent image for bystander effect experiment with red boxes

denoting cells which were hit by an alpha particle. White arrows denote positive

signals which were not associated with direct irradiation. ........................................ 42

VI

List of Tables

Table 1: Angular spread calculation for different diameter and thickness of collimator

................................................................................................................................. 12

Table 2: Dose and percentage of nuclear hits delivered to cells .............................. 19

Table 3: Thickness of air gap travelled by alpha particles at different angles of

incidence .................................................................................................................. 31

Table 4: Initial trial conditions with their corresponding bulk etching rates ............... 34

Table 5: Surface roughness measurement results for different concentration of

KOH/ethanol ............................................................................................................. 36

Table 6: Calculation results of angle of incidence from the track’s aspect ratio and

extra distance travelled by alpha particle ................................................................. 40

1

Chapter 1: Introduction

Radiation is the transmission of energy through space and it is broadly classified into

ionising radiation and non-ionising radiation. Ionising radiation such as X-ray,

gamma, alpha and beta radiation has high energy which can cause ionisation in

atoms, while non-ionising radiation such as radio waves and microwaves is less

harmful as it has lesser energy. The motivation for this project draws from the

importance to understand the impact of different types of radiation in our daily lives

and the potential health risks involved.

1.1. Motivation

Humans are exposed to radiation in our everyday lives such as the sun’s ultraviolet

radiation, radio waves used in television signal, and X-rays used in radiotherapy.

Certain types of radiation are harmful to the human body, depending on its energy

level. When cells in our body are exposed to high energy radiation, known as

ionising radiation, the deoxyribonucleic acid (DNA) molecules will be ionised and

damaged. Since DNA is made of two complementary strands, the DNA damage can

be in the form of single strand break (SSB) or double strand break (DSB). While the

former can be easily repaired by the cells themselves, the latter is usually more

difficult to repair and prone to errors. Mutation of cells will occur if the errors are not

rectified properly, which can lead to tumour development. Or, cell death can occur if

DSBs fails to be rectified. As such, ionising radiation can have a devastating effect

on humans.

On the other hand, radiation effect on cells can be put to good use in the medical

field. Radiation is often used in radiotherapy for cancer patients to target the tumour

and kill the mutated cells before they spread to other parts of the body. However, the

down side of conventional radiotherapy is that healthy living cells are also exposed

to radiation. When a cell is exposed to radiation, DNA damage will occur in the cell.

Yet, experiments have shown that unirradiated cells near the irradiated cell also

exhibit effects of radiation. This phenomenon is known as radiation-induced

bystander effect where radiation effects have been induced in unirradiated cells

which are close to irradiated cells. This effect has an undesirable consequence as

2

healthy living cells around the targeted mutated cell are also exposed to

unnecessary radiation effects which are a source of carcinogen. Hence, this

motivates researchers to explore means to minimise bystander effect and deliver the

optimum radiation dose to the target tumour without damaging surrounding healthy

cells.

In the National University of Singapore, the Centre of Ion Beam Application (CIBA)

research centre has the cutting edge technology in focusing particle beams to 20 nm

spot sizes [2]. This inspires us to use a beam of targeted charged particle radiation

on single cells to investigate the phenomenon of radiation-induced bystander effect.

However, some fundamental problems such as quantification of radiation effects on

cells and discrimination of cells hit by charged particles need to be solved first. As

such, this leads us to the aim of this project: to understand the effects of alpha

particle radiation on cells before advancing to more sophisticated ion beam setup in

the future and to develop protocols using CR39 nuclear track detector in identifying

the irradiated cells so as to study the phenomenon of bystander effect.

The subsequent sections in this chapter will describe the background information

specifically on ionising radiation and how accelerated charged particles interacts with

matter; the effects of radiation in cells; and the principles of using CR39 to locate

alpha particles.

1.2. Alpha Particle Radiation

The origin of ionising radiation is from the nuclei of atoms. When an atom is unstable,

it will try to achieve a stable state by emitting excess energy as radiation via a

process called radioactive decay. The emitted radiation can be in the form of gamma

rays, alpha particles or beta particles, which are high energy waves or particles [3].

More elaboration will be done on alpha particles as the project mainly uses alpha

particles as the source of radiation.

Alpha particles are made up of two protons and two neurons in the atomic nuclei.

Due to the two positively-charged protons, alpha particles are known to be doubly

charged and the mass is relatively heavier than electrons. Alpha particles usually

interact with matter by coulomb force between the positively-charged protons and

3

the negatively-charged electrons in the matter [4]. As the energy levels of atoms are

quantised, only certain amount of energy can be transferred from the alpha particles

to the atoms in the matter. In general, the transfer of energy occurs by exciting

electrons in an atom to a higher energy level or ionising the electron from the atom.

The charged alpha particles will then lose kinetic energy and decelerate. Since the

charged particle is much heavier than electrons, it will only lose small amount of

energy in its initial path and mostly travel in a straight line through the matter. There

may be occurrences of the charged particle colliding with the nuclei in the matter,

resulting in a significant deviation from its straight path. However, such incidents are

usually rare. At the end of the particle’s range, it will lose all its energy and deposit

into the matter.

A way to describe the ionising properties of the matter is to use a physical quantity

called stopping power. The linear stopping power (𝑆) of a matter is defined to be the

loss of kinetic energy of the alpha particle within the matter per unit path length.

(0) 𝑆 = −

𝑑𝐸

𝑑𝑥 (1)

The loss in kinetic energy 𝑑𝐸 of the alpha particle after travelling a distance of 𝑑𝑥 is

transferred into the matter and this is known as the Linear Energy Transfer (LET).

For most matter, the stopping power is usually very high for alpha particles and

penetration power is low, which can be easily stopped by a sheet of paper or human

skin layer. However, when cells are exposed to alpha particles directly, severe

biological damage can be inflicted on the cells due to the particles’ high ionising

power.

Dedicated software programs have been written to calculate the stopping power of

different materials and the particle’s range. For example, the most commonly used

software is the Stopping and Range of Ions in Matter (SRIM) developed by James F.

Ziegler in 1983 [5].

4

1.3. Radiation Effects on Cells

In the previous section, we have seen how the alpha particle interacts with matter. In

this section, we will examine how alpha particles interact with cells and the

consequential radiation effects.

Alpha particles are considered as ionising radiation which has the capability to ionise

the DNA molecules in cells and cause DNA damage in the form of DSBs. This

triggers the cells’ DNA repair mechanism to repair the DSBs. As soon as DSBs

occur, phosphorylation of histone H2AX will follow rapidly at serine 139 (S139) and

tyrosine142 (Y142) residues which forms -H2AX. This process is fast and the

amount of -H2AX is found to correlate well with each DSB [6]. A primary antibody

will be used to recognise the -H2AX and subsequently labelled (or tagged) with

fluorescent dyes using a fluorophore-conjugated secondary antibody. In this way,

DNA damage can be visualised by immunofluorescence. Therefore, by examining

the amount of H2AX phosphorylation after cell irradiation, we will be able to quantify

the amount of DNA damage done to the cells [7].

Radiation effects are not limited to cells that are irradiated by alpha particles.

