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Using laser correlation spectroscopy to study gold nanoparticles diffusion dynamics for photodynamic therapy

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Using laser correlation spectroscopy to study gold nanoparticles diffusion dynamics for photodynamic therapy
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Alizaidan, Al Ogaidi Marwah ( author )
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English
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Photochemotherapy ( lcsh )
Nanoparticles ( lcsh )
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bibliography ( marcgt )
theses ( marcgt )
non-fiction ( marcgt )

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The dynamic behaviour of gold nanoparticles of different shapes (sphere and rod) were studied under different experimental conditions to be used for drug delivery in photodynamic therapy. Fluorescence correlation spectroscopy (FCS) using single photon excitation had been used to study the diffusion dynamics of gold nanoparticles of different shapes in water solution to utilize these results for further enhancement for the photodynamic therapy. We analyzed four different colloidal gold solutions (colloidal gold nanosphere solution, colloidal gold nanosphere-photosensitizer solution, colloidal gold nanoshpere- Rhodamine B solution, and colloidal gold nanorods-Rhodamine B solution) using the custom software (MAZ-2015) we made to analyze the results for these samples by calculating and fitting the autocorrelation. We found that we could get a reliable correlation when laser power was higher than 4.2 µW. The phosphate buffered saline shows the best results over the nanopure water and culture media when used to dilute the colloidal gold nanoparticles solutions, and the (MAZ-2015) software provides a comparable results to commercial software provided by the microscope vendors. The results indicated that gold nanosphere conjugated with Rhodamine-B can be a potential candidate to study drug transport to cells in photodynamic therapy treatment.
Thesis:
Thesis (M.S.)--University of Colorado Denver.
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Includes bibliographic references
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Department of Electrical Engineering
Statement of Responsibility:
by Al Odaidi Marweh Alizaidan.

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|University of Colorado Denver
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|Auraria Library
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951917321 ( OCLC )
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Full Text
USING LASER CORRELATION SPECTROSCOPY TO STUDY GOLD
NANOPARTICLES DIFFUSION DYNAMICS FOR PHOTODYNAMIC THERAPY
By
AL OGAIDI MARWAH ALIZ AID AN
B.S. University of Technology, Baghdad, 2010
A thesis submitted to the
Faculty of the Graduate school of the
University of Colorado in partial fulfilment
of the requirements for the degree of
Master of Science
Electrical Engineering
2015


11
This thesis for the Master of Science degree by
A1 Ogaidi Marwah Ali Zaidan
Has been approved for the Electrical Engineering program by
Tim C. Lei, Chair
Mark Golkowski
Yiming J. Deng
November 20, 2015


Ill
A1 Ogaidi, Marwah Ali Zaidan (MS, Electrical Engineering)
Using Laser Correlation Spectroscopy to Study Gold Nanoparticles Diffusion Dynamics
for Photodynamic Therapy.
Thesis directed by Associate Professor Tim C. Lei
ABSTRACT
The dynamic behaviour of gold nanoparticles of different shapes (sphere and rod)
were studied under different experimental conditions to be used for drug delivery in
photodynamic therapy. Fluorescence correlation spectroscopy (FCS) using single photon
excitation had been used to study the diffusion dynamics of gold nanoparticles of different
shapes in water solution to utilize these results for further enhancement for the
photodynamic therapy. We analyzed four different colloidal gold solutions (colloidal gold
nanosphere solution, colloidal gold nanosphere-photosensitizer solution, colloidal gold
nanoshpere- Rhodamine B solution, and colloidal gold nanorods-Rhodamine B solution)
using the custom software (MAZ-2015) we made to analyze the results for these samples
by calculating and fitting the autocorrelation. We found that we could get a reliable
correlation when laser power was higher than 4.2 pW. The phosphate buffered saline shows
the best results over the nanopure water and culture media when used to dilute the colloidal
gold nanoparticles solutions, and the (MAZ-2015) software provides a comparable results
to commercial software provided by the microscope vendors. The results indicated that
gold nanosphere conjugated with Rhodamine-B can be a potential candidate to study drug
transport to cells in photodynamic therapy treatment.
The form and content of this abstract is approved. I recommend its publication.
Approved: Tim C. Lei


ACKNOWLEDGEMENT
IV
Much appreciation and gratitude to God for giving me the strength, health and
capacity to do this research work. Secondly, I take this opportunity to express my profound
gratitude and deep regards to my supervisor Dr. Tim Lei for his exemplary guidance,
monitoring and constant encouragement throughout the course of this thesis. I also, would
like to thank Saif Al-Juboori for the time and the efforts he puts in helping with setting the
experiment and doing the tests .In addition to the valuable advices. I would also thank Dr.
Zheng Huang and his group who helped in clarifying some points about the photodynamic
therapy and providing the samples. Much thanks to Mr. Gregory Glazner who helped in
doing the characterization experiment and explaining some concepts about it. I also thank
my parents, brothers, sisters and friends for their constant encouragement without which
this research would not be possible.


V
TABLE OF CONTENTS
I. INTRODUCTION...............................................................1
Correlation...............................................................3
Microscopy................................................................4
Confocal Microscopy.......................................................5
Fluorophores..............................................................5
LASERS....................................................................6
Stokes Shift..............................................................7
Fluorescence Correlation Spectroscopy.....................................8
The History of Fluorescence Correlation Spectroscopy.....................11
Principles and Theories of Fluorescence Correlation Spectroscopy.........11
Theoretical Concepts of FCS..............................................13
Fluorescence Correlation Spectroscopy and Nanoparticles..................14
Nanoparticles............................................................15
The History of Nanoparticles.............................................15
The Properties of Nanoparticles that Differ to Bulk Materials............17
The Utilities of Different Properties of Nanoparticles...................18
Photodynamic Therapy.....................................................19
Introduction.............................................................19
The Principles of PDT....................................................21
Applications of Photodynamic Therapy.....................................21
Photosensitizers.........................................................23
Works Done on Nanoparticles..............................................24
Motivation of the thesis.................................................25
II INSTRUMENTS AND METHODS...................................................27
Gold Nanoparticles Characterization......................................27
UV- visible Spectroscopy.................................................27
Fluorescence Correlation Spectroscopy....................................28


vi
III. RESULTS.............................................36
IV. DISCUSSION...........................................40
V. CONCLUSIONS..........................................43
VI FUTURE WORK...........................................44
REFERENCES...............................................46


LIST OF TABLES
Table
1: Results from autocorrelation calculations for different colloidal gold nanoparticles
solutions................................................................ 38


Vlll
LIST OF FIGURES
Figure
1: Stokes shift................................................................. 7
2: Conceptual diagram of a fluctuating fluorescence signal...................... 8
3: Typical confocal FCS system.................................................. 9
4: Process of Photodynamic Therapy............................................. 21
5: UV-visible spectra for four different colloidal gold solutions.............. 29
6: Flow Chart for (MAZ-2015) software.......................................... 32
7: Graphic user interface for custom software (MAZ-2015)....................... 33
8: The working software when computing the autocorrelation for file 1.......... 34
9: The selective feature to remove unwanted fluctuations....................... 35
10: Cross correlation.......................................................... 35
11: RMSE results using different concentrations................................ 39
12: RMSE results using different laser powers.................................. 40
13: RMSE results using different samples....................................... 40
14: RMSE results when diluting the colloidal gold solution in three different
solvents................................................................... 41


1
CHAPTERI
INTRODUCTION
While the applications of photodynamic therapy are indisputably well established
within a number of fields, its use in treatments for cancer and other diseases is still in a
developmental phase. It works by producing a reactive species, typically singlet oxygen,
through transferring energy from light-excited dye molecules in target tissues. Recently,
wide-ranging strategies were proposed, seeking to enhance the efficiency of photodynamic
therapy through the use of nanoparticles. This thesis, therefore, studies the diffusion
dynamics for colloidal gold nanoparticles.
Fluorescent correlation spectroscopy is a commonly used technique to study the
dynamics of molecules due to its high sensitivity; it can also be employed in biomedical,
chemical, and biological fields (Weiss, 1999).
Gold nanostructures attract particular interest because of their favourable emission
properties in terms of good photo stability under continuous irradiation, there are no
blinking effects (the light and dark periods the single particles experience when exposed to
continuous laser illumination), and good biocompatibility. The likelihood of tuning the
absorption band by modifying the particles shape is also another advantage for their
application in different fields. A detailed investigation on the luminescence behaviour,
under one and two-photon excitation at increasing laser power, of citrate capped gold
nanoparticles (Au NP) with increasing diameter (up to 50 nm) has been reported by
Loumaigne and Colleagues.


According to the fluorescence correlation spectroscopy (FCS) analysis over the past
years researchers found that there is a linear relationship between the diameters of gold nano
spheres and the diffusion time. They also found that the size, shape, and surrounding surface
environment are crucial in the optical properties of nanoparticles
In addition to that the nanoparticles are less prone to photobleaching when excited
with strong laser light. In accordance with all the above mentioned reasons, the
nanotechnology becomes widely used in medical applications like drug delivery and
specifically the photodynamic therapy and diagnosis (De Jong and Borm, 2008).
FCS experiments are run under increasing the laser power, using different samples
of colloidal gold solutions, using different wavelengths, and using different solutions to be
dispersed in. A review for the fluorescence correlation spectroscopy, nanoparticles,
photodynamic therapy, and discussion for the results will be presented in the following
sections of this thesis.
There has been a high growth of research and applications in the area of nano science
as well as nanotechnology in the past years. Recently there is an increasing optimism that
nanotechnology while applied to medicine will bring important developments in the
diagnosis as well as the treatment of the disease. Applications in medicine that are
anticipated are such as drug delivery, in vitro and in vivo diagnostic, as well as production
of improved biocompatible materials. Engineered nanoparticles are an important tool since
they help one to realize a number of these applications.
It has also been identified that not all particles that are used for medical purposes
comply with the acceptable definition that has been recently proposed by the Royal Society and
Royal Academy of Engineering of a size lOOnm. This has not proven to have any impact on


3
the functionality of medical applications. Moreover, for drug delivery not only engineered
particles may be used as carriers. The drug may be formulated on itself at a nanoscale and then
function on itself as a carrier.
Correlation
Correlation is referred to as a statistical measurement, which is utilized to describe
the relation between two fluctuating signals (Cross-Correlation) or the signal with itself
(Autocorrelation). The autocorrelation function G (t) can be determined using the following
equation, (Gratton, 2005):
G(Z>------F(t)2 (1)
Where:
F(t) refers to the fluorescence signal
t refers to the real time
r refers to the time difference between two intensity measurements
(t) refers to the variance where 8F(t)= F(t)
< (SF(t). (t + t)) > refers to the autocorrelation of F(t)
(< (t) >)2 refers to the square of the average value of F(t)
Whereas the cross-correlation function for the fluorescence fluctuations
from two different channels 1 and 2 (Gn (r)) can be calculated using the following
equation, (Gratton, 2005):
G12 (t) -
{SF^t). 8F2(t+r))

(2)
Where: Fi (t) and F2 (t): are the fluorescence signals from channel 1 and 2, respectively.


On the other hand, correlation can be defined in different ways according to the field
of study. In essence, the correlation being a statistical measurement portrays the relationship
and degree to which two variables or more fluctuate together. In signal processing, the
correlation used to analyse functions or series of values like the time domain signals.
Subsequently, correlation is mainly defined in two terminologies, which are a positive
correlation and a negative correlation (Rigler & Elson, 2012).
Hence, a positive correlation shows the level at which variables decrease or increase
in parallel. On the other hand, a negative correlation shows the level at which one of the
variables increases while the other variable decreases. Cross-Correlation in signal
processing refers to a similarity measure of two series mainly as a lag function of one
relative series to the other (Berezin, 2014).
Microscopy
Microscopy refers to a noble scientific practice, which comprises of magnifying
objects that the unaided eye cannot see. Thus, the main objective of this scientific discipline
is to be able to magnify the object so that it is visible for studying. This allows researchers
to conduct their study and learn essential things about the invisible objects, as well as how
they work. In addition, microscopy utilizes microscopes to view these objects and samples.
There are mainly three major branches of microscopy which include scanning probe,
electron and optical microscopies (Berberan -Santos, 2008).
On the same note, electron and optical microscopy involve the refraction, reflection
or diffraction of electromagnetic radiation beams that interact with the specimen. It also
interacts with the scattered radiation collection or any other signal so that it can create an


image. Conversely, this process can be performed through the sample wide-field irradiation
or through scanning the samples fine beam. Scanning probe microscopy comprises of the
scanning probe interaction with the samples surface (Berezin, 2014).
Confocal Microscopy
Confocal Microscopy refers to an optical imaging method, which is used to increase
the optical resolution and the difference of a micrograph through an additional spatial
pinhole that is placed on the lens confocal plane. Confocal Microscopy has gradually gained
popularity particularly in the industrial and scientific communities. Its distinctive
applications are in materials science, life sciences and semiconductor (Rigler & Elson,
2012).
In this sense, this technique provides numerous advantages as compared to the
conventional optical microscopy. These advantages include out of focus glare elimination,
and narrow depth of field (Rege & Medintz, 2009).
Fluorophores
A fluorophore is also known as a fluorochrome, and it is similar to a chromophore.
A fluorophore is mainly a fluorescent chemical substance which can re-emit light when
light excitation takes place. Hence, a fluorophore refers to a section of a molecule, which
leads to the creation of a fluorescent emission specifically in the observable light spectrum.
These fluorophores absorb different light wavelengths, and this creates the visible light.
These fluorophores can be introduced through artificial methods, or they can exist naturally.