Surrounding cells of an irradiated cell can also experience radiation effect which is

known as bystander effect. This phenomenon was first discovered in 1992 by

Nagasawa and Little where they experimented on V79 Chinese hamster ovary cells

with extremely low dose of alpha particles [1]. The results had shown that instead of

the radiated 1% cell nuclei that exhibit DNA damage, 30% of the cells had shown

positive DNA damage which could only be explained by bystander effect. Although

bystander effect has been studied over many years, the actual mechanism for

bystander response is still not fully understood as it involves multiple pathways. One

of the pathways suggested by Little in 2006 is that the bystander signal has been

transmitted directly via cell-to-cell contact where damage signal has been

transmitted from irradiated cells to unirradiated cells through the gap junctions, or by

soluble factors in the culture medium [8]. As such, bystander can be mediated by

different modes.

5

1.4. Principle of Track Detector CR39

A nuclear track detector CR39 is employed to track the location of alpha particles so

that irradiated cells can be differentiated from the unirradiated cells before bystander

effect can be established. Thus, this section will explain how CR39 works as an

alpha particle track detector and the mechanism behind the track formation.

CR39 is a polyallyl diglycol carbonate (PADC) which is commonly used for nuclear

track detection. When heavy charged particles such as alpha particles penetrate into

the CR39, they will lose energy along their path. Ionisation and excitation of the

molecules occur close to the alpha particle’s path in the material which creates a

damaged zone. This damaged zone is known as a latent track [9].

When a CR39 with latent tracks is immersed in a chemical etchant solution such as

sodium hydroxide (NaOH), more intensive chemical reaction will occur along the

latent tracks. During the process, two etching mechanisms take place: bulk etching

and track etching. Bulk etching is the removal of CR39 surface layer and the

thickness is 𝑉𝐵𝑡 where 𝑉𝐵 refers to the bulk etching rate and 𝑡 is the duration of

etching as shown in Figure 1. Track etching is the preferred etching that takes place

along the latent track and is faster than the bulk etching which creates a deeper

etching depth into the CR39, forming an etched cone. The thickness of this

preferential etching is 𝑉𝑇𝑡 where 𝑉𝑇 refers to the track etching rate. The end of the

particle’s trajectory in the CR39 is known as the particle range (𝑅). An upright etch

cone will be formed if the angle of incidence is normal whereas an oblique cone will

be formed if the angle of incidence is slanted. Under the microscope, circular tracks

will be observed for normal incidence particles and elliptical tracks are observed for

oblique incidence.

Other etch cone parameters include the particle range (𝑅) from the CR39 surface

and the cone dip angle (𝛿). At very low dip angle, the post etched surface will lie

beyond the end of particle range (overetching), resulting in a tear-drop structure as

shown in Figure 2. Any further etching will cause the disappearance of the track and

information on the location history of the alpha particle will be lost.

6

Figure 1: Schematic diagram of etched track for normal angle of incidence alpha particle (left) and oblique angle of incidence alpha particle (right) [10]

Figure 2: Variation in etch cone structure [11]

Also, at small values of 𝑅, the post etched surface is way beyond the limit of particle

range and the track has been overetched, leaving behind the remainder of the

original cone structure [11]. Accurate information about the track will be lost and one

7

can observe that the tail of the track will start to disappear under the microscope.

The ideal track is formed when the post etched surface coincides with the end of the

particle’s range.

The dimension and shape of the tracks on CR39 are dependent on several factors.

Firstly, it is dependent on the incoming energy of alpha particles. Particles which

possess higher energy will penetrate deeper into the CR39, resulting in a large 𝑅

value. Assuming the ideal track formation where the post etched surface

corresponds to the end of the particle’s range, higher energy particles will form

bigger tracks on the CR39. Secondly, the track size is dependent on the angle of

incidence. Particles with normal angle of incidence on CR39 will result in a circular

track formation and slanted angles will result in an ellipse track formation.

Furthermore, higher angle of incidence will have a higher risk of overetching as the

post etched surface reaches the end of particle range very quickly. Lastly, the track

size is dependent on the etching conditions used as they can affect 𝑉𝐵 and 𝑉𝑇. The

three main conditions are concentration of etchant, time duration for etching

procedure, and the etchant temperature. Higher concentration and longer etching

temperature will increase the bulk and track etching rates which result in bigger track

formations on the CR39 for a fixed amount of time. For longer etching time, more

layers are allowed to etch away, increasing the thickness 𝑉𝐵𝑡 and 𝑉𝑇𝑡 and hence,

track size increases. In general, these relationships between etching conditions and

track size are valid before overetching occurs.

To conclude this introduction chapter, we have understood the following things.

Ionising radiation is harmful to our body and radiation interacts with matter mainly

through ionisation or excitation of molecules in the matter. When cells are exposed

to radiation, radiation effects such as DNA damage will occur which trigger the

formation of -H2AX. The -H2AX can be used as a biomarker to visualise DNA

damage in cells using immunofluorescence staining. Nuclear track detector CR39

can be used to differentiate irradiated cells from the unirradiated cells so that

bystander effect can be observed.

The following chapter of this work will cover the different experimental methods on

CR39 and cells to understand bystander effect. Results and discussion will be

presented in Chapter 3 and finally, Chapter 4 will conclude the findings of this project.

9

Chapter 2: Experiment Methodology

The aim of this project is to study the phenomenon of radiation-induced bystander

effect. This requires the knowledge of identifying the irradiated cells from the location

of alpha particles. Thus, CR39 is a suitable material since it is a transparent, clear

solid state nuclear track detector (SSNTD). In addition, CR39 is resistant to

electromagnetic pulse (EMP) and X-rays commonly present in our environment,

ensuring a good alpha particle signal in the experiment. In this work, CR39 was

purchased from Track Analysis Systems Ltd whose appearance is clear, colourless

and rigid. Its density is 1.30 g/cm3 and dimensions are 25mm 25mm 1.5mm for

the bulk samples [12] and 25mm 25mm 100m for the thin samples [13].

However, the commercially available thickness was still too thick for the experiment.

The CR39 was required to be thin enough so that the incoming alpha particle could

penetrate and hit the cells behind it. Thus, a bulk etching experiment had to be

carried out to optimise the condition for etching down CR39 to the desired thickness.

In addition, the track etching conditions were optimised so that valuable track

information such as the track area and major/minor axis could be extracted. By

correlating the track properties with the particle properties, a relationship could be

established between the alpha particle’s angle of incidence and the aspect ratio of

minor to major axis. Lastly, methods such as staining protocol of cells and

visualisation techniques of DNA damage were used to identify the bystander effect.

This chapter will be structured according to the following sequence – optimisation of

track etching procedures; track angle variation experiment; methods in preparing thin

CR9 and lastly, the bystander effect experiment.

2.1. Optimisation of Track Etching Conditions

This section covers the track etching procedure developed to achieve the optimum

track size for image analysis under MATLAB and a simulation was done to confirm

the optimum conditions derived.

By exposing CR39 to alpha particles, latent tracks formed on the CR39 would be

visible under an optical microscope after immersing in an etchant NaOH solution. As

10

mentioned in the introduction, there are several factors involved in the etching

process: etching time, concentration of NaOH, and etching temperature. Generally,

the degree of etching is proportional to the magnitude of factors involved. For

example, higher concentration of NaOH would lead to more etching of the CR39. As

detailed mechanism of the etching process was not required in this project, two of

the factors, concentration of NaOH and etching temperature, were fixed at the

recommended conditions by TASTRACK: 6.25M NaOH and 75C respectively. Then

the etching time was varied from 2 hours to 6 hours. The alpha radiation source used

in this project was a mixed source containing: Americium-241, Curium-244 and

Plutonium-239 with different peaks in their energy distribution [14]. The alpha source

was placed on the source holder shown in Figure 3 and covered with Kapton film.