6
It is paramount to note that many rocks and fish maintain some natural levels of these
fluorophores (Berberan Santos, 2008).
Nevertheless, the fluorophores are utilized widely in the scientific fraternity for
research purposes since they assist in analyzing certain material properties. Hence,
researchers can identify changes and reactions in the biochemistry fields, as well as protein
study. Besides, the immunofluorescence discipline utilizes this technique to assist in
labelling antibodies and antigens at the level of subcellular (Rege & Medintz, 2009).
LASERS
LASER is a short for Light Amplification by Stimulated Emission of Radiation.
Theodore Maiman invented it in 1964. The laser device can produce monochromatic,
directive, and coherence light, which could aid many useful inventions. The laser device is
composed of an active medium (number of atoms or molecules emit electromagnetic
radiation by stimulated emission after being stimulated by population inversion), an
excitation mechanism (supplies energy to the active medium), an optical feedback, a
resonator (the place between the two laser mirrors where the laser action occurs), an output
coupler (partially reflecting mirror located at the end part of the optical cavity which enables
the light to exit the laser). Lasers can be classified according to the active medium into gas
lasers, solid-state lasers, excimer lasers, dye lasers, or semiconductor lasers. A laser is
different from other light sources because it emits its light coherently whereby spatial
coherence enables it to be focused to one tight spot. Ideally, the invention of these powerful
focused light sources enables various applications such as lithography, laser cutting, and
more advanced inventions like confocal laser scanning microscopes (Rege & Medintz,
2009). It also has many further applications and can be used in laser printers, laser surgery,


barcode scanners, optical disk drives, free-space and fiber-optic optical communication, as
well as skin treatments, laser lighting displays, welding and cutting materials (Berberan -
Santos, 2008).
Stokes Shift
When absorbing light, the atom or the molecule undergoes a transition into an
excited electronic state accompanies with losing small amount of absorbing energy before
releasing the rest of its energy as luminescence (thermal energy in most cases). The
difference between the band maxima of the absorption and luminescence spectra within the
same electronic state is known as stokes shift and it could be represented in frequency or
wavelength units (Tuite, 2013).
Figure 1: Stokes shift: The difference between the band maxima of the
absorption and luminescence spectra in wavelength units (Tuite, 2013).


8
Fluorescence Correlation Spectroscopy
Fluorescence correlation spectroscopy (FCS) is a correlation of temporal
fluctuations of the fluorescence intensity. The fluctuation of the signal is used to calculate
the autocorrelation (G (x)) where x is the lag time from the original signal. The amplitude
of the autocorrelation function (typically G (0)) is inversely proportional to the average
number of molecules in the probe volume ().
A B
Figure 2: Conceptual diagram of a fluctuating fluorescence signal, (A) Fluctuated fluorescence
signal as a function of time. (B) The autocorrelation, G (x), where x is the lag time from the original
signal. The amplitude of the autocorrelation function (typically G (0)) is inversely proportional to
the average number of molecules in the probe volume (), (NCBI.gov, Figurel, 2012).
FCS is one of the many different modes of high resolution spatial and temporal
analysis of extremely low concentrated biomolecules. It measures the fluctuations of
fluorescence intensity in a sub-femtoliter volume to detect such parameters as the number
of molecules and the diffusion time. The temporal changes in the fluorescence emission
intensity is recorded which is caused by single fluorophores that pass through the detection
volume. Eventually, important biochemical parameters can be determined as the
concentration, size, and shape of the particle or the viscosity of the environment changes
(Lakowicz, 2006).


9
Sample
Dichroic
Mirror
Inteference
Filter tZ
xz
> Tube Lens
Pinhole
Avalanche
Photodiode
Figure (3): A typical confocal FCS system. Laser light is focused by an objective (usually
with high numerical aperture) to a diffraction limited spot. Fluorescence is collected by
the same objective and filtered by an interference filter. A pinhole placed in the
conjugate image plane reduces out of focus light. The pinhole is usually omitted in two-
photon excitation (NCBI.gov, Figure2, 2012).
FCS is a sensitive form of analytical tools due to the fact that it is able to observe
a small number of molecules that is nanomolar to picomolar concentrations in a small
volume. This in turn makes FCS the perfect method to provide quantitative answers on
diffusing molecules from within unperturbed compartments such as cells. FCS was
developed in the early seventies as a special case of relaxation analysis. Classical relation
methods induce a certain level or kind of external perturbation like pressure or temperature
jumps to a reaction system and records information about the kinetic parameters by
observing the way the system jumps back to equilibrium. FCS just as classical techniques
takes advantage of the spontaneous minute fluctuations of physical parameters that are
reflected by the fluorescence emission of the molecules. These fluctuations are continually
occurring at ambient temperature and are represented as noise patterns of measured signal


in fluorescence. This autocorrelation analysis provides a measure for self-similarity of a
time series signal that describes the persistence of the information carried. The information
processes governing the molecular dynamics can thus be derived from temporal patterns
display by fluorescence fluctuations decay and arise (Rigler & Elson, 2001). The main
factors that affect the autocorrelation function and as a result the rate of diffusion are, the
viscosity of the solvent, the size of the fluctuated particles and the temperature as shown
in the following Stokes-Einstein formula which can be used when the fluid is a liquid and
brownian particles are spheres of radius r, (Sri Balaji et al., 2011)

K T
6nrri
(3)
Where,
r| is the viscosity of the solvent.
T is the temperature.
K is Boltzmann constant.
r is the particle hydrodynamic radius.
Fluorescence Cross-Correlation Spectroscopy (FCCS) is a daughter technique that
correlates signals originating from two different fluorophores detected in two channels with
each other. When two different spectral fluorophores are attached to two molecules they
form a dual colour FCCS results. This information of the degree of coinciding appearance
in the optical volume is used to learn about the degree of interaction between fluorophores.
FCCS therefore offer binding kinetics in unperturbed systems and also in low molecular
concentrations in solutions (Lakowicz, 2006).


11
The History of Fluorescence Correlation Spectroscopy
Almost 40 years since its introduction FCS has evolved from a mysterious and
difficult measurement to a technique that is routinely used in the research technology. FCS
value in biological and physical sciences consists in the measurements that it makes possible
and the concepts that it illustrates and that form its basis. FCS provides the window for the
field of single molecule measurement and microscopic world (Graslund, Rigler &
Widengren, 2010).
FCS was first introduced by Madge, Elson and Webb in 1972, where it was applied
to measure diffusion and chemical dynamics of DNA-drug interaction. The term FCS was
coined by the Webb lab. The main breakthrough of the technique was the introduction of
the confocal optics by Rigler and co-worker in the early 1990s which resulted in increased
sensitivity to sample fluorescence at the single molecule level. These pioneering studies
were then followed by a number of other applications by many different groups describing
translation and rotational mobility in two or three dimensions, attempting to determine the
particle concentration even in the cellular environment. These early measurements suffered
from poor signal to noise ratios, which was mainly because of the low detection efficiency
and insufficient background suppression (Magde, Webb & Elson, 1978).
Principles and Theories of Fluorescence Correlation Spectroscopy
FCS Experiments commonly involve sample volumes as low as a few microliters
and the measurement focal volume is in the order of femtoliters. FCS measurements can be
performed in solutions and living cells. FCS is based on the analysis of fluctuations of
fluorescence. The molecules typically originate from Brownian motion, the random motion
of particles suspended in a fluid (a liquid or a gas) resulting from their collision with the


quick atoms or molecules in the gas or liquid of dye labelled molecules through small laser
spot (Rigler & Elson, 2001). These molecules stay within the laser spot depending on their
size and if a small dye tagged molecule binds to a large one it emits photons and slows down
during its diffusion time (Rigler & Elson, 2001).
A sensitive detector records single photons emitted by the molecule of dye.
Correlation functions are applied to extract information about the number of molecules
(concentration). Physical modes are fitted to the correlation data to quantify the information
on the source of the fluctuations. When one detector and one type of fluorescent dye are
used the method is known as auto-correlation. To increase the flexibility of the method, two
dyes and detectors are used and the method is called cross-correlation (Rigler & Elson,
2001).
The principles of FCS are marked by different stages in the exploitation of fluctuations,
as identified and explained below:
1. Source of Fluctuations
Source of Fluctuations involves the movement of small particle which is free
diffusion in Brownian motion. Fluorescent dye tagged molecules emit a burst of photons
while they diffuse through a small laser spot (Rigler & Elson, 2001).
2. Create a Small Measurement Volume
Measurement of fluctuations of different samples in solutions forms the basis for
FCS. Low number of molecules produces a fluctuation signal that is higher. For a higher
concentration measurement, the measurement of volume as small as possible should be
created. The measurement of volume is produced using diverse methods like Multi-photon
excitation and confocal volume (Rigler & Elson, 2001).


13
3. Detect Single Photons
During the passing of the measurement volume, very few photons are emitted by the
chromophore that is attached to the protein of interest. Sensitive photon counting detectors
are needed for the detection of the photons. Characteristically Avalanche Photo Detectors
(APD) are used (Rigler & Elson, 2001).
4. Calculate Correlation Function
The transformation of the data form the measure time domain to the correlation time
domain is done by the auto-correlation function. To compute the auto-correlation function,
one multiplies the measured data with a time-shifted data. If there is no time-shift and both
data traces are the same, then the correlation is high. If the shift is large and the two traces
are very different, then the correlation is low (Rigler & Elson, 2001).
Theoretical Concepts of FCS
a. Autocorrelation Analysis
Autocorrelation analysis is performed when the focus is on one species of
fluorescent particles. Fluctuations in the fluorescence signal are quantified by temporally
auto correlating the recorded intensity signal. In principle, this autocorrelation routine
provides a measure for the self-similarity of a time signal and highlights characteristic time
constants of underlying processes (Schwille & Haustein, 2004).
b. Cross-correlation Analysis
In autocorrelation analysis, one effectively compares a measured signal with itself
at some later time and looks for recurring patterns. In cross-correlation analysis two
different signals are correlated and thus a measure of crosstalk is obtained. It involves


looking out for common features of independently measured signals (Schwille & Haustein,
2004).
14
Fluorescence Correlation Spectroscopy and Nanoparticles
The prospects of using nanoparticles as superior sensors and labels that do not
photobleach in biological and environmental studies has sparked wide spread interest in the
science community. For the application of nanoparticles in FCS, the diffusion of
nanoparticles in a liquid environment must be studied and understood. This is because when
applying the correlation techniques, the diffusion constants of nanoparticles are extracted
from solutions mainly using the FCS. FCS measures spontaneous intensity fluctuations
which in the case of nanoparticles are caused by small deviations from the equilibrium
which in turn are caused by nanoparticles entering and leaving the detection area. Just like
in the ordinary FCS, FCS that use nanoparticles obtain the largest fluctuations when there
are only few molecules or particles in a small detection volume with the ultimate limit being
a single molecule or particle at a given time. The sufficient signal to noise for a single
molecule or particle in FCS can be achieved through minimizing the detection volume by
focusing a laser beam to a diffraction limited spot combined with high quantum yield
photodetectors (Tetin, 2013).
FCS can be used to measure the diffusion of nanoparticles to understand their
mobility under different conditions like different shapes and core sizes, an applied external
field, or varying surface capping materials. The hydrodynamic radius of the nanoparticles
is determined using FCS. FCS also allows for follow up on binding and dissociation
between the nanoparticle surface and molecular targets or substrates as well as among
nanoparticles including nanoparticle aggregation. FCS that uses nanoparticles enables


researchers to understand the heterogeneous systems, which contain mixtures of different
nanoparticles (Tetin, 2013).
Nanoparticles
Nanoparticles are particles that range from 1 to 100 nanometers in size. They are
mostly the same size as biomolecules which include proteins, antibodies and membrane
receptors. Due to their size nanoparticles are primarily used to mimic biomolecules and
other particles therefore giving them a huge potential of use in the biomedical field. The
properties of many conventional materials change when they are formed from nanoparticles
because nanoparticles have greater surface area per unit weight than large particles which
makes them more reactive to other molecules. This makes the use of nanoparticles or the
evaluation of their use to be applicable in many fields such as medicine, manufacturing of
materials, implementation in the environment and application in optical energy and
electronic fields. Nanotechnology describes a particle as a small object with its transport
and properties that behaves as a whole unit. Nanoparticles fall under the ultrafine category
of particles while those particles above 100 nanometers fall under fine particles (100 to
2,500 nanometers) and coarse particles (2,500 and 10,000 nanometers) (Schmid, 2011).
The History of Nanoparticles
Nanoparticles have a long history that includes results not only from modern
research and man-made material but also from naturally occurring nanoparticles. Naturally
occurring nanoparticles include organic particles like proteins and viruses and inorganic
particles like oxyhydroxides and metals that have been produced by volcanic eruptions,
weathering, wildfires, and microbial processes. Nanoparticles are obviously not only
provided by modem synthesis laboratories but also have been in existence since the


prehistoric times. Nanoparticles use was evident in the ancient time in both natural and man-
made forms. More than 4500 years ago there is evidence of use of nanoparticles of clay
minerals and ceramic matrix with natural asbestos used to create different commodities in
the ancient times. There was also the use of metal nanoparticles to form color pigments in
lustre and glass technology in Mesopotamia during the 9th century. Gold nanoparticles were
also utilized in the 4th century by Romans to introduce a striking red color to ruby glass that
appears green in daylight. This trait by the Romans had been forgotten until the 1679 and
1689 when Johann Kunckel used it in his glass factory to manufacture ruby glass. The same
character was described by Rudolph years before Kunckel but there was no evidence to
show that he applied the trait. The next big forward step in nanoparticles history was made
by Michael Faraday approximately 150 years ago. In 1857, he presented his work on
experimental relations of gold and other metals to light to the Royal Society of London
which marked the emergence of nanoscience and nanotechnology (Bulte & Modo, 2008).
These led to the principle motivation to perform research on nanoparticles. These
observations of the size effects raised expectation for the superior performance of
nanomaterials compared to bulk materials in many applications if the shape and size of the
particles could have been optimized in a rational way. In the 1980s, the systematic work
on the photocatalytic properties of colloidal cadmium sulphide (CCS) and the results in the
description of the quantum size effect began. The findings from this work opened up new
possibilities of tailoring the physical and chemical properties of materials by altering the
crystallite size and shape on a nanometer scale rather than changing the composition of the
material. Over the years the development of advanced synthesis route of nanoparticles has
offered control not only over the composition of nanomaterials but also over the particles
size distribution, size, shape and surface properties The development has paved way to the


17
study and the application of size dependent properties of nanomaterials and nanoparticles
(Shah, 2014).
The Properties of Nanoparticles that Differ to Bulk Materials
Nanoparticle research is currently a significant area of scientific research due to the
diverse potential applications in optical, biomedical and electronic science fields. The property
that makes nanoparticles have great scientific interest is their ability to actually act as the bridge
amid molecular or atomic structures and bulk materials. Nanoparticles have constant physical
properties at its current nano-scale unlike bulk materials. Bulk materials only have constant
physical properties at its usual normal size but when they are reduced to their nano-scale size
they lose their constant physical properties like their size, shape, and conductivity. Size-
dependent properties that change in bulk materials while they are in their nano-size include
quantum confinement in semiconductor particles, super paramagnetism in magnetic materials
and surface Plasmon resonance in some metal particles. These properties of bulk materials
change as their size approaches the nanoscale and as the percentage of atoms at the surface of
the bulk material increase or decrease. The number of atoms at the surface of the bulk material
is significant to the physical property of the bulk material since it determines the physical
property of the material that is going to change and what it is going to change to. For bulk
materials which are larger than one micrometer, the percentage of the surface atoms is
minuscule compared to the total number of atoms of the material. This makes the number of
surface atoms in bulk materials that are larger than one micrometer not able to affect the
physical properties of the material. Properties of nanoparticles on the other hand are not partly
due to the aspects of the surface atoms of the material dominating the properties unlike in bulk
material (Schmid, 2011).


Nanoparticles differ from the bulk material in many ways including the bending of
bulk copper material that occurs when the atoms in the copper move at about 50 nm scale.
The same does not apply to nanoparticles of copper since they smaller than 50 nm and are
super hard material thus do not display the same ductility and malleability of bulk copper.
Nanoparticles can change properties, for example, ferroelectric materials that are smaller than
10 nm can change their magnetism direction using the room temperature thermal energy.
Nanoparticles can suspend themselves in solvents since their interaction with the solvent is
strong enough to overcome the differences in density that cause floating and sinking in bulk
materials. Nanoparticles like gold that appears deep red and black in solutions, have unexpected
visible properties due to their small size that enable them to confine their electrons and produce
quantum effects, unlike bulk materials. Nanoparticles have high surface area to volume ratio
that provides a tremendous driving force for their diffusion especially at elevated temperatures
and also reduces their incipient melting temperature (Thanh, 2012).
The Utilities of Different Properties of Nanoparticles
The main reason behind the use of different shapes and sizes of nanoparticles is that
the changes in nanoparticles size and shape or surface composition can result in changes in
the chemical and physical properties of the nanoparticles. When producing nanoparticles of
a size within a certain range of between 1 to 100 nm, their chemical, electronic and physical
properties change. These changes usually depend on the nanoparticles size and
characteristics such as magnetic behavior, reductive oxidation potential, melting
temperature and their colour. All these features can be controlled by altering the
nanoparticles shape and size (Gurunathan et al., 2009). Over the years, changes in these
characteristics that occur in nanoparticles due to their size and shape have attracted a lot of


attention because of their good conductivity, chemical stability, uses as catalysts, and their
application in various industries like the food industries and medical industry, and
nanoparticles unique electrical and optical qualities. Studies of nanoparticles over the years
have shown that the morphology, size, stability and chemical and physical properties of
nanoparticles are influenced strongly by the experimental conditions they have been put
under. Conditions like the kinetics of interaction of the nanoparticles ions with reducing
agents and adsorption process of stabilizing agent with nanoparticles, cause changes in the
chemical and physical properties of nanoparticles (Ghorbani et al., 2011). This shows that
accurate control of the size, shape and distribution of the produced nanoparticles is achieved
by changing stabilizing and reducing factors, and the method of synthesis. Incredible
properties of nanomaterials strongly depend on size and shape of the nanoparticles, their
interaction with the surrounding media, preparation method and stabilizers. All this
incredible properties of nanoparticles can be best achieved through the synthesis of
nanocrystals. The synthesis of different sizes and shapes of nanocrystals makes it possible
for each nanoparticle synthesised to reach its best-applied capability explaining why
different sizes and shapes of nanoparticles are used (El-Khesheshen and El-Rab, 2012).
Photodynamic Therapy
Introduction
The battle against oncological and non oncological diseases cannot be described
without describing the impact of Photodynamic therapy (PDT) in saving lives. PDT can be
described as the treatment that uses a photosynthesizing agent or a photosensitizer and a
special type of light to activate and be effective. Photodynamic therapy refers to use of
nontoxic light sensitive drugs that activate when exposed to particular light and become


20
toxic to diseased cells in the body. PDT is effective in killing microbial cells, viruses,
bacteria and fungi. PDT can also be referred to as photo-chemotherapy, photo radiation or
phototherapy. Photosensitizer is either put into the bloodstream through the skin or a vein
(Abdel-Kader, 2014). The drug is absorbed by the cancerous cells, due to constriction of
the blood vessels and tissues in the cancerous cells the drug stays longer and is released
faster in healthier cells. The light is applied to the area of treatment whereby the drug
reacts with the light and oxygen to become toxic to the cells. PDT is also effective in
destroying blood vessels that nourish the cancer cells and alter the immune system to attack
cancer cells. PDT has played an active role in shaping cancer treatments. Doctors use
specific photosensitizers and different wavelengths that will help to trigger the
photosensitizers to ensure treatment of specific body parts (Abdel-Kader, 2014). PDT has
recently proven to be a form of therapy that can be specifically applied to only harmful cells
and tissues; this is in contrast to alternative treatments which lack specificity in their targets.
Because of this, PDT is a better treatment method for disease, as it can bypass lots of the
negative side effects that other, nonspecific treatments bring. The study of PDT is important
in the treatment of oncological and non-oncological diseases as a non invasive treatment
option.
Step''
Inject
Step3
Activated by
light
photosensitizer
Concentrates in
the tumor
Tumoris selectively
destroyed
Figure 4: The process of photodynamic Therapy (photolitec, 2013)


21
The Principles of PDT
A photosensitizer (PS) is administered systemically or topically. After a period of
systemic PS distribution it selectively accumulates in the tumor. Irradiation activates the PS
and in the presence of molecular oxygen triggers a photochemical reaction that culminates
in the production of 1C>2. Irreparable damage to cellular macromolecules leads to tumor cell
death via an apoptotic, necrotic or autophagic mechanism, accompanied by induction of an
acute local inflammatory reaction that participates in the removal of dead cells, restoration
of normal tissue homeostasis and sometimes in the development of systemic immunity
(Agosgtinis et al., 2011).
Applications of Photodynamic Therapy
Photodynamic therapy has gained a lot of attention over the years as an effective
treatment option for various cancers. PDT uses light to trigger photosensitive agents that
release chemical oxygen that destroys cancerous cells. The use of PDT in treating cancer
has been very effective in various cases and experiments.
PDT has proven successful in the treatment of many skin diseases. For example,
PDT can be used for the treatment of severe acne. Serious forms of acne can be treated by
use of photosensitizers that are applied to the face and scalp and later radiated by specific
light to kill the bacterial cells which cause acne within the patient. PDT causes sensitivity
and, therefore, should only be used on the face and scalp during severe situations of acne
that cannot be controlled by other therapies (Allison, Moghissi, Downie, & Dixon, 2011).
The greatest use of PDT is in the treatment of cancer. PDT has proven to be effective
in both limiting the metastases of cancer cells, while also being able to directly cause cancer
cell death. A photosensitizing agent, any material that is triggered by light, is injected into


the body of the patients, followed by absorption in body tissues. One of the most striking
results of this is that the agent stays for a longer period in cancer cells than in normal cells.
This is because the tumor presses the blood vessels and reduces the circulation of blood
within it. With limited circulation the drug takes longer to be removed from the cancerous
cells by the lymphatic system as compared to healthy cells. After 24 to 72 hours, the agent
has left the normal body cells but still present in the cancer cells. When the cancerous cells
or tumors are exposed to light, they absorb it and produce an active form of oxygen capable
of destroying the nearby cancer cells (American Cancer society, 2015).
The light used in activation of PDT may come from many sources; the first being
laser light that is transmitted to the body by use of fiber optic cables. In this example, the
fiber optic cable can be inserted into the body through an endoscope and into the esophagus
or lungs to treat cancer in these areas (American Cancer society, 2015). Other sources of
light that can be used include light emitting diodes (LEDs) that can be used in treatment of
surface tumors such as skin cancer.
PDT is mainly performed as an outpatient procedure and can be repeated with the
use of other therapies such as radiation, chemotherapy and surgery to maximize the
effectiveness of the treatment. The PDT treatment can be performed outside the confines of
a hospital with the patient receiving both the photosensitizers and light treatments at their
homes (American Cancer society, 2015).
Extracorporeal photoresist (ECP) is a form of PDT whereby a machine collects
blood cells from the patient, treats them outside the body by exposing them to light and then
returns them to the patient. The US Food and Drug Administration (FDA) have approved
the use of ECP to lessen the severity of skin symptoms of T-cell Lymphoma that have


previously not been affected by other therapies. ECP studies have also been carried out to
investigate its use on blood cancers and to reduce rejection after transplants (American
Cancer society, 2015).
Photosensitizers
A photosensitizer is a chemical compound capable of undergoing a chemical change
in another molecule during a photochemical process upon illumination. A photosensitizer
reacts with light to undergo a chemical change, resulting in the release of oxygen. There are
many photosensitizers for PDT that include aminolevulinic acid. Aminolevulinic acid is
applied directly to the skin and is used for the treatment of actinic keratosis, a skin condition
that can easily develop to cancer and is used on the scalp and face (Abdel-Kader, 2014). A
special blue light is used for the activation of this drug and not laser light. Porfimer sodium
is the most commonly used photosensitizer and is activated by red light from a laser. It is
approved for the treatment of cancer of the esophagus and when it cannot be treated by laser
therapy alone. It is effective in treating Barretts esophagus with dysplasia; a disease that
can lead to esophageal cancer if surgery is not conducted. Porfimer sodium is also used for
the treatment of non-small lung cancer that affects inner lining of the bronchus called
endotracheal cancer (American Cancer society, 2015).
Levulan is photosensitizer that is used for the treatment of lesions. It is applied to
the lesions and given 6 hours to penetrate the skin. It is then irradiated with blue light for
15 minutes. This generates the reactive oxygen that kills cells. The patients must protect
themselves from the bright sunshine for 40 hours since the skins are sensitive (Pazurek &
Malecka Panas, 2005).