The Kapton film had a 6mm diameter hole which matched the source active area

and CR39 bulk sample was placed on top of it for irradiation as shown in Figure 4.

The exposure time was 10s.

Figure 3: Schematic diagram of source holder (left) and actual experiment source holder with Kapton orange film (right)

Figure 4: Schematic diagram for track etching experiment setup (side view)

11

After exposure, the CR39 was placed into 6.25M NaOH etchant solution at 75C.

Samples of CR39 were removed at intervals of 1 hour, varying the etching time from

2 hours to 6 hours. The CR39 samples were cleaned with distilled water and

isopropanol before being air dried. The tracks on the CR39 were viewed under the

Olympus Microscope BX51 and images were taken at 10x magnification under

reflective mode.

The images were then processed in MATLAB using Image Processing Toolbox.

Statistical data such as the mean track area, major and minor axis of the tracks, and

density of tracks were extracted from the images. The mean track area would be

used to determine the optimum track etching condition.

As the track area increases, the probability of overlapped tracks increases which is

not ideal for image analysis. Therefore, to prove that the track area produced by the

optimum condition did not have a high probability of overlapped tracks, a Monte

Carlo simulation was conducted to find out this probability. MATLAB code was

written to simulate random alpha particles hitting an area of 1 mm2 of CR39. The

track area and track density formed within this area were pre-set to the track area

and track density derived earlier to determine the probability of overlapped tracks.

The tracks were assumed to be circular for simplicity. An algorithm called Density-

Based Spatial Clustering of Applications with Noise (DBSCAN) was applied to all the

tracks to count the number of overlapped tracks. The frequency of overlapped tracks

was grouped together and accumulated over 1000 trials before plotting into a

histogram. The probability of overlapped tracks was determined by dividing the

number of overlapped tracks over the total number of tracks. The probability of non-

overlapped tracks would be very high if the track area produced by the etching

condition were optimal.

2.2. Track Angle Variation Experiment

Besides analysing the track area and density, the relationship between the aspect

ratio of the track’s minor to major axis with the angle of incidence of the alpha

particle were investigated. From the background information on CR39 track

formation, it was understood that alpha particles coming from oblique (normal)

incidences will create an oblique (upright) etch cone, thereby forming an elliptical

12

(circular) shape tracks. Therefore, this section will describe the track angle variation

experiment where the angle of incidence of alpha particles onto the CR39 was varied

and MATLAB was used to determine the aspect ratio. The relationship between the

aspect ratio to the angle of incidence then serves as a calibration to determine the

angle of incidence of the alpha particle that hit the cells.

As alpha particles were emitted in random directions from the radioactive source, a

collimator had to be designed to collimate the alpha particles before the angle of

incidence could be varied. The parameters to be considered were the diameter of

the collimator (d) and the thickness of the collimator (t). Angular spread of the

collimated beam had been taken into consideration in the calculation to ensure that it

was minimal. As the diameter of the radioactive source was 6mm, d should be less

than 6mm to achieve its collimation effect and the angular spread was given by

(0) tan−1 (

𝑑

𝑡) (2)

The various possible combinations are presented in Table 1. The ideal collimator

dimensions were chosen to be d = 1.5mm and t = 10mm as the angular spread is

less than 10 and alpha particles would not deviate more than 10 after exiting from

the collimator. The actual collimator was manufactured using a 3-D printer and

consisted of 7 holes. The dimension of the holes was measured to be about 1.0 mm

as shown in Figure 5.

Diameter of hole (mm) Thickness of Collimator (mm) Angular Spread ()

1.0 10 5.71

1.5 10 8.53

2.0 10 11.31

Table 1: Angular spread calculation for different diameter and thickness of collimator

Hence the radiation exposure time had to be re-estimated. The activity of the alpha

radioactive source was 2500 Bq obtained from the manufacturer with half-life

consideration. With d = 1.0 mm and t = 10 mm, one could calculate the exposure

time (T) by

13

Figure 5: Schematic diagram of collimator (left) and actual experiment collimator (right)

(0) 𝑇 =

𝜌𝜋𝑟2

𝐴= 𝜌𝜋𝑟2 (

Ω

2𝜋𝐴0)

−1

(3)

where 𝜌 is the density of tracks derived from Section 2.1, 𝑟 is the radius of collimator

hole, 𝐴 is the surface activity on top of the collimator, Ω is the solid angle subtended

on top of the collimator and 𝐴0 is the base activity for the collimator hole. The

exposure time was calculated to be about 20 minutes.

Figure 6: Schematic diagram for track angle variation experiment setup

The collimator would be placed on top of the source holder as shown in the

experimental setup in Figure 6 to allow particles to pass through at normal incidence.

To vary the angle of incidence, the CR39 was pivoted at different angles by stacking

glass slides on one side of the CR39 above the collimator. With the width (𝑤 )

between the pivot point and the stack of glass slides of height (ℎ) known, the angle

of incidence (𝜃) could be calculated using Equation (4). 𝜃 was varied from 0 to 60.

14

(0) 𝜃 = tan−1 (𝑤

ℎ) (4)

As the alpha particle had to pass through some distance in air from the source to the

CR39 through the collimator, SRIM simulation had been carried out to calculate the

particle’s energy at the end of its trajectory. The width (𝑤) and height (ℎ) were

adjusted to ensure that the particles had sufficient energy to reach the CR39 in the

process of varying the angle of incidence.

After irradiation, the CR39 was etched under the optimum conditions derived in

Section 2.1. Images were taken using Olympus Microscope BX51 and post-

processed using the same MATLAB codes to obtain information about the track

minor and major axis, track area and density of tracks. The aspect ratio of minor axis

to major axis of the tracks was calculated and plotted against the angle of incidence

to obtain the calibration curve for the angle variation.

2.3. Thin CR39 Preparation

After investigating the different track properties, preparing thin CR39 is the final issue

to be resolved. The final goal of observing bystander effect was planned such that

the alpha particles had to pass through an air gap before reaching the CR39,

penetrate through CR39 and an additional layer of polypropylene (PP) film where

cells were grown on before hitting the cells successfully. Hence the alpha particles

had to pass through several media and each medium has its own stopping power

depending on the thickness and density. As such, the thickness of CR39 was crucial

as it must be thin enough so that the alpha particles had enough energy to cause

radiation damage to cells after passing through the media.

This section describes the usage of SRIM to calculate the desired thickness of thin

CR39 and methods on etching CR39 to our desired thickness. The optimum bulk

etching condition would be derived from the experiment and used to prepare thin

CR39.

Firstly, the desired thickness of thin CR39 had to be determined. Using the SRIM

programme, one could input multiple choices of media with its respective density and

thickness and simulate alpha particles passing through them. SRIM would calculate

15

the final output energies at the end of the particles’ trajectory and the desired

thickness of CR39 was determined from the data which would be presented in the

next chapter. For easy reference, the desired thickness was found to be ~20 m.

Since the thinnest commercially available CR39 was 100 m, a bulk etching

procedure had to be developed to etch away 80 m of CR39. Trials were done on

the bulk sample of CR39 (25mm 25mm 1.5mm) to determine the optimum bulk

etching condition and subsequently, applied on the thin sample CR39 (25mm

25mm 100 m) to produce our desired thin CR39 of thickness 20 m. The

experiment procedure was carried out by applying half of the bulk sample CR39 with

a layer of Araldite (epoxy resin) to mask its surface, leaving the other half exposed

as shown in Figure 7.

Figure 7: Masked CR39 a) before etching and b) after etching

The half masked CR39 was immersed in an etchant solution where the exposed

region (top and bottom) was etched away according to the etchant’s bulk etching

rate 𝑉𝐵 while the masked region was protected. As a result, a step-like structure was

formed on the top surface of CR39. The height of the step (Η) was measured using a

profilometer (Alpha-Step® 500 Surface Profiler) and the etchant’s 𝑉𝐵 of CR39 was

deduced by

(0) 𝑉𝐵 =

Η

𝜏 (5)

where 𝜏 is the duration of bulk etching. Various etchants were tested such as

potassium hydroxide (KOH) and NaOH with the addition of ethanol at different

concentrations to determine the optimum bulk etching condition. Surface roughness

of the samples were measured using Atomic Force Microscopy (AFM) to check the

surface quality of the CR39. Images from AFM were analysed using computer

software, NanoScope Analysis 1.5. Smooth surface were generally required so that

the CR39 could maintain its transparency for tracks to be observable under the

16

optical microscope. A bulk etching rate of 20m/h to 30m/h was desirable so that

the target thickness of 20 m could be achieved within hours. The optimum bulk

etching condition was then used to produce thin CR39 of thickness 20 m from the

thin CR39 sample (25mm 25mm 100 m). The actual thickness of thin CR39 was

measured using a micrometre screw gauge.

2.4. Bystander Effect Experiment

With the availability of thin CR39, the bystander effect experiment could be executed

by assembling the different layers together. An overview of the experiment setup is

shown in Figure 8. Cells were grown on a 4m thick polypropylene (PP) film in a

custom-designed cell irradiation dish and the CR39 was attached below the PP film.

The CR39 was separated from the radiation source by a 2mm air gap. During

irradiation, alpha particles emitted from the source had penetrated through CR39

and PP film before hitting the cells. Latent tracks were formed on the CR39 and the

cells experienced DNA damage. After irradiation, the cells were fixed and a set of

pre-staining cell images were taken with the CR39 to denote the cell locations with

respect to the CR39 boundary. Then the CR39 was removed from the PP film for

track etching and track images were taken using an optical microscope. Meanwhile,

the cells had undergone immunofluorescence staining and images of DNA damage

were taken under a fluorescent microscope. Three sets of images namely, pre-

staining images, track images and fluorescent images were digitally matched

together to observe bystander effect.

Figure 8: Bystander effect experiment setup

17

In this section, major steps of the experiment are outlined. Section 2.4.1 describes

the preparation of the custom cell dish and thin CR39 while Section 2.4.2 describes

the cell irradiation and calculation of dose delivered to the cell. Section 2.4.3 shows

the steps in fixing the cells and methods in taking the pre-staining cell images.

Section 2.4.4 illustrates the immunofluorescence staining procedure. Section 2.4.5

explains the etching procedure for CR39 and cell imaging methods. Lastly, Section

2.4.6 shows how the three set of images were matched to observe bystander effect.

2.4.1. Preparation of Custom Cell Dish and Thin CR39

The custom cell dish comprises three segments: the base, the chamber and the cap

as shown in Figure 9. The bottom layer of the chamber was mounted with PP film

and secured tightly with an o-ring. The top layer was also fitted with an o-ring to seal

the chamber when necessary. The CR39 was pre-cut into an asymmetric shape and

an intentional cut was made into the CR39 for reference purpose as shown in Figure

10. Then CR39 was attached to the bottom of PP film before inserting into the base

and the cap was screwed tightly to the base.

Figure 9: Schematic diagram of the custom cell dish

Figure 10: Thin CR39 with a cut for reference purpose

18

200 l of gelatine was added to the PP film inside the chamber and incubated for an

hour at 37C. Excess gelatine was removed, washed once with phosphate-buffered

saline (PBS) and air dried for at least an hour.

After that, about 25,000 cells were seeded onto the membrane in Dulbecco’s

Modified Eagle Medium (DMEM) supplemented with fetal bovine serum (10%),

penicillin (100 U/ml) and streptomycin (100 μg/ml) (200 l) and incubated overnight

at 37C. Two more custom cell dishes were also prepared in the same way as

described above without using CR39, setting the positive and negative control of the

experiment.

2.4.2. Cell Irradiation and Dose Calculation

Before irradiation, the custom cell dishes were filled completely with the medium and

sealed with a coverslip. To prevent any possible leakage of the medium onto the

radioactive source, the custom cell dishes were inverted and radiated from the top

instead. The positive control sample was irradiated for 1 hour. The custom cell dish

with CR39, which was meant for bystander effect experiment, was irradiated for 30

seconds. The negative control sample was placed together with the other two

samples in the same environment. Following irradiation, the cells were incubated for

another hour at 37C prior to fixation.

The dose delivered to the cell is calculated by finding the energy deposited in the cell

divided by the mass of the cell. The unit for dose is J/kg.

(0) 𝐷𝑜𝑠𝑒 =

∆𝐸 × 𝑁

𝑚𝑐𝑒𝑙𝑙=

∆𝐸 × 𝜌𝑡𝑟𝑎𝑐𝑘𝑠

𝜌𝑐𝑒𝑙𝑙 × ℎ𝑐𝑒𝑙𝑙(

𝐴𝑛

𝐴𝑐) (6)

where ∆𝐸 is the energy deposited per alpha particle in, 𝑁 is the number of alpha

particle hit per cell, 𝑚𝑐𝑒𝑙𝑙 is the mass of the cell, 𝜌𝑡𝑟𝑎𝑐𝑘𝑠 is the density of tracks

derived in Section 2.1, 𝜌𝑐𝑒𝑙𝑙 is the density of cell, ℎ𝑐𝑒𝑙𝑙 is the thickness of cell, 𝐴𝑛 𝐴𝑐⁄

is the ratio of nucleus area to cell area. For a simplistic estimation, 𝜌𝑐𝑒𝑙𝑙 is taken to be

the density of water (1 g/cm3), ℎ𝑐𝑒𝑙𝑙 is taken to be 3.770.87 m [15] and 𝐴𝑛 𝐴𝑐⁄ was

measured using ImageJ software on the cell image areas. The dose delivered for the

bystander effect and positive control samples is shown in Table 2 below.

19

Another piece of useful information was the percentage of nuclear hits within the

exposed time. This is given by

(0) % 𝑁𝑢𝑐𝑙𝑒𝑎𝑟 ℎ𝑖𝑡𝑠 = (

𝐴𝑛 × 𝜌𝑡𝑟𝑎𝑐𝑘𝑠

105) 𝑇 (7)

where 𝐴𝑛 is the cell nucleus area in m2, 𝜌𝑡𝑟𝑎𝑐𝑘𝑠 is the density of tracks in units of

tracks/mm2 and 𝑇 is the exposure time in seconds. 𝐴𝑛 was measured using ImageJ

on the cell nucleus.