Other types of photosensitizers used include allumera, visudeneyne, cysview,
foscan, metrix and laserphyrin. Although the photosensitizers have different treatments they
have certain similar characteristics such as high absorption at long wavelengths, high
chemical stability, low dark toxicity, high singlet oxygen yield and natural fluorescence
(Pazurek & Malecka Panas, 2005).
Works Done on Nanoparticles
The nanoparticles play a supplementary and complementary role to the in PDT
(Abdel-Kader, 2014). The active nanoparticles are self-illuminating and, therefore, can
reach deep tissues where light cannot reach and destroy the dangerous cancerous cells
without the use of external light sources. Nanoparticles can be used for the simultaneous
radiation and therapeutic treatment of cancer. Some luminescent nanoparticles such as
CuCy nanoparticles do not require photosensitizers to be effective, therefore, the treatment
is convenient, has a lower cost and more efficient. Secondary illuminating nanoparticles are
capable of responding to limited amount of light and this means that they are capable of
being activated in deep tissues where small amounts of light can reach. Additionally, they
activate and destroy cells as effectively as self-illuminating nanoparticles. Researchers have
discovered that Cu-Cy nanoparticles are effective in treating prostate and breast cancer
when exposed to x-ray light. The research showed that a tumor treated with Cu-Cy
nanoparticles stayed the same size for close to two weeks while that which was not treated
tripled in its size (Pazurek & Malecka Panas, 2005). These specific nanoparticles have
various advantages, the biggest being that they have lower toxicity to the healthy cells
compared to other nanoparticle approaches. This means that the luminescent nanoparticles
are safer to use as they have less negative side effects to healthy tissues. The intense


photoluminescence of the nanoparticles and the x-ray luminescence can be used to produce
cell images. The nanoparticles can penetrate large tissue and kill the surrounding cancerous
cells with greater specificity and efficiency. Research is being conducted to reduce the size
of Cu-Cy nanoparticles, to help increase its absorption into tumor tissues. Nanoparticles can
be used to expand the reach of PDT and to ensure that treatment can be done on deep tissues
due to self-illuminating nanoparticles. Nanoparticle research is meant on increasing the
applicability for PDT and is the key to ensuring that the treatment of deep tissue cancers is
possible (Pazurek & Malecka Panas, 2005).
Motivation of the thesis
In 2008, cancer accounted for around 13% of all deaths worldwide. With nearly 13
million cancer new cases annually, deaths are projected to rise reaching 13.1 million in 2030
(Lucky et al., 2015). Intervention measures such as screening and surveillance programs are
good at improving the outcome and survival, though there is need for effective, efficient,
affordable, and acceptable cancer therapeutic options.
Available treatment options include chemotherapy, radiotherapy, surgery, and small molecule-
based therapies and immunotherapy although chemotherapy has systemic side effects, and
radiation therapy has limitation of the cumulative radiation dose. Refining existing treatment
modalities is important but research also focuses on safe, potent but cost effective alternate
treatment modalities such as Photodynamic therapy (PDT).
PDT uses a combination of light, light-sensitive drugs, which are harmless and
nondamaging. Upon irradiation by an appropriate wavelength of light, the drugs become
excited transferring energy to surrounding molecular oxygen to generate cytotoxic reactive


oxygen species. These oxidize critical cellular macromolecules induce cellular permeability
alterations resulting to cell death by necrosis or apoptosis or both.
Despite its success, PDT treatment is yet to gain clinical acceptance as a first-line
oncological intervention mainly due to certain limitations. However, the use of
nanoparticles has been a major stride in resolving some challenges associated with classic
PS. Its use in cancer therapy is attractive as it promises better tumor selectivity accessible
to light and a lower systemic toxicity with fewer side effects in comparison to radiation and
chemotherapy. Though proved effective in cancer treatment, it is unfortunate that most of
the photosensitizers used in PDT can only be activated by visible light, which cannot pass
through a thick tissue thus hindering its full therapeutic potential. Whereas using
nanoparticles as a drug carrier makes it possible to treat tumors that are deeper under the
skin or in body tissues because it can be activated by the near-infrared light which can afford
penetration depths greater than those of the visible light besides being less harmful to cells
and tissues (Scholz and Dedic, 2012). An approach to study the diffusion dynamics of rod
and ball shaped gold nanoparticles using fluorescence correlation spectroscopy is presented.


27
CHAPTER II
INSTRUMENTS AND METHODS
In this work an upright Olympus FV1000 microscope for confocal fluorescence
microscopy has been used to get the fluorescence correlation spectroscopy traces for
different colloidal gold nanoparticles solutions. This will aid in the study their behaviour to
know which of the samples is best suited to be used in Photodynamic Therapy as it relates
to medical treatment.
Gold Nanoparticles Characterization
UV- Visible Spectroscopy
UV- visible characterization of (colloidal gold nanosphere, colloidal gold
nanosphere Photosensitizer, colloidal gold nanoshpere RB, and colloidal gold
nanorodsRB) solutions was done by using Cary 100 UV-Visible spectrophotometer. The
absorbance measurements were done over the wavelength of 300- 900 nm using 1 cm path
length quartz cuvettes.


28
Figure (5): Absorption spectra for four different colloidal gold solutions abbreviations,Ball 3:
colloidal gold nanosphere conjugated with Rhodamine B in water,Rod3: colloidal gold nanorod
conjugated with Rhodamine B in water, Ball 1: is the colloidal gold nanospheres solution, Rod 2:
colloidal gold nanorod solution.
Fluorescence Correlation Spectroscopy
The FV 1000 confocal microscope has six laser lines. These include UV/Violet (405
nm), Multi-line Argon laser with 458, 488, and, 514 nm wavelengths, green He-Ne laser
(543 nm), and red He-Ne laser (635 nm). Two wavelengths 488 nm and the 534 nm were
used to excite the colloidal gold nanoparticles solutions dispersed on a cover slip using a
water immersion microscope objective lens (60x, 1.2 NA). FCS instrument was calibrated
with Rhodamine-B with radial dimension of 0.282 pm, 0.293 pm, and 0.307 pm. The
scientific computations in Python can be done using Scipy library which contains modules
for Fourier transforms, optimization, linear algebra, signal and image processing, and other
modules. Numpy is another package for scientific computing in python. The plots were


generated with the help of matplot library which generates 2D plots. To generate the graphic
user interface the Pyside library was used. The code can be divided into three important
steps the first is generating a horizontal data, the second is computing the autocorrelation,
and the third is filtering the autocorrelation. The program generates a horizontal data which
may contain around eight million elements and then divide this ID array into slices,
compute the autocorrelation for each slice using Fourier transform average all of the
autocorrelation curves for all the slices to get the final autocorrelation curve. The final
autocorrelation curve has just particular number of points to be chosen by the programmer
in order to get smooth curve because plotting all the points would result in unwanted
fluctuations within the final representation. The code also has a feature that allows the user
to remove any unwanted autocorrelation curve to enhance the final autocorrelation result.
Measurements for fluorescence correlation spectroscopy were accomplished using
a custom software (MAZ-2015) written in Python programming language. Equation 1 was
used to measure the fluorescence fluctuations for the system, whereas, equation 2 used to
calculate the cross correlation function. To avoid the computational complexity involved in
the autocorrelation computation, the autocorrelation was calculated by means of the
Wiener-Khinchin theorem. This theorem uses the Fourier transform to compute the
autocorrelation, resulting in a more convenient method, because it has several efficient
algorithms, such as the Fast Fourier Transform. An exponential axis is generated for the
time delay, with a fixed number of points to avoid the unnecessary fluctuations in the
resulted autocorrelation and cross correlation curves. A correction on the values of this array
when the difference between each subsequent point is less than the sampling time is also


30
accounted for. Normalized autocorrelation of a 3D freely diffusing sample can be expressed
as: (Schwille & Haustein, 2004)
1
Where,
t = delay time.
td = characteristic diffusion time.
N= average number of particles within the light focal volume.
Wxy = beam waist of the laser focal volume in the XY direction.
wz = beam waist in the Z direction.
These last two parameters can be obtained from calibration measurements. Equation
(3) can be used as model that can be fitted to the autocorrelation computed from equation
(1). Once the normalized autocorrelation function has been computed, it can be used to find
the parameters N and td in equation (3) using an appropriate fitting procedure. MAZ-2015
uses the SciPy Python library and its optimization package, which provides the least
function that allows to minimize the square error between the values from the computed
autocorrelation and a parametric model given by equation (3), whose parameters are G (0)
and td, where (Lakowicz, 2006)
c()-£
Once the Go and N parameters have been found via least squares minimization, the diffusion
coefficient (D) is calculated by Ficks law (Sri Balaji et al., 2011) as follows:


31
p (Wxy)2
4rD
(4)
Flowchart in Figure (6) summarizes the flow of the software:
Figure (6): Flow chart represents the steps for the custom software (MAZ-2015).


32
Compute and fit
autocorrelation
buttons for file 1
Select file 1:
Open.,,
Select file 2:
Open...
Aquisition Frequency (kHz) ioo
Size of division: 256000
Focal spot size (urn): 0.282516
Autocorrelation
Compute
Compute
fit
fit

Cross-correlation
frOO +
Compute
D
r ft O O *
ef
Compute and fit
autocorrelation
buttons for file 2
Compute and fit
cross correlation
buttons for selected
files
Figure 7: Graphic user interface for the custom software (MAZ-2015)


Select file 1:
Open...
Select file 2:
File 1 selected.
Aquisition Frequency (kHz) 100
Focal spot s2e (urn): 0.282516
Autocorrelation
Compute
Fit
Compute
T
Autocorrelation 1
Cross-correlation
fcOO
0.010
0.005
0.000
0.005
Results from fitting Autocorrelation 1:
Go =0.00614049900686
D (umAVsec) 306.242345935
Open...
Size of division: 256000
Compute
Fit
Average Correlation
0.008
0.006
0.004
0.002
0.000
0.002
0.004
acl
102 10'1
Td (sec) = 6,51569674438e-05
10
Figure 8: The working software when computing the autocorrelation for the
fluctuations of the signal for the colloidal gold nanosphere solution.


34
Select fie l:
Flelselected.
Aquisttwi Frequency (kHz) 100
Focal spot sne (um): 0.292516
Autocorrelation
Cross-correlation
Fie 2 selected.
Sia ofdrnsion: 256000
| Compute ~] [ Fit
frOOSJ.'Blsr ftOOffi- lBr
Go =0,00614049900686
0 (unA/sec) = 306.24234S935
Results from fitting Autocorrelation t
Go =0.210690641227
D {umAJ/sec) = 191.093920814
Results from fitting Cross-correlation:
Go > 1,52990518116-19
D (cmAtysec) = 199.538224494
Id feet) =6.51569674438e-05
Td(sec) =0.000104418929074
Tdfsec) =0.000100000000574
Aquation Frequency (|d4z) 100
Focal spot sot (um): 0.282516
Autocorrelation
Cross-correla bon
C
^00 + BHr
Autocorrelation 2
Results from fitting Autocorrelation 1:
Go = 0.00614049900686
O (umAVsec) = 306.242345935
Results from fitting Autocorrelation 2:
GO = 0.210690641227
0 (umA*/sec) = 191.093920814
Results from fitting Cross-correlation:
Go = l.S299051811e-19
O (cmAVsec) = 199.538224494
File 2 selected.
Sere ofdrnsion: 256000
^00 4*
lr
Td (sec) = 6.51569674438e-05
Td (sec) =0.000104418929074
Td(sec) =0.000100000000574
a) Before removing the unwanted trace b) After removing the unwanted trace
Figure 9: shows the selective feature to remove unwanted fluctuations using the colloidal gold
nanoball solution with unwanted fluctuations, the yellow line at the left side, and the
result after removing it appearing at the right side.
Figure 10: Cross correlation for the data from two different solutions ( colloidal
gold nanoballs and colloidal gold nanorods ), it shows that there is no
correlation between them.


35
The quantification of the results done by calculation the root mean square error (RMSE)
values for each result using the following equation, the lower RMSE is the best the result,
(Kaggle, 2015).
RMSE =i£?=, ef = - C{)2 (5)
Where
n refers to the number of elements e refers to the error i.e. the difference between the
fitted values and the measured ones.
Gi refers to the calculated correlation at i.
GfLtrefers to the fitted values according to equation (3).