Dose (cGy) % of Nuclear hit

Bystander Effect 0.3780.003 5.62

Positive Control 29.3000.003 674.61

Negative Control 0 0

Table 2: Dose and percentage of nuclear hits delivered to cells

2.4.3. Fixing of Cells and Pre-staining Cell Imaging

The medium was removed from the custom cell dishes and washed once with

phosphate-buffered saline (PBS). 200 l of 4% Paraformaldehyde solution (PFA)

was added into each custom cell dish and fixed for 20 minutes at room temperature

(RT). After that, PFA was removed and the cells were washed with PBS for 5

minutes, three times. Finally, pre-chilled 200 l of 70% ethanol was added and

incubated at RT for 20 minutes. PBS was used to wash the cells once before taking

them for pre-staining cell imaging.

The purpose of pre-staining cell imaging was to know the location of the cells with

respect to the CR39 reference line for image matching later. Therefore, only the

custom cell dish with CR39 would require pre-staining cell imaging. Nikon Eclipse

TE2000-U fluorescent microscope was used to view the cells against the CR39

background under optical transmission mode. Images of cells near the CR39

reference line were taken at 10x and 20x magnification. As PP film has its own

natural stress line markings on the film, it was important to focus on these stress

lines together with the CR39 reference line for easy comparison.

20

2.4.4. Immunofluorescence Staining Procedure

After pre-staining imaging, immunofluorescence staining was performed on the cells

using -H2AX as the biomarker for DNA damage so that the damage could be

visualised under a fluorescent microscope. Detailed step-by-step procedure can be

found in [6] but the major steps are outlined here. Firstly, 200 l of 0.1% Triton X-100

was added into each custom cell dish and incubated at RT for 15 minutes, followed

by incubating the samples with 200 l of 0.1 M glycine at RT for 2 minutes twice. The

cells were then washed with PBS for 5 minutes, 3 times. Secondly, 200 l of 10%

goat serum was added into each custom cell dish and incubated at RT for 60

minutes and washed once with PBS for 5 minutes. Thirdly, primary antibody, mouse

monoclonal anti--H2AX, was diluted in 10% goat serum at 1:100 and 100 l was

added into each custom cell dish. Then, they were covered with glass coverslips and

incubated for 2 hours at RT. After that, the custom cell dishes were washed with

PBS for 5 mins, four times. Next, secondary antibody, Alexa FluorTM 568 Goat anti-

mouse IgG, was diluted in 10 % fetal bovine serum (FBS) at 1:400 and 200 l was

added into each custom cell dish. They were incubated for 1 hour at RT with

aluminium foil wrapped around the custom cell dishes. Then, PBS was used to wash

the cells for 5 minutes, 4 times. Lastly, 200 l of 1 g/ml DAPI was added into each

custom cell dish and incubated for 10 minutes at RT. One last wash with PBS for 5

minutes was done and the custom cell dishes were stored in the refrigerator with

aluminium foiled wrapped around them.

2.4.5. Etching of CR39 and Cell Imaging

The CR39 was removed from the bystander effect custom cell dish and immersed

into the etchant solution 6.25M NaOH, at 75C, for 1.5 hours. Track images near the

CR39 reference line were then taken using Olympus Microscope BX51 at 20x

magnification under reflective mode.

All three samples were taken for fluorescent imaging using the Nikon Eclipse

TE2000-U fluorescent microscope at 20x magnification using the NIS-Elements BR

3.0 software. For a particular cell region, a bright field image of the cells was first

taken. Next, Nikon G-2A green filter cube was used to take -H2AX image which

showed regions of DNA damage and Nikon UV-2A filter cube was used to take the

21

DAPI image which showed the stained cell nuclei. The whole imaging process was

repeated to take 5 different cell regions in the positive control, negative control and

bystander effect samples.

When the bystander effect sample was imaged, additional effort was made to locate

the same cell regions as the pre-staining cell images with the help of the natural

stress lines of the PP film. The bright field, -H2AX and DAPI images were taken

around the CR39 reference line and superimposed in Adobe Photoshop CS6 to

match the fluorescent DNA damage with the location of CR39 tracks.

2.4.6. Image Matching Method for Bystander Effect

The three sets of images (pre-staining cell images, CR39 track images and

fluorescent images) were imported into Adobe Photoshop CS6 to match along the

CR39 reference line. The aim of matching is to associate irradiated cells with the

CR39 tracks. Hence, cells that have not been irradiated but exhibit DNA damage as

detected by the -H2AX fluorescence staining display the bystander effect. Location

of the cells and natural PP stress lines were used as matching points for the images

as well. Images could be stitched together to form a bigger sample area. After

matching successfully, the DAPI images were compared with the CR39 track images

to determine the nuclei being hit by an alpha particle. The nuclear hits should have

corresponding bright spots in the -H2AX images to indicate that DNA damage had

occurred. Non-irradiated cells in proximity to the irradiated cell that showed positive

signal in the -H2AX image were evidence of cells undergoing bystander effect. The

-H2AX images of positive and negative control were used to differentiate between a

positive signal and a negative signal in this bystander effect experiment. Repeated

experiments were conducted with different conditions in attempt to improve the

signal to noise ratio.

In addition, a modification was done to the positive control in a separate experiment

to test for bystander effect in the cells. Half of the custom cell dish base was covered

with a Kapton film of thickness 50 m to block out the alpha particles while the other

half was irradiated for 1 hour. -H2AX images and DAPI images were then taken for

the positive signal region, the negative signal region and the middle region. The cells

22

are expected to display positive fluorescent signal in the middle region if bystander

effect occurred in the cells.

To conclude this experimental methodology, a series of experiments had been

conducted to study bystander effect in the cells. Firstly, the optimum track etching

condition was found from the best track area after image analysis was done in

MATLAB. Secondly, the aspect ratio of minor to major axis of the tracks was

determined in relation to the angle of incidence of the alpha particle. A calibration

curve was obtained from this relationship. Thirdly, thin CR39 of thickness below 20

m was achieved by carrying out the bulk etching procedure. Finally, the thin CR39

was used in the bystander effect experiment to provide information on which cell had

been irradiated and immunofluorescence techniques were employed to observe

DNA damage within the cell. Track images and fluorescent images were compared

to determine the presence of bystander effect in non-irradiated cells which were in

close proximity to irradiated cells. The next chapter shows the results from the

experiments mentioned above.

23

Chapter 3: Results and Discussion

In this chapter, the results from the various experiments are presented and

discussed in details. The results presented are the optimised track etching conditions;

a calibration curve for the angle of incidence of alpha particles in the track angle

variation experiment; optimised bulk etching conditions used to prepare thin CR39;

and lastly the combined cell, track and fluorescent images from the bystander effect

experiment.

3.1. Optimisation of Track Etching Conditions

In this experiment, CR39 (25mm 25mm 1.5mm) was exposed to alpha particles

for 10 seconds and latent tracks formed on the CR39 were etched in a chemical

solution for the tracks to be visible under the microscope. This experiment was

carried out to determine the optimum conditions used to etch CR39 in order to

produce a track area favourable for image analysis.

Three main factors that affect the etching rate are the concentration of etchant,

temperature of the etchant and the etching duration. Concentration and temperature

of etchant were fixed at 6.25M NaOH and 75C as recommended by the

manufacturing company TASTRACK. The duration of etching was varied from 2

hours to 6 hours and images of the tracks taken are shown in Figure 11.

A total of 6 images were taken for various etching durations and processed using

MATLAB to obtain the average track area. The average track area was plotted

against the etching time as shown in Figure 12. The average density of tracks was

also determined for the 10 seconds exposure time by counting the number of tracks

per image area.

Track areas were observed to be increasing with longer etching time because the

thickness of bulk etching (𝑉𝐵𝑡) and track etching (𝑉𝑇𝑡) is proportional to time. Longer

etching time results in larger thickness being etched away, forming a bigger etch

cone. Hence larger track areas were observed under the optical microscope.

24

Figure 11: Images of tracks after etching duration of a) 2 hours b) 3 hours c) 4 hours d) 5 hours and e) 6 hours

As a sizeable track area was required for accurate MATLAB image analysis, the

optimum etching time was chosen at 4 hours from Figure 12 as it produced an

average track area of 898 m2 which was good enough for MATLAB to process.

Although bigger track areas resulting from longer etching time would be desirable, it

would not be time efficient in terms of experiment execution as the track areas

produced by 4 hours etching time were sufficient for data analysis. The standard

deviation of track areas was observed to be increasing with etching time because

longer etching time would induce greater uncertainty in the track etching. The

percentage error of track area for 4 hours was calculated to be 9% which is within an

25

acceptable range of 10% uncertainty. The average density of tracks formed on the

CR39 within 10 seconds of exposure was found to be 14517 tracks/mm2.

Figure 12: Graph of track area against the duration of etching. The optimum etching time of

4 hours produced an average track area of 898 m2.

Figure 13: Simulated probability distribution of overlapped tracks using circular track area of

89m2 and track density of 145 tracks/mm2

26

To affirm that the track area produced by 4 hours of etching was the optimum

condition, a Monte Carlo simulation was conducted to determine the probability of

non-overlapped tracks and the probability distribution is shown in Figure 13. The

simulation results showed that the probability of zero overlapped tracks was 0.95.

This means that 95% of the tracks formed was single tracks with no overlapping and

the chance of one or more overlapped tracks was very low. This provided evidence

that the track area of 89 m2 produced by 4 hours of etching was ideal for our

experiment as it produced a relatively high percentage of single non-overlapped

tracks. Therefore it was concluded from this experiment that the optimum track

etching conditions were 6.25M NaOH at 75C and an etching time of 4 hours.

3.2. Track Angle Variation Experiment

Besides studying the track area and density of tracks, the relationship between the

aspect ratio of minor to major axis of the tracks and the alpha particle’s angle of

incidence was also investigated. The aim of this experiment is to obtain a calibration

curve that could be used to determine the angle at which a cell had been hit by an

alpha particle in the bystander effect experiment later.

A collimator was used to collimate the alpha particles to vary the angle of incidence

from 0 to 60C onto the CR39. The CR39 was etched using the conditions of 6.25M

NaOH, 75C, 4 hours of etching and the resultant track images are shown in Figure

14. It was observed that the tracks transformed from circular shape at normal

incidence to elliptical shape at higher angles of incidence due to the oblique etch

cone formed after etching. These images were processed in MATLAB to obtain

statistical data on the tracks’ aspect ratio of minor to major axis, track area and

density of tracks. These data were plotted against the angle of incidence and the

graphs are shown in Figure 15, Figure 16 and Figure 17 respectively.

27

Figure 14: Images of track formation on CR39 at different angles of incidence

a) 10 b) 20 c) 30 d) 40 e) 50 f) 60

28

Figure 15: Graph of aspect ratio of tracks’ minor to major axis against angle of incidence

Figure 16: Graph of track area against angle of incidence

29

Figure 17: Graph of track density against angle of incidence

In Figure 15, the aspect ratio of tracks at normal incidence was found to be

0.980.01 which is very close to 1 as circular tracks were formed. The aspect ratio

declined as the angle of incidence was increased because elliptical tracks were

formed with minor axis shortened and major axis lengthened.

An empirical curve was fitted onto the data points and the empirical equation is

(0) 𝑦 = 0.984 + 𝐵1𝑥 + 𝐵2𝑥2 + 𝐵3𝑥3 (8)

where 𝐵1 = −0.019, 𝐵2 = 8.844 × 10−6 and 𝐵3 = 2.369 × 10−6 for 0 < 𝑥 < 60. This

equation serves as an angle of incidence calibration curve to determine the angle

which a cell had been hit by an alpha particle.

Besides the aspect ratio, the track area and density of tracks were also analysed.

Figure 16 depicts the graph of track area against the angle of incidence. At small

angle of incidence (0 to 30), the track area was observed to be increasing but

fluctuated at higher angle of incidence (40 to 60). The initial increase in track area

was expected as larger elliptical tracks were formed for oblique incidence. But the

fluctuations in track areas at high angles of incidence might be due to overetching.

30

With increasing angle of incidence, the depth which the alpha particle penetrates into

the CR39 reduces. With a constant etching condition applied to all angles, tracks

produced by high angle of incidence were more susceptible to overetching, causing

variability in the track areas. This was supported by the optical images in Figure 14

where long tail-like structures were observed at low angles of incidence but started

to disappear at 50 and 60. This indicates that the post etched surface might be

lying beyond the end of particle range and hence overetching had occurred. As such,

the track area data obtained for high angle of incidence, especially at 60 in Figure

16 was not reliable.

The density of tracks shown in Figure 17 was generally decreasing with increasing

angle of incidence. This was because the exposure area of alpha particles on the

CR39 increased with increasing angle of incidence. Since the density of tracks is

defined as the number of tracks per unit area, an increased in exposure area would

lead to a decrease in track density.

Another reason for the decrease in density was the disappearance of tracks on the

CR39, possibly due to overetching. If the tracks were overetched completely at high

angle of incidence, the tracks would disappear and not be detected during image

analysis which explained the drop in density. The disappearance of tracks

phenomenon was supported by comparing the density of tracks obtained in Figure

17 for 0 angle of incidence (17217 tracks/mm2) and the density of tracks obtained

in Section 3.1 (14517 tracks/mm2) when a collimator was not used. The source’s

activity was calculated for Section 3.1 and found to be 1000 Bq instead of the

original activity 2500 Bq obtained from the manufacturer. This proves that tracks had

been lost and one can attribute this to the overetching of high angle incidence tracks

since a wide range of incidence angles were involved when a collimator was not

utilised in Section 3.1.

As the angle variation increased the distance which the alpha particle had to travel in

the air before hitting the CR39, the particle would lose energy along its pathway.

SRIM simulation was carried out to determine the final energy of alpha particles after

travelling through the varoius thickness of air gap. Using simple trigonometry, the

thickness of air gap was calculated for the different angles of incidence as shown in

Table 3.

31

Angle of

incidence ()

Thickness of air

gap (mm)

0 10.0

10 10.8

20 11.7

30 12.6

40 13.5

50 14.9

60 17.0

Table 3: Thickness of air gap travelled by alpha particles at different angles of incidence

As the radiation source used in this work consisted of 3 different elements

(Americium-241, Curium-244 and Plutonium-239), three distinct energy profiles

existed in the emitted alpha particles. However, only the lowest energy profile would

be used for reasons explained below.

Figure 18 shows the output energy distribution of alpha particles from SRIM after

passing through 10.0 mm of air gap at zero angle of incidence. Clearly, there were

three different energy peaks corresponding to the 3 different elements.