36
CHAPTER III
RESULTS
No X X * ^ X
1 Colloidal gold nanoballs-RB solution 543 100 nM in PBS 7.8 10 377.67 52.8
2 Colloidal gold nanoballs-RB solution 543 100 nM in PBS 7.8 20 230.49 86.56
3 Colloidal gold nanoballs-RB solution 543 100 nM in PBS 7.8 10 409.49 48.7
4 Colloidal gold nanoballs-RB solution 543 100 nM in culture media 7.8 10 392.24 50.8
5 Colloidal gold nanoballs-RB solution 543 100 nM in culture media 2.3 10 552.8 36.09
6 Colloidal gold nanoballs-RB solution 543 100 nM in PBS 1.6 10 24533.12 0.813
7 Colloidal gold nanoballs-RB solution 543 lOOnM in nanopure water 2.3 10 1181.56 16.88
8 Colloidal gold nanoballs-RB solution 543 Full strength 7.8 10 770920.7 0.030


37
9 Colloidal gold nanoballs-RB solution 488 100 nM in PBS 10.2 10 5869.81 4.014
10 Colloidal gold nanoballs-RB solution 488 100 nM in PBS 33.1 10 631.98 37.28
11 Colloidal gold nanoballs-RB solution 543 100 nM in PBS 1.6 10 5719.536 4.119
12 Colloidal gold nanoballs-RB solution 543 100 nM in PBS 4.2 10 618.74 38.08
13 Colloidal gold nanorod solution 488 Full strength 10.2 10 24.936 860.67
14 Colloidal gold nanorod solution 543 2uL in 998uL PBS 1.6 10 33020190.13 0.0006
15 Colloidal gold nanoballsPhotosensitize r solution 543 Full strength 7.8 10 29275421.52 0.00068
16 Colloidal gold nanoballs solution 543 Full strength 7.8 10 74982283.2 0.00026
Table 1: Results from autocorrelation for different colloidal gold nanoparticles solutions
(nanoshpere, nanorod, nanosphere conjugated with Rhodmine B in water, nanorod
conjugated with Rhodmine B in water) the correlation analysis shows that the nanosphere
conjugated with Rhodmine B in water with laser wavelength of 543 nm and power of 7.8
micro-watt.


38
According to the RMSE observations we got the following:
Concentrations
Figure 11: Varying concentrations of colloidal gold nanosphere conjugated with
Rhodamine B in water solution.
1.6 fixW 4.2 nW 7.8 /xH
Laser Power
Figure 12: Varying laser powers of colloidal gold nanoballs conjugated with
Rhodamine B in water solution.


RMSE x 10"
39
50
40
T
I
o
^ 30
x
LU
U~)
§ 20
10
0
Figure 13: Using different colloidal gold nanoparticles (nanosphere and nanorod )
under the same experimental conditions, the colloidal gold nanosphere -RB
solution shows the best results.
3.0
2.5
2.0
1.5
1.0
0.5
0.0
Different Solutions for Dilution
12 3
PBS Nanopure water Culture media
Figure 14: Diluting the colloidal gold solution in PBS nanopure water and
culture media it is obvious that the PBS shows the best RMSE value over the
other two solvents.


40
CHAPTER IV
DISCUSSION
In this thesis, a software called MAZ-2015 was successfully implemented
which is comparable to the commercial software used for the fluorescence correlation
spectroscopy calculations, with this software one can do the correlation and cross
correlation calculations with a special feature to remove the unwanted fluctuations caused
by impurities or other sample fluctuations in order to get more accurate results, the user
could specify the frequency, the focal spot size, and the size of division over which we are
computing the correlation or the cross correlation. The tiny differences in the results
between the custom software and the MAZ-2015 may relate to the approximations for the
two different programming languages because the commercial software FVI000 uses
Pascal whereas MAZ-2015 uses Python programming language. The colloidal gold
nanoparticles samples have been synthetized by Dr. Zheng's group at Fujian normal
university in China in which three samples are manufactured with ball and rod shapes. Some
of them were conjugated with photosensitizers, some with Rhodamine-B, while the others
were not conjugated with neither molecules. All these were studied using FCS while the
correlation curves were obtained to track the behaviour of each of these samples to
determine which of them would be of the best choice as well as which are the best
experimental conditions to implement such experiment to make use of these results in the
Photodynamic Therapy. The correlation results for the colloidal gold nanorod solutions by
changing the concentrations and excitation wavelengths at each time were inconclusive
which gives astronomical diffusion coefficients values that cannot be considered as real


values and this maybe because they are too heavy to diffuse so what we got is just a noise.
The correlation results for the colloidal gold nanoball solution were also unreliable because
there were problems with their synthesizing. The same problem occurred with the colloidal
gold nanoballs solution and the gold nanoballs conjugated with photosensitizer solution.
The best result was obtained from the gold nanoballs conjugated with Rhodamine-B and
laser power of 7.8 pW with average diffusion coefficient of 352.4725 cm2/sec and standard
deviation of 71.32. Whereas, using lower laser powers did not provide reliable results given
that the concentration is in the nanomolar range. The RMSE was used to quantify our
observations, in accordance with the RMSE values we found that it is better to use low
concentrations for the fluorescence correlation spectroscopy experiments. High
concentration of the sample means high number of particles in the measurement volume
which will lead to small correlation function (Go) value at time (x = 0) and may not lead to
reliable results, given that the number of particles within the observation volume should not
be too few or too large. The other comparison we did with the aid of RMSE values was
between different samples under the same experimental conditions it shows that the gold
nanoparticles conjugated with Rhodamine-B was of the best behaviour and that may return
to the effects of size. This also proves that the conjugation with the Rhodamine-B is better
than conjugation with the photosensitizer and both of them are better than using the gold
nanoparticles without any kind of conjugation. Our results showed that diluting the colloidal
gold solution in PBS shows the best behaviour for these particles over diluting them in
nanopure water or culture media. Using different solutions under the same experimental
conditions showed that the RMSE value colloidal gold nanosphere solution was the lowest
which indicates that this sample is of the best behaviour. This may be related to the different
sizes and shapes but unfortunately we were not able to do the size and shape characterization


experiment. We also found that as the excitation power increases the RMSE value
decreases; this is because as the power increases the number of observed photons from each
fluorophore increases due to the Brownian motion and this result in increasing the strength
of the recorded intensity signal given that the concentration in the nanomolar range. We
also found that when we used 100 nM and full strength concentrations of colloidal gold
nanoparticles-RB solution with the same laser power, excitation wavelength of 543 nm, and
sampling time of 10 microsecond per pixel. We may notice that the RMSE value is much
lower for the 100 nM concentration value. This can be connected with the number of
fluorophores in the observation volume, as the number of fluorophores increases the value
of the autocorrelation function at t = 0 decreases. Note that Go = (1/N), where N is the
number of fluorophores in the observation volume and hence the amplitude of the
correlation function will be too small for reliable measurements.


43
CHAPTER V
CONCLUSIONS
In light of the aforementioned results, the following conclusions could be reached:
the customized software (MAZ-2015) works well and we can use it for autocorrelation and
cross correlation analysis. Moreover, its selective feature enables the users to remove
unwanted fluctuations for more accurate calculations, increases the laser power also
enhances the accuracy of the correlation results. This occurs because increasing the laser
power increases the fluorescence intensity of the sample which, in turn, increases the
number of photons obtained by the detector. Diluting the colloidal gold nanoparticles in
water solution with the Phosphate Buffered Saline (PBS) yields good signals for analysis,
compared to diluting them in nanopure water and culture media to obtain the diluted
colloidal gold nanoparticles solution. The concentration of 100 nM of colloidal gold
nanoparticles solution also yielded better results, compared to the tests conducted using
higher concentrations. The green He-Ne laser (543 nm) was the most suitable wavelength
for this experiment, as was duly demonstrated using the absorption spectra. The colloidal
gold nanoparticles solution diluted in PBS of nanospheres conjugated with RB yielded the
most promising results.


44
CHAPTER VI
FUTURE WORK
The optical behavior of colloidal nanoparticles can be tuned by changing their
dimensions, shape, and surrounding environment. Moreover, different sizes of
nanoparticles can be used, since the gold nanoparticles are plasmonic particles. When the
light propagates near the colloidal nanoparticles, it interacts with the free electrons at the
surface of the metal and induces them to vibrate collectively creating an electric field. When
the electric field of the vibrated free electrons resonate with the electric field of the incident
light, we call this phenomena surface plasmon resonance. The oscillations of the free
electrons on the surface of the metal affect the absorption of light. For instance, the
absorption of light for small gold nanoparticles occurs in the blue green portion of the
spectrum, but as their size increase the light absorption of the surface plasmon resonance
wavelength shifts to the red portion of the spectrum. Regarding nanorods, as the aspect ratio
(length/width) increases the fluorescence bands also drastically increase, given that the
nanorods have two plasmon resonance bands that correspond to the length and width of the
rod shaped nanoparticle. It has been showed that increasing the length of these particles
leads to improvements of the fluorescence quantum efficiency (Li et al., 2005). We may
also use different shapes of nanoparticles like the nano urchins to further improve the
results in which the absorption and emission from rough surfaces of nano urchins are
better than from smooth ones. In order to reduce the aggregation to the lowest level, specific
materials must be added to the colloidal nanoparticles solution. These characteristics help
in tailoring the optical properties for nanoparticles to meet the requirements of different


applications. For photodynamic therapy, it is important to have the absorption in the
therapeutic window (around 650-1000 nm) where light can penetrate deeper layer of the
skin, compared to other portions of the spectra (Scholz and Dedic, 2012). Probes other than
the Rhodamine-B and silicon phthalocyanine (Pc4) photosensitizer can be conjugated to the
nanoparticles when doing the FCS experiment to determine which one is the efficient to
enhance the fluorescence of the observed particles. Furthermore, nanoparticles made of
different materials can be tested in the future. Silicon nanoparticles could yield positive
results given that it has been found that they are able to generate cytotoxic singlet oxygen
upon illumination when used in photodynamic therapy. We may also try to use
concentrations lower than 100 nM and determine if it would yield better results because the
FCS works well for solutions concentrations in the nanomolar range.


46
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Full Text

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USING LASER CORRELATION SPECTROSCOPY TO STUDY GOLD NANOPARTICLES DIFFUSION DYNAMICS FOR PHOTODYNAMIC THERAPY By AL OGAIDI MARWAH ALI ZAIDAN B.S. University of Technology, Baghdad, 2010 A thesis submitted to the Faculty of the Graduate school of the University of Colorado in partial fulfilment of the requirements for the degree of Master of Science Electrical Engineering 2015 of the requirements for the degree of

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ii This thesis fo r the Master of Science degree by Al Ogaidi Marwah Ali Zaidan Has been approved for the Electrical Engineering program by Tim C. Lei, Chair Mark Golkowski Yiming J. Deng November 20, 2015

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iii Al Ogaidi, Marwah Ali Zaidan (MS, Electrical Engineering) U sing L aser C orrelation S pectroscopy to S tudy G old N anoparticles D iffusion D ynamics for P hotodynamic Th erapy Thesis directed by Associate Professor Tim C. Lei ABSTRACT The dynamic behaviour of gold nanoparticles of different shapes (sphere and rod) were studied under different experimental conditions to be used for drug delivery in photodynamic therapy. Fluorescence correlation spectroscopy (FCS) using single photon excitation had been used to study the diffusion dynamics of gold nanoparticles of different shapes in water solution to utilize these results for further enhancement for the photodynamic thera py. We analyzed four different colloidal gold solutions (colloidal gold nanosphere solution, colloidal gold nanosphere photosensitizer solution, colloidal gold nanoshpere Rhodamine B solution, and colloidal gold nanorods Rhodamine B solution) using the cu stom software (MAZ 2015) we made to analyze the results for these samples by calculating and fitting the autocorrelation. We found that we could get a reliable correlation when laser power was higher than 4.2 W. The phosphate buffered saline shows the bes t results over the nanopure water and culture media when used to dilute the colloidal gold nanoparticles solutions, and the (MAZ 2015) software provides a comparable results to commercial software provided by the microscope vendors. The results indicated t hat gold nanosphere conjugated with Rhodamine B can be a potential candidate to study drug transport to cells in photodynamic therapy treatment. The form and content of this abstract is approved. I recommend its publication. Approve d: Approved: Tim C. Lei

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iv ACKNOWLEDGEMENT Much appreciation and gratitude to God for giving me the strength, health and capacity to do this research work. Secondly, I take this opportunity to express my profound gratitude and deep regards to my supervisor Dr. Tim Lei for his exemplary guidance, monitoring and constant encouragement throughout the course of this thesis. I also, would like to thank Saif Al Juboori for the time and the efforts he puts in helping with setting the experiment and do ing the tests .In addition to the valuable advices. I would also thank Dr. Zheng Huang and his group who helped in clarifying some points about the photodynamic therapy and providing the samples. Much thanks to Mr. Gregory Glazner who helped in doing the c haracterization experiment and explaining some concepts about it. I also thank my parents, brothers, sisters and friends for their constant encouragement without which this research would not be possible.