Figure 18: Output energy distribution from SRIM after passing through 10.0 mm of air gap at normal incidence

32

However, image analysis on the CR39 track areas for zero angle of incidence

showed that there was only a single peak in its track area distribution as shown in

Figure 19. Therefore, it was evident that CR39 was not sensitive enough to

distinguish the three energy profiles. As such, only the lowest emitted energy profile

from Plutonium-239 was considered in subsequent SRIM calculations to find the

minimum energy possessed by the alpha particles. The output energies from SRIM

were plotted against the angle of incidence as shown in Figure 20. It was observed

that even at a high angle of incidence of 60, the emitted alpha particles still

possessed a minimum energy of 3.410.03 MeV to create latent tracks on the CR39.

In summary, the importance of the track angle variation experiment was to acquire

the empirical calibration curve for the aspect ratio with respect to the angle of

incidence, represented by Equation (8).

Figure 19: CR39 track area distribution for normal angle of incidence

33

Figure 20: SRIM output energies of alpha particles at different angles of incidence

3.3. Thin CR39 Preparation

The last component required for the bystander effect experiment was the thin CR39.

The thickness of CR39 must be small enough so that the alpha particles still possess

sufficient energy to cause radiation damage to cells after passing through the

different media in the experimental setup. In this section, the results from SRIM that

was used to determine the desirable thickness of CR39 are presented. Next, the

optimum bulk etching condition used to thin down commercially available CR39 to

the target thickness is established.

The CR39 thickness was varied from 0 m to 25 m and the output data from SRIM

is shown in Figure 21. It was observed that CR39 with thickness greater than 25 m

would result in alpha particles losing all of its energies before reaching the cells.

Hence the target thickness of CR39 should at least be smaller than 20 m so that

the alpha particles still possess at least 1.370.06 MeV of energy to inflict radiation

damage onto the cells. As the thinnest commercially available CR39 was 100 m, a

bulk etching procedure had to be conducted to etch away 80 m of CR39 to achieve

the thin CR39.

34

Figure 21: SRIM output energies of alpha particles after passing through varying CR39 thickness

Trials were tested on the bulk CR39 sample masked with Araldite (epoxy resin)

using different etchants and conditions. The bulk etching rate of CR39 was

determined by measuring the surface profile of CR39. Using Equation (5), the initial

trial conditions with their corresponding bulk etching rates are shown in Table 4.

Etchant Ratio Etching

Condition

Bulk Etching

Rate (m/h)

aq NaOH (10M) + Methanol 8:1 60C, 2 hours 1.0370.151

aq NaOH (10M) + Ethanol 8:1 60C, 2 hours 3.2910.084

aq NaOH (10M) + H20 + Ethanol 1:1:3 50C, 2 hours 1.4160.073

aq NaOH (6.25M) - 50C, 2 hours 0.6910.052

Table 4: Initial trial conditions with their corresponding bulk etching rates

Although the bulk etching rates for the initial conditions were slow, it was observed

that the addition of ethanol into the etchant could produce a faster bulk etching rate.

The difference in bulk etching rates could be explained in terms of the solubility of

the etched products in ethanol. As most of the etchant products produced during

35

bulk etching were polar compounds [16], they would be highly soluble in polar

solvents such as ethanol. This prevented any accumulation of etchant products on

the surface of CR39 during bulk etching which would otherwise hinder the etching

process. Hence, it was concluded that the addition of ethanol was necessary for the

choice of etchants.

Figure 22 shows the bulk etching data obtained by varying the concentrations of

KOH/ethanol and NaOH/ethanol at 50C. Bulk etching rates were observed to be

increasing with the concentration of etchants, as they varied from 0.5M to 2.5M. The

achieved bulk etching rates were much higher than the initial trials as KOH and

NaOH were dissolved directly in ethanol without any aqueous solution. The bulk

etching rate of KOH/ethanol was observed to be much faster than NaOH/ethanol

because KOH is more reactive than NaOH.

Figure 22: Bulk etching rates for NaOH/ethanol (blue) and KOH/ethanol (red) at 50C with concentration varied from 0.5M to 2.5M. Repeated experiments were carried out for

KOH/ethanol 1.0M and 1.5M (black).

The surface roughness of CR39 samples etched using KOH/ethanol was measured

using Atomic Force Microscopy (AFM) and the root mean square roughness (Rq)

data are presented in Table 5. The results indicated that the CR39 became relatively

36

smoother after bulk etching as compared to the original sample which was ideal for

optical microscopy. To achieve a desirable bulk etching rate of 20m/h to 30m/h

such that the target thickness of 20 m thin CR39 could be achieved within hours,

1.0M or 1.5M KOH/ethanol would be suitable.

Etchant Rq (nm)

KOH/ethanol (0.5M) 3.13

KOH/ethanol (1.0M) 3.91

KOH/ethanol (1.5M) 3.95

KOH/ethanol (2.0M) 4.10

KOH/ethanol (2.5M) 4.14

Original Sample 4.45

Table 5: Surface roughness measurement results for different concentration of KOH/ethanol

Repeated experiments were conducted for both concentration levels and the bulk

etching rates were reported to be consistently higher than before as shown in Figure

22. This suggested that there was an experimental error during the bulk etching

process. This error was introduced when multiple CR39 samples were loaded into

the etchant as more time would be required to load the samples as compared to

loading a single sample in the repeated experiment. As the etchant would be away

from the heating source during loading, an increase in loading time would result in a

drop in initial etching temperature which explained the decrease in bulk etching rate.

Nevertheless, a more reliable and consistent rate of 20.930.91 m/h and

28.170.52 m/h were achieved for 1.0M and 1.5M KOH/ethanol respectively in the

repeated experiments.

The optimum bulk etching condition was decided to be 1.0M KOH/ethanol at 50 C

as milder etching conditions were usually preferred since they produce a more even

etching of CR39. Using this condition, commercially available CR39 of 100 m

thickness would be etched below the target thickness of 20 m in about 2 hours to

produce the thin CR39. The average thickness of thin CR39 was measured to be

162 m and these thin CR39 were used in the bystander effect experiment.

37

3.4. Bystander Effect Experiment

In this experiment, two approaches were used to demonstrate the bystander effect in

cells. The first approach was a proof-of-principle experiment to test for the presence

of bystander effect in the positive sample. The second approach was a more detailed

investigation in which thin CR39 prepared from the previous section was used to

differentiate irradiated cells from the non-irradiated cells such that bystander effect

could be distinguished between neighbouring cells.

In this section, images from the positive control, negative control, and bystander

effect samples are presented using 15 m in thickness of CR39. Figure 23 shows -

H2AX fluorescent images for both the positive control and negative control samples

of the experiment. The bright areas in the positive control image denote regions with

positive signal where DNA damage had occurred in the cells. In contrast, some

background staining in the negative control was observed, corresponding to inherent

genetic instability of the Hela cells used. These two images were used to

differentiate a positive signal and negative signal in the bystander effect experiment.

The first approach involved a modification to the positive control setup. Half of the

cells were irradiated for 1 hour while the other half was shielded with a layer of

Kapton film. -H2AX fluorescent images from the exposed, shielded and middle

regions are shown in Figure 24. A significant difference in positive signals was

clearly observed between the middle region and the shielded region. This difference

denotes radiation damage in the middle region cells and thus suggests the presence

of bystander effects in these cells.

38

Figure 23: -H2AX fluorescent images for a) positive control and b) negative control. Red bright spots denote regions of DNA damage.

39

Figure 24: -H2AX fluorescent images for a) exposure region b) shielded region c) middle region

40

In the second approach, images of CR39 tracks were matched with -H2AX and

DAPI fluorescent images in Adobe Photoshop to examine bystander effect in

unirradiated cells. The bright field pre-staining images were first matched with the

CR39 track images along the reference line as shown in Figure 25 before

superimposing on the post-staining images. The DAPI image, which shows the

stained cell nuclei, were then overlapped with the images of CR39 tracks in Figure

26 to determine which cell nucleus had been irradiated based on the track locations.

As the alpha particle had to pass through a layer of PP film of 4 m thickness after

exiting the CR39, the exact hit locations of the alpha particle on the cells could be

accurately determined by analysing the aspect ratio of minor to major axis of the

tracks using MATLAB and using the calibration curve obtained in Section 3.2. For

example, Figure 27 shows three cropped areas from Point X, Y and Z in Figure 26.

The aspect ratio of the tracks was measured and shown in Table 6 below. The

corresponding angle of incidence was calculated using Equation (8) and the extra

distance which the alpha particle had to travel after passing through 4 m thick of PP

film was found using simple trigonometry.

Aspect Ratio Angle of incidence () Extra distance (m)

X 0.69 15.95 1.14

Y 0.42 36.35 2.94

Z 0.50 28.63 2.18

Table 6: Calculation results of angle of incidence from the track’s aspect ratio and extra distance travelled by alpha particle

The exact hit location could be determined by plotting the extra distance from the

centroid of the tracks as shown in Figure 27. It can be seen that although the tracks

were overlapping with the cell nuclei for Y and Z, the exact hit location was outside

the cell nuclei and thus they were not considered as a nuclear hit. This method of

analysis was applied to all other possible overlapping tracks in Figure 26 and the

nuclear hits were marked out by the red boxes. The percentage of nuclear hits in

Figure 26 was calculated to be about 7.07% which was in close agreement with our

initial calculation of 5.62% from Table 2.

41

Figure 25: Overlaying of pre-staining bright field image onto CR39 along the reference line.

Figure 26: DAPI image superimposed on CR39 track image for bystander effect experiment. The red boxes denote cell nuclei which were hit by an alpha particle.

42

Figure 27: Cropped image from Point X, Y and Z in Figure 26. White cross denotes the exact hit location of the alpha particle on the cell.

With the knowledge of nuclear hits, the corresponding -H2AX fluorescent image

was studied and the same red boxes in Figure 28 were used to denote positive -

H2AX signals due to direct irradiation. The surrounding cells which were not

irradiated but yet display positive -H2AX signals were identified with the white

arrows in Figure 28. By examining this image alone, it seemed that the unirradiated

cells in close proximity with radiated cells had experienced radiation damage due to

bystander effect.

Figure 28: -H2AX fluorescent image for bystander effect experiment with red boxes denoting cells which were hit by an alpha particle. White arrows denote positive signals

which were not associated with direct irradiation.

43

However, the noticeable background -H2AX staining in the negative control (Figure

23) made the interpretation of data in Figure 28 less straightforward. The two images

were similar in terms of their -H2AX signals which caused difficulty in differentiating

the positive signals from the negative signals in Figure 28. The error of having

positive signals (bright spots) appearing in the negative control could be explained

by two reasons.

Firstly, the choice of cell line for this experiment may not be suitable. As HeLa cells

are tumour cells, they are generally more genetically unstable than normal cells

which result in higher number of DSB within the cells. Thus, more positive -H2AX

signal will be displayed even though no radiation has been delivered.

The second reason could be due to some non-specific staining. As the primary and

secondary antibodies were meant to bind to specific sites where DNA damage had

occurred, there might be non-specific binding to other sites that caused the increase

in -H2AX signals. Methods such as reducing the concentration of primary antibody

or using a signal enhancer reagent called Image-iT® FX had been adopted to reduce

non-specific staining but no significant improvements were observed.

One may argue that positive signals in Figure 28 may be due to the background

signals instead of attributing to bystander effect. As such, more efforts need to be

taken for the second approach experiment to improve the signal to noise ratio.

Possible improvements for the experiment include testing on an alternative cell line

which is not cancerous in nature, and also re-optimising the immunofluorescence

staining protocol to reduce non-specific staining. However, we can still conclude that

the bystander effect exists between irradiated cells and non-irradiated cells as the

proof-of-principle experiment had supported the phenomenon of bystander effect.

45

Chapter 4: Conclusion

In summary, we have established a few optimised conditions and developed several

procedures to study radiation-induced bystander effect. The track areas of CR39

were first investigated to determine the optimum track etching condition, which was

found to be 6.25M NaOH, 75C and 4 hours of etching time. This condition produced

an average track area of 898 m2 and a track density of 14517 tracks/mm2 which

was good for image analysis using MATLAB.

The aspect ratio of the track’s minor to major axis was also examined to determine

the empirical relationship between the aspect ratio and the alpha particles’ angle of

incidence represented by Equation (8). This equation serves as a calibration curve

for us to determine the angle of incidence of the alpha particles onto the cells. The

exact hit location of the alpha particles on the cells can then be accurately identified

in the bystander effect experiment.

As CR39 was used in the experiment to provide information on cells which had been

irradiated based on the track locations, the thickness of CR39 was required to be

less than 20 m in order for the alpha particles to possess sufficient energy to

penetrate the different layers in the experimental setup. A bulk etching procedure

was developed to etch down commercially CR39 of thickness 100 m to the desired

thickness using the optimum bulk etching condition of 1.0M KOH/ethanol at 50C.

Lastly, the proof-of-principle experiment suggests that bystander effect could be

detected in unirradiated cells. Results from the experiments using thin CR39 showed

several cells appeared to display bystander effect but the background -H2AX

staining in the negative control made the interpretation of these results less

straightforward. This was possibly due to the limitations mentioned above and further

work will be required.

47

Chapter 5: Future Work

As a mixed radiation source was used in this work with no targeting ability, one

possible future work will be using the ion beam accelerator to target microbeam on

specific cells which allows better study of the bystander effect phenomenon.

Furthermore, other particles such as protons can be used to irradiate cells to

investigate the difference in radiation effects on cells from alpha particles. The

energy of the particles can also be better controlled using an accelerator to vary the

dose delivered to the cells in a single particle hit. All these work may be helpful in the

future development of proton therapy as means to cure cancer patients.

49

Chapter 6: Acknowledgement

Firstly, I would like to thank God for the completion of this final year projection.

Secondly, I would like to thank Assoc. Prof Andrew A. Bettiol and Dr Chen Ce-belle

for the opportunity and guidance for this project. Also, I would like to express my

deepest gratitude to my mentor, Tao Ye, who has taught me many skills and

knowledge. Thirdly, I would like to thank all other CIBA staffs who provided me

assistance in one way or another with special mention of Tan Hong Qi and Dr

Shuvan Prashant Turaga who has helped me in the Monte Carlo simulations and

microscopy respectively. Lastly, I would like to thank my family and girlfriend who

have provided me with the strength and encouragement throughout the year.

51

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