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v TABLE OF CONTENTS I. I I NTRODUCTION ................................ ................................ ................................ ....... 1 Correlation ................................ ................................ ................................ ................... 3 Microscopy ................................ ................................ ................................ ........................... 4 Confocal Microscopy ................................ ................................ ................................ .. 5 Fluorophores ................................ ................................ ................................ ................ 5 LASERs ................................ ................................ ................................ ....................... 6 Stokes Shift ................................ ................................ ................................ ................. 7 Fluorescence Correlation Spectroscopy ................................ ................................ ...... 8 The History of Fluorescence Correlation Spectroscopy ................................ ........... 11 Principles and Theories of Fluorescence Correlation Spectroscopy ......................... 11 Theoretical Concepts of FCS ................................ ................................ .................... 13 Fluorescence Correlation Spectroscopy and Nanoparticles ................................ ...... 14 Nanoparticles ................................ ................................ ................................ ............. 15 The History of Nanoparticles ................................ ................................ .................... 15 The Properties of Nanoparticles that Differ to Bulk Materials ................................ 17 The Utilities of Different Properties of Nanoparticles ................................ .............. 18 Photodynamic Therapy ................................ ................................ ............................. 19 Introduction ................................ ................................ ................................ ......................... 19 The P rinciples of PDT ................................ ................................ ............................... 21 Applications of Photodynamic Therapy ................................ ................................ .... 21 Photosensitizers ................................ ................................ ................................ ......... 23 Works Done on Nanop articles ................................ ................................ .................. 24 Motivation of the thesis ................................ ................................ ............................. 25 II i INSTRUMENTS AND METHODS ................................ ................................ .......... 27 Gold Nanoparticles Characterization ................................ ................................ ........ 27 UV visible Spectroscopy ................................ ................................ .......................... 27 Fluorescence Correlation Spectroscopy ................................ ................................ .... 28

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vi III. RESULTS ................................ ................................ ................................ ................... 36 IV. DISCUSSION ................................ ................................ ................................ ............. 40 V. CONCLUSIONS ................................ ................................ ................................ ......... 43 VI FUTURE WORK ................................ ................................ ................................ ........ 44 REFERENCES ................................ ................................ ................................ ................. 46

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vii LIST OF TABLES Table 1: Results from autocorrelation calculations for different colloida l gold nanoparticles

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viii LIST OF FIGURES Figure 1: Stokes shift.................................................................. ........................................ 7 2: Conceptual diagram of a fluctuating fluorescence signal.................................... 8 3: Typical confocal FCS system.............................................................................. 9 4: Process of Photodynamic Therapy....................................................................... 21 5: UV visible spectra for four different colloidal gold solutions............................. 29 6: Flow Chart for (MAZ 2015) software............ ..................................................... 32 7: Graphic user interface for custom software (MAZ 2015)................................... 33 8: The working software when computing the autocorrelation for file 1. ................ 34 9: The sel ective feature to remove unwanted fluctuations....................................... 35 10: Cross correlation.................................................................................................. 35 11: RMSE results using different concentra tions....................................................... 39 12: RMSE results using different laser powers.......................................................... 40 13: RMSE results using different samples........................................... ...................... 40 14: RMSE results when diluting the colloidal gold solution in three different ........................................................................................................ 41

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1 INTRODUCTION While the applications of photodynamic therapy are indisputably well established within a number of fields, its use in treatments for cancer and other d iseases is still in a developmental phase. It works by producing a reactive species, typically singlet oxygen, through transferring energy from light excited dye molecules in target tissues. Recently, wide ranging strategies were proposed, seeking to enhan ce the efficiency of photodynamic therapy through the use of nanoparticles. This thesis, therefore, studies the diffusion dynamics for colloidal gold nanoparticles. Fluorescent correlation spectroscopy is a commonly used technique to study th e dynamics of molecules due to its high sensitivity; it can also be employed in biomedical, chemical, and biological fields (Weiss, 1999). Gold nanostructures attract particular interest because of their favourable emission properties in terms of good pho to stability under continuous irradiation, there are no blinking effects ( the light and dark periods the single particles experience when exposed to continuous laser illumination), and good biocompatibility. The likelihood of tuning the absorption band by modifying the particles shape is also another advantage for their application in different fields. A detailed investigation on the luminescence behaviour, under one and two photon excitation at increasing laser power, of citrate capped gold nanoparticles (Au NP) with increasing diameter (up to 50 nm) has been reported by Loumaigne and Colleagues.

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2 According to the fluorescence correlation spectroscopy (FCS) analysis over the past years researchers found that there is a linear relationship between the diam eters of gold nano spheres and the diffusion time. They also found that the size, shape, and surrounding surface environment are crucial in the optical properties of nanoparticles In addition to that the nanoparticles are less prone to photobleaching whe n excited with strong laser light. In accordance with all the above mentioned reasons, the nanotechnology becomes widely used in medical applications like drug delivery and specifically the photodynamic therapy and diagnosis (De Jong and Borm, 2008). FCS experiments are run under increasing the laser power, using different samples of colloidal gold solutions, using different wavelengths, and using different solutions to be dispersed in. A review for the fluorescence correlation spectroscopy, na noparticles, photodynamic therapy, and discussion for the results will be presented in the following sections of this thesis. There has been a high growth of research and applications in the area of nano science as well as nanotechnology in the past years Recently there is an increasing optimism that nanotechnology while applied to medicine will bring important developments in the diagnosis as well as the treatment of the disease. Applications in medicine that are anticipated are such as drug delivery, in vitro and in vivo diagnostic, as well as production of improved biocompatible materials. Engineered nanoparticles are an important tool since they help one to realize a number of these applications. It has also been identified that not all particles that are used for medical purposes comply with the acceptable definition that has been recently proposed by the Royal Society and Royal Academy of Engineering of a size 100nm. This has not proven to have any impact on

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3 the functionality of medical applications. Moreover, for drug delivery not only engineered particles may be used as carriers. The drug may be formulated on itself at a nanoscale and then function on itself as a carrier. Correlation Correlation is referred to as a statistical measur ement, which is utilized to describe the relation between two fluctuating signals (Cross Correlation) or the signal with itself (Autocorrelation). The autocorrelation function can be determined using the following equation, (Gratton, 2005): G (1) Where: F ( ) refers to the fluorescence signal refers to the real time refers to the time difference between two intensity measurements ( ) refers to the variance where ( ) = F(t) < ( ( ) ( + )) > refers to the autocorrelation of F(t) (< ( ) >) 2 refers to the square of the average value of F(t) Whereas the cross correlation function for the fluorescence fluctuations from two different channels 1 and 2 (G 12 )) can be calculated using the following equation, (Gratton, 2005): G (2) Where: F 1 (t) and F 2 (t) : are the fluorescence signals from channel 1 and 2 respectively.

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4 On the other hand, correlation can be defined in different ways according to the field of study. In essence, the correlation being a statistical measurement portrays the relationship and degree to which two variables or more fluctuate together. In signal processing, the corr elation used to analyse functions or series of values like the time domain signals. Subsequently, correlation is mainly defined in two terminologies, which are a positive correlation and a negative correlation (Rigler & Elson, 2012). Hence, a positive cor relation shows the level at which variables decrease or increase in parallel. On the other hand, a negative correlation shows the level at which one of the variables increases while the other variable decreases. Cross Correlation in signal processing refer s to a similarity measure of two series mainly as a lag function of one relative series to the other (Berezin, 2014). Microscopy Microscopy refers to a noble scientific practice, which comprises of magnifying objects that the unaided eye cannot see. Thus the main objective of this scientific discipline is to be able to magnify the object so that it is visible for studying. This allows researchers to conduct their study and learn essential things about the invisible objects, as well as how they work. In a ddition, microscopy utilizes microscopes to view these objects and samples. There are mainly three major branches of microscopy which include scanning probe, electron and optical microscopies (Berberan Santos, 2008). On the same note, electron and optica l microscopy involve the refraction, reflection or diffraction of electromagnetic radiation beams that interact with the specimen. It also interacts with the scattered radiation collection or any other signal so that it can create an

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5 image. Conversely, thi s process can be performed through the sample wide field irradiation Confocal Microscopy Confocal Microscopy refers to an optical imaging method, which is used to increase the optical resolution and the difference of a micrograph through an additional spatial pinhole that is placed on the lens confocal plane. Confocal Microscopy has gradually gained po pularity particularly in the industrial and scientific communities. Its distinctive applications are in materials science, life sciences and semiconductor (Rigler & Elson, 2012). In this sense, this technique provides numerous advantages as compared to th e conventional optical microscopy. These advantages include out of focus glare elimination, and narrow depth of field (Rege & Medintz, 2009). Fluorophores A fluorophore is also known as a fluorochrome, and it is similar to a chromophore. A fluorophore is mainly a fluorescent chemical substance which can re emit light when light excitation takes place. Hence, a fluorophore refers to a section of a molecule, which leads to the creation of a fluorescent emission specifically in the observable light spectrum. These fluorophores absorb different light wavelengths, and this creates the visible light. These fluorophores can be introduced through artificial methods, or they can exist naturally.

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6 It is paramount to note that many rocks and fish maintain some natural levels of these fluorophores (Berberan Santos, 2008). Nevertheless, the fluorophores are utilized widely in the scientific fraternity for research purposes since they assist in analyzing certain material properties. Hence, researchers can identify chan ges and reactions in the biochemistry fields, as well as protein study. Besides, the immunofluorescence discipline utilizes this technique to assist in labelling antibodies and antigens at the level of subcellular (Rege & Medintz, 2009). LASERs LASER is a short for Light Amplification by Stimulated Emission of Radiation. Theodore Maiman invented it in 1964. The laser device can produce monochromatic, directive, and coherence light, which could aid many useful inventions. The laser device is composed of an active medium (number of atoms or molecules emit electromagnetic radiation by stimulated emission after being stimulated by population inversion), an excitation mechanism (supplies energy to the active medium), an optical feedback, a resonator (the place between the two laser mirrors where the laser action occurs), an output coupler (partially reflecting mirror located at the end part of the optical cavity which enables the light to exit the laser). Lasers can be classified according to the active medium i nto gas lasers, solid state lasers, excimer lasers, dye lasers, or semiconductor lasers. A laser is different from other light sources because it emits its light coherently whereby spatial coherence enables it to be focused to one tight spot. Ideally, the invention of these powerful focused light sources enables various applications such as lithography, laser cutting, and more advanced inventions like confocal laser scanning microscopes (Rege & Medintz, 2009). It also has many further applications and can b e used in laser printers, laser surgery,

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7 barcode scanners, optical disk drives, free space and fiber optic optical communication, as well as skin treatments, laser lighting displays, welding and cutting materials (Berberan Santos, 2008). Stokes Shift W hen absorbing light, the atom or the molecule undergoes a transition into an excited electronic state accompanies with losing small amount of absorbing energy before releasing the rest of its energy as luminescence (thermal energy in most cases). The difference between the band maxima of the absorption and luminescence spectra within the same electronic state is known as stokes shift and it could be represented in frequency or wavelength units (Tuite, 2013). Figure 1: Stokes shift: The difference between the band maxima of the absorption and luminescence spectra in wavelength units (Tuite, 2013).

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8 Fluorescence Correlation Spectroscopy Fluorescence correlation spectroscopy (FCS) is a correlation of temporal fluctuations of the fluorescence intensity. The fluctuation of the signal is used to calculate of the autocorrelation function (typical ly G (0)) is inversely proportional to the average number of molecules in the probe volume (). Figure 2: Conceptual diagram of a fluctuating fluorescence signal, (A) Fluctuated fluorescence signal. The amplitude of the autocorrelation function (typically G (0)) is inversely proportional to the average number of molecules in the probe volume (), (NCBI.gov, Figure1, 2012). FCS is one of the man y different modes of high resolution spatial and temporal analysis of extremely low concentrated biomolecules. It measures the fluctuations of fluorescence intensity in a sub femtoliter volume to detect such parameters as the number of molecules and the di ffusion time. The temporal changes in the fluorescence emission intensity is recorded which is caused by single fluorophores that pass through the detection volume. Eventually, important biochemical parameters can be determined as the concentration, size, and shape of the particle or the viscosity of the environment changes (Lakowicz, 2006).

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9 FCS is a sensitive form of analytical tools due to the fact that it is able to observe a small number of molecules that is nanomola r to picomolar concentrations in a small volume. This in turn makes FCS the perfect method to provide quantitative answers on diffusing molecules from within unperturbed compartments such as cells. FCS was developed in the early seventies as a special case of relaxation analysis. Classical relation methods induce a certain level or kind of external perturbation like pressure or temperature jumps to a reaction system and records information about the kinetic parameters by observing the way the system jumps b ack to equilibrium. FCS just as classical techniques takes advantage of the spontaneous minute fluctuations of physical parameters that are reflected by the fluorescence emission of the molecules. These fluctuations are continually occurring at ambient tem perature and are represented as noise patterns of measured signal Figure (3): A typical confocal FCS system. Laser light is focused by an objective (usually with high numerical apert ure) to a diffraction limited spot. Fluorescence is collected by the same objective and filtered by an interference filter. A pinhole placed in the conjugate image plane reduces out of focus light. The pinhole is usually omitted in two photon excitation ( NCBI.gov, Figure2, 2012).

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10 in fluorescence. This autocorrelation analysis provides a measure for self similarity of a time series signal that describes the persistence of the information carried. The information proce sses governing the molecular dynamics can thus be derived from temporal patterns display by fluorescence fluctuations decay and arise (Rigler & Elson, 2001). The main factors that affect the autocorrelation function and as a result the rate of diffusion ar e, the viscosity of the solvent, the size of the fluctuated particles and the temperature as shown in the following Stokes Einstein formula which can be used when the fluid is a liquid and brownian particles are spheres of radius r, (Sri Balaji et al., 2011) D (3) Fluorescence Cross Correlation Spectroscopy (FCCS) is a daughter technique that correlates signals originating from two differ ent fluorophores detected in two channels with each other. When two different spectral fluorophores are attached to two molecules they form a dual colour FCCS results. This information of the degree of coinciding appearance in the optical volume is used to learn about the degree of interaction between fluorophores. FCCS therefore offer binding kinetics in unperturbed systems and also in low molecular concentrations in solutions (Lakowicz, 2006). Where, is the viscosity of the solvent. T is the temperature. K is Boltzmann constant r is the particle hydrodynamic radius.

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11 The History of Fluorescence Correlation Spectroscopy Almost 40 years since its introduction FCS has evolved from a mysterious and difficult measurement to a technique that is routinely used in the research technology. FCS value in biological and physical sciences consists in the measurements that it makes possible and the concepts that it illustrates and that form its basis FCS provides the window for the field of single molecule measuremen Widengren, 2010). FCS was first introduced by Madge, Elson and Webb in 1972, where it was applied to measure diffusion and chemical dynamics of DNA drug interaction. The term FCS was coined by the Webb lab. The main breakthrough of the technique was the introduction of the confocal optics by Rigler and co sensitivity to sample fluorescence at the single molecule level. These pioneering studies were then foll owed by a number of other applications by many different groups describing translation and rotational mobility in two or three dimensions, attempting to determine the particle concentration even in the cellular environment. These early measurements suffere d from poor signal to noise ratios, which was mainly because of the low detection efficiency and insufficient background suppression (Magde, Webb & Elson, 1978). Principles and Theories of Fluorescence Correlation Spectroscopy FCS Experim ents commonly involve sample volumes as low as a few microliters and the measurement focal volume is in the order of femtoliters. FCS measurements can be performed in solutions and living cells. FCS is based on the analysis of fluctuations of fluorescence. The molecules typically originate from Brownian motion, the random motion of particles suspended in a fluid (a liquid or a gas) resulting from their collision with the

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12 quick atoms or molecules in the gas or liquid of dye labelled molecules through small l aser spot (Rigler & Elson, 2001). These molecules stay within the laser spot depending on their size and if a small dye tagged molecule binds to a large one it emits photons and slows down during its diffusion time (Rigler & Elson, 2001). A sensitive dete ctor records single photons emitted by the molecule of dye. Correlation functions are applied to extract information about the number of molecules (concentration). Physical modes are fitted to the correlation data to quantify the information on the source of the fluctuations. When one detector and one type of fluorescent dye are used the method is known as auto correlation. To increase the flexibility of the method, two dyes and detectors are used and the method is called cross correlation (Rigler & Elson, 2001). The principles of FCS are marked by different stages in the exploitation of fluctuations, as identified and explained below: 1. Source of Fluctuations Source of Fluctuations involves the movement of small particle which is free diffusio n in Brownian motion. Fluorescent dye tagged molecules emit a burst of photons while they diffuse through a small laser spot (Rigler & Elson, 2001). 2. Create a Small Measurement Volume Measurement of fluctuations of different samples in solutions forms th e basis for FCS. Low number of molecules produces a fluctuation signal that is higher. For a higher concentration measurement, the measurement of volume as small as possible should be created. The measurement of volume is produced using diverse methods lik e Multi photon excitation and confocal volume (Rigler & Elson, 2001).

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13 3. Detect Single Photons During the passing of the measurement volume, very few photons are emitted by the chromophore that is attached to the protein of interest. Sensitive photon counti ng detectors are needed for the detection of the photons. Characteristically Avalanche Photo Detectors (APD) are used (Rigler & Elson, 2001). 4. Calculate Correlation Function The transformation of the data form the measure time domain to the correlation ti me domain is done by the auto correlation function. To compute the auto correlation function, one multiplies the measured data with a time shifted data. If there is no time shift and both data traces are the same, then the correlation is high. If the shift is large and the two traces are very different, then the correlation is low (Rigler & Elson, 2001). Theoretical Concepts of FCS a. Autocorrelation Analysis Autocorrelation analysis is performed when the focus is on one species of fluorescent particles. Fl uctuations in the fluorescence signal are quantified by temporally auto correlating the recorded intensity signal. In principle, this autocorrelation routine provides a measure for the self similarity of a time signal and highlights characteristic time con stants of underlying processes (Schwille & Haustein, 2004). b. Cross correlation Analysis In autocorrelation analysis, one effectively compares a measured signal with itself at some later time and looks for recurring patterns. In cross correlation analysis two different signals are correlated and thus a measure of crosstalk is obtained. It involves

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14 looking out for common features of independently measured signals (Schwille & Haustein, 2004). Fluorescence Correlation Spectroscopy and Nanoparticles The prosp ects of using nanoparticles as superior sensors and labels that do not photobleach in biological and environmental studies has sparked wide spread interest in the science community. For the application of nanoparticles in FCS, the diffusion of nanoparticle s in a liquid environment must be studied and understood. This is because when applying the correlation techniques, the diffusion constants of nanoparticles are extracted from solutions mainly using the FCS. FCS measures spontaneous intensity fluctuations which in the case of nanoparticles are caused by small deviations from the equilibrium which in turn are caused by nanoparticles entering and leaving the detection area. Just like in the ordinary FCS, FCS that use nanoparticles obtain the largest fluctuati ons when there are only few molecules or particles in a small detection volume with the ultimate limit being a single molecule or particle at a given time. The sufficient signal to noise for a single molecule or particle in FCS can be achieved through mini mizing the detection volume by focusing a laser beam to a diffraction limited spot combined with high quantum yield photodetectors (Tetin, 2013). FCS can be used to measure the diffusion of nanoparticles to understand their mobility under different condit ions like different shapes and core sizes, an applied external field, or varying surface capping materials. The hydrodynamic radius of the nanoparticles is determined using FCS. FCS also allows for follow up on binding and dissociation between the nanopart icle surface and molecular targets or substrates as well as among nanoparticles including nanoparticle aggregation. FCS that uses nanoparticles enables

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15 researchers to understand the heterogeneous systems, which contain mixtures of different nanoparticles ( Tetin, 2013). Nanoparticles Nanoparticles are particles that range from 1 to 100 nanometers in size. They are mostly the same size as biomolecules which include proteins, antibodies and membrane receptors. Due to their size nanoparticles are primarily us ed to mimic biomolecules and other particles therefore giving them a huge potential of use in the biomedical field. The properties of many conventional materials change when they are formed from nanoparticles because nanoparticles have greater surface area per unit weight than large particles which makes them more reactive to other molecules. This makes the use of nanoparticles or the evaluation of their use to be applicable in many fields such as medicine, manufacturing of materials, implementation in the environment and application in optical energy and electronic fields. Nanotechnology describes a particle as a small object with its transport and properties that behaves as a whole unit. Nanoparticles fall under the ultrafine category of particles while th ose particles above 100 nanometers fall under fine particles (100 to 2,500 nanometers) and coarse particles (2,500 and 10,000 nanometers) (Schmid, 2011). The History of Nanoparticles Nanoparticles have a long history that includes results not only from m odern research and man made material but also from naturally occurring nanoparticles. Naturally occurring nanoparticles include organic particles like proteins and viruses and inorganic particles like oxyhydroxides and metals that have been produced by vol canic eruptions, weathering, wildfires, and microbial processes. Nanoparticles are obviously not only provided by modern synthesis laboratories but also have been in existence since the

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16 prehistoric times. Nanoparticles use was evident in the ancient time i n both natural and man made forms. More than 4500 years ago there is evidence of use of nanoparticles of clay minerals and ceramic matrix with natural asbestos used to create different commodities in the ancient times. There was also the use of metal nanop articles to form color pigments in lustre and glass technology in Mesopotamia during the 9 th century. Gold nanoparticles were also utilized in the 4 th century by Romans to introduce a striking red color to ruby glass that appears green in daylight. This tr ait by the Romans had been forgotten until the 1679 and 1689 when Johann Kunckel used it in his glass factory to manufacture ruby glass. The same character was described by Rudolph years before Kunckel but there was no evidence to show that he applied the trait. The next big forward step in nanoparticles history was made by Michael Faraday approximately 150 years ago. In 1857, he presented his work on experimental relations of gold and other metals to light to the Royal Society of London which marked the em ergence of nanoscience and nanotechnology (Bulte & Modo, 2008). These led to the principle motivation to perform research on nanoparticles. These observations of the size effects raised expectation for the superior performance of nanomaterials compared to bulk materials in many applications if the shape and size of the on the photocatalytic properties of colloidal cadmium sulphide (CCS) and the results in the descript ion of the quantum size effect began. The findings from this work opened up new possibilities of tailoring the physical and chemical properties of materials by altering the crystallite size and shape on a nanometer scale rather than changing the compositio n of the material. Over the years the development of advanced synthesis route of nanoparticles has offered control not only over the composition of nanomaterials but also over the particles size distribution, size, shape and surface properties The develop ment has paved way to the

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17 study and the application of size dependent properties of nanomaterials and nanoparticles (Shah, 2014). The Properties of Nanoparticles that Differ to Bulk Materials Nanoparticle research is currently a significant area of scien tific research due to the diverse potential applications in optical, biomedical and electronic science fields. The property that makes nanoparticles have great scientific interest is their ability to actually act as the bridge amid molecular or atomic stru ctures and bulk materials. Nanoparticles have constant physical properties at its current nano scale unlike bulk materials. Bulk materials only have constant physical properties at its usual normal size but when they are reduced to their nano scale size th ey lose their constant physical properties like their size, shape, and conductivity. Size dependent properties that change in bulk materials while they are in their nano size include quantum confinement in semiconductor particles, super paramagnetism in ma gnetic materials and surface Plasmon resonance in some metal particles. These properties of bulk materials change as their size approaches the nanoscale and as the percentage of atoms at the surface of the bulk material increase or decrease. The number of atoms at the surface of the bulk material is significant to the physical property of the bulk material since it determines the physical property of the material that is going to change and what it is going to change to. For bulk materials which are larger than one micrometer, the percentage of the surface atoms is minuscule compared to the total number of atoms of the material. This makes the number of surface atoms in bulk materials that are larger than one micrometer not able to affect the physical proper ties of the material. Properties of nanoparticles on the other hand are not partly due to the aspects of the surface atoms of the material dominating the properties unlike in bulk material (Schmid, 2011).

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18 Nanoparticles differ from the bulk material in ma ny ways including the bending of bulk copper material that occurs when the atoms in the copper move at about 50 nm scale. The same does not apply to nanoparticles of copper since they smaller than 50 nm and are super hard material thus do not display the s ame ductility and malleability of bulk copper. Nanoparticles can change properties, for example, ferroelectric materials that are smaller than 10 nm can change their magnetism direction using the room temperature thermal energy. Nanoparticles can suspend themselves in solvents since their interaction with the solvent is strong enough to overcome the differences in density that cause floating and sinking in bulk materials. Nanoparticles like gold that appears deep red and black in solutions, have unexpected visible properties due to their small size that enable them to confine their electrons and produce quantum effects, unlike bulk materials. Nanoparticles have high surface area to volume ratio that provides a tremendous driving force for their diffusion es pecially at elevated temperatures and also reduces their incipient melting temperature (Thanh, 2012). The Utilities of Different Properties of Nanoparticles The main reason behind the use of different shapes and sizes of nanoparticles is that the changes in nanoparticles size and shape or surface composition can result in changes in the chemical and physical properties of the nanoparticles. When producing nanoparticles of a size within a certain range of between 1 to 100 nm, their chemical, electronic and physical properties change. These changes usually depend on the nanoparticles size and characteristics such as magnetic behavior, reductive oxidation potential, melting temperature and their colour. All these features can be controlled by altering the nan oparticles shape and size (Gurunathan et al., 2009). Over the years, changes in these characteristics that occur in nanoparticles due to their size and shape have attracted a lot of

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19 attention because of their good conductivity, chemical stability, uses as catalysts, and their application in various industries like the food industries and medical industry, and nanoparticles unique electrical and optical qualities. Studies of nanoparticles over the years have shown that the morphology, size, stability and che mical and physical properties of nanoparticles are influenced strongly by the experimental conditions they have been put under. Conditions like the kinetics of interaction of the nanoparticles ions with reducing agents and adsorption process of stabilizing agent with nanoparticles, cause changes in the chemical and physical properties of nanoparticles (Ghorbani et al., 2011) This shows that accurate control of the size, shape and distribution of the produced nanoparticles is achieved by changing stabilizin g and reducing factors, and the method of synthesis. Incredible properties of nanomaterials strongly depend on size and shape of the nanoparticles, their interaction with the surrounding media, preparation method and stabilizers. All this incredible proper ties of nanoparticles can be best achieved through the synthesis of nanocrystals. The synthesis of different sizes and shapes of nanocrystals makes it possible for each nanoparticle synthesised to reach its best applied capability explaining why different sizes and shapes of nanoparticles are used (El Khesheshen and El Rab, 2012 ). Photodynamic Therapy Introduction The battle against oncological and non oncological diseases cannot be described without describing the impact of Photodynamic therapy (PDT) in saving lives. PDT can be described as the treatment that uses a photosynthesizing agent or a photosensitizer and a special type of light to activate and be effective. Photodynamic therapy refers to use of nontoxic light sensitive drugs that acti vate when exposed to particular light and become

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20 toxic to diseased cells in the body. PDT is effective in killing microbial cells, viruses, bacteria and fungi. PDT can also be referred to as photo chemotherapy, photo radiation or phototherapy. Photosensiti zer is either put into the bloodstream through the skin or a vein (Abdel Kader, 2014). The drug is absorbed by the cancerous cells, due to constriction of the blood vessels and tissues in the cancerous cells the drug stays longer and is released faster in healthier cells. The light is applied to the area of treatment whereby the drug reacts with the light and oxygen to become toxic to the cells. PDT is also effective in destroying blood vessels that nourish the cancer cells and alter th e immune system to attack cancer cells. PDT has played an active role in shaping cancer treatments. Doctors use specific photosensitizers and different wavelengths that will help to trigger the photosensitizers to ensure treatment of specific body parts (A bdel Kader, 2014). PDT has recently proven to be a form of therapy that can be specifically applied to only harmful cells and tissues; this is in contrast to alternative treatments which lack specificity in their targets. Because of this, PDT is a better t reatment method for disease, as it can bypass lots of the negative side effects that other, nonspecific treatments bring. The study of PDT is important in the treatment of oncological and non oncological diseases as a non invasive treatment option. Fi gure 4: The process of photodynamic Therapy (photolitec, 2013)

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21 The P rinciples of PDT A photosensitizer (PS) is administered systemically or topically. After a period of systemic PS distribution it selectively accumulates in the tumor. Irradiation activ ates the PS and in the presence of molecular oxygen triggers a photochemical reaction that culminates in the production of 1 O 2 Irreparable damage to cellular macromolecules leads to tumor cell death via an apoptotic, necrotic or autophagic mechanism, acc ompanied by induction of an acute local inflammatory reaction that participates in the removal of dead cells, restoration of normal tissue homeostasis and sometimes in the development of systemic immunity (Agosgtinis et al., 2011). Applications of Photody namic Therapy Photodynamic therapy has gained a lot of attention over the years as an effective treatment option for various cancers. PDT uses light to trigger photosensitive agents that release chemical oxygen that destroys cancerous cells. The use of PD T in treating cancer has been very effective in various cases and experiments. PDT has proven successful in the treatment of many skin diseases. For example, PDT can be used for the treatment of severe acne. Serious forms of acne can be treated by use of photosensitizers that are applied to the face and scalp and later radiated by specific light to kill the bacterial cells which cause acne within the patient. PDT causes sensitivity and, therefore, should only be used on the face and scalp during severe sit uations of acne that cannot be controlled by other therapies (Allison, Moghissi, Downie, & Dixon, 2011). The greatest use of PDT is in the treatment of cancer. PDT has proven to be effective in both limiting the metastases of cancer cells, while also bei ng able to directly cause cancer cell death. A photosensitizing agent, any material that is triggered by light, is injected into

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22 the body of the patients, followed by absorption in body tissues. One of the most striking results of this is that the agent s tays for a longer period in cancer cells than in normal cells. This is because the tumor presses the blood vessels and reduces the circulation of blood within it. With limited circulation the drug takes longer to be removed from the cancerous cells by the lymphatic system as compared to healthy cells. After 24 to 72 hours, the agent has left the normal body cells but still present in the cancer cells. When the cancerous cells or tumors are exposed to light, they absorb it and produce an active form of oxyge n capable of destroying the nearby cancer cells (American Cancer society, 2015). The light used in activation of PDT may come from many sources; the first being laser light that is transmitted to the body by use of fiber optic cables. In this example, the fiber optic cable can be inserted into the body through an endoscope and into the esophagus or lungs to treat cancer in these areas (American Cancer society, 2015). Other sources of light that can be used include light emitting diodes (LEDs) that can be u sed in treatment of surface tumors such as skin cancer. PDT is mainly performed as an outpatient procedure and can be repeated with the use of other therapies such as radiation, chemotherapy and surgery to maximize the effectiveness of the treatment. The PDT treatment can be performed outside the confines of a hospital with the patient receiving both the photosensitizers and light treatments at their homes (American Cancer society, 2015). Extracorporeal photoresist (ECP) is a form of PDT whereby a machine collects blood cells from the patient, treats them outside the body by exposing them to light and then returns them to the patient. The US Food and Drug Administration (FDA) have approved the use of ECP to lessen the severity of skin symptoms of T cell Ly mphoma that have

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23 previously not been affected by other therapies. ECP studies have also been carried out to investigate its use on blood cancers and to reduce rejection after transplants (American Cancer society, 2015). Photosensitizers A photosensitizer is a chemical compound capable of undergoing a chemical change in another molecule during a photochemical process upon illumination. A photosensitizer reacts with light to undergo a chemical change, r esulting in the release of oxygen. There are many photo sensitizers for PDT that include aminolevulinic acid. Aminolevulinic acid is applied directly to the skin and is used for the treatment of actinic keratosis, a skin condition that can easily develop to cancer and is used on the scalp and face (Abdel Kader, 2014). A special blue light is used for the activation of this drug and not laser light Porfimer sodium is the most commonly used photosensitizer and is activated by red light from a laser. It is approved for the treatment of cancer of the esophagus and when it cannot be treated by laser can lead to esophageal cancer if surgery is not conducted. Porfimer sodium is also used for the treatment of non small lung ca ncer that affects inner lining of the bronchus called endotracheal cancer (American Cancer society, 2015). Levulan is photosensitizer that is used for the treatment of lesions. It is applied to the lesions and given 6 hours to penetrate the skin. It is th en irradiated with blue light for 15 minutes. This generates the reactive oxygen that kills cells. The patients must protect themselves from the bright sunshine for 40 hours since the skins are sensitive (Pazurek & Malecka Panas, 2005).

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24 Other types of p hotosensitizers used include allumera, visudeneyne, cysview, foscan, metrix and laserphyrin. Although the photosensitizers have different treatments they have certain similar characteristics such as high absorption at long wavelengths, high chemical stabil ity, low dark toxicity, high singlet oxygen yield and natural fluorescence (Pazurek & Malecka Panas, 2005). Works Done on Nanoparticles The nanoparticles play a supplementary and complementary role to the in PDT (Abdel Kader, 2014). The active nanopar ticles are self illuminating and, therefore, can reach deep tissues where light cannot reach and destroy the dangerous cancerous cells without the use of external light sources. Nanoparticles can be used for the simultaneous radiation and therapeutic treat ment of cancer. Some luminescent nanoparticles such as CuCy nanoparticles do not require photosensitizers to be effective, therefore, the treatment is convenient, has a lower cost and more efficient. Secondary illuminating nanoparticles are capable of resp onding to limited amount of light and this means that they are capable of being activated in deep tissues where small amounts of light can reach. Additionally, they activate and destroy cells as effectively as self illuminating nanoparticles. Researchers h ave discovered that Cu Cy nanoparticles are effective in treating prostate and breast cancer when exposed to x ray light. The research showed that a tumor treated with Cu Cy nanoparticles stayed the same size for close to two weeks while that which was not treated tripled in its size (Pazurek & Malecka Panas, 2005). These specific nanoparticles have various advantages, the biggest being that they have lower toxicity to the healthy cells compared to other nanoparticle approaches. This means that the lumine scent nanoparticles are safer to use as they have less negative side effects to healthy tissues. The intense

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25 photoluminescence of the nanoparticles and the x ray luminescence can be used to produce cell images. The nanoparticles can penetrate large tissue and kill the surrounding cancerous cells with greater specificity and efficiency. Research is being conducted to reduce the size of Cu Cy nanoparticles, to help increase its absorption into tumor tissues. Nanoparticles can be used to expand the reach of PD T and to ensure that treatment can be done on deep tissues due to self illuminating nanoparticles. Nanoparticle research is meant on increasing the applicability for PDT and is the key to ensuring that the treatment of deep tissue cancers is possible (Pazu rek & Malecka Panas, 2005). Motivation of the thesis In 2008, cancer accounted for around 13% of all deaths worldwide. With nearly 13 million cancer new cases annually, deaths are projected to rise reaching 13.1 million in 2030 (Lucky et al ., 2015). Intervention measures such as screening and surveillance programs are good at improving the outcome and survival, though there is need for effective, efficient, affordable, and acceptable cancer therapeutic options. Available treatment options include chemotherapy, radiotherapy, surgery, and small molecule based therapies and immunotherapy although chemotherapy has systemic side effects, and radiation therapy has limitation of the cumulative radiation dose. Refining existing treatment modalities is important but research also focuses on safe, potent but cost effective alternate treatment modalities such as Photodynamic therapy (PDT). PDT uses a combination of light, light sensitive drugs, which are harmless and nondamaging. Upon i rradiation by an appropriate wavelength of light, the drugs become excited transferring energy to surrounding molecular oxygen to generate cytotoxic reactive

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26 oxygen species. These oxidize critical cellular macromolecules induce cellular permeability altera tions resulting to cell death by necrosis or apoptosis or both. Despite its success, PDT treatment is yet to gain clinical acceptance as a first line oncological intervention mainly due to certain limitations. However, the use of nanoparticles has been a major stride in resolving some challenges associated with classic PS. Its use in cancer therapy is attractive as it promises better tumor selectivity accessible to light and a lower systemic toxicity with fewer side effects in comparison to radiation and chemotherapy. Though proved effective in cancer treatment, it is unfortunate that most of the photosensitizers used in PDT can only be activated by visible light, which cannot pass through a thick tissue thus hindering its full therapeutic potential. Wher eas using nanoparticles as a drug carrier makes it possible to treat tumors that are deeper under the skin or in body tissues because it can be activated by the near infrared light which can afford penetration depths greater than those of the visible light besides being less harmful to cells and tissues (Scholz and Dedic, 2012). An approach to study the diffusion dynamics of rod and ball shaped gold nanoparticles using fluorescence correlation spectroscopy is presented.

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27 CHAPTER II INSTRUMENTS AND METHODS In this work an upright Olympus FV1000 microscope for confocal fluorescence microscopy has been used to get the fluorescence correlation spectroscopy traces for different colloidal gold nanoparticles solutions. This will aid in the study their behaviour to know which of the samples is best suited to be used in Photodynamic Therapy as it relates to medical treatment. Gold Nanoparticles Characterization UV V isible Spectroscopy UV visible characterization of (colloi dal gold nanosphere, colloidal gold nanosphere Photosensitizer, colloidal gold nanoshpere RB, and colloidal gold nanorodsRB) solutions was done by using Cary 100 UV Visible spectrophotometer. The absorbance measurements were done over the wavelength of 300 900 nm using 1 cm path length quartz cuvettes.

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28 Figure (5): Absorption spectra for four different colloidal gold solutions ;abbreviations,Ball 3: colloidal gold nanosphere conjugated with Rhodamine B in water,Rod3: colloidal gold nanorod conjugated with Rhodamine B in water, Ball 1: is the colloidal g old nanospheres solution, Rod 2 : colloidal gold nanorod solution. Fluorescence Correlation Spectroscopy The FV 1000 confocal microscope has six laser lines. These include UV/Violet (405 nm) Multi line Argon laser with 458, 488, and, 514 nm wavelengths, green He Ne laser (543 nm), and red He Ne laser (635 nm). Two wavelengths 488 nm and the 534 nm were used to excite the colloidal gold nanoparticles solutions dispersed on a cover slip using a water immersion microscope objective lens (60x, 1.2 NA). FCS instrument was calibrated with Rhodamine B with radial dimension of 0.282 m, 0.293 m, and 0.307 m. The scientific computations in Python can be done using Scipy library which contains module s for Fourier transforms, optimization, linear algebra, signal and image processing, and other modules. Numpy is another package for scientific computing in python. The plots were

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29 generated with the help of matplot library which generates 2D plots. To gene rate the graphic user interface the Pyside library was used. The code can be divided into three important steps the first is generating a horizontal data, the second is computing the autocorrelation, and the third is filtering the autocorrelation. The prog ram generates a horizontal data which may contain around eight million elements and then divide this 1D array into slices, compute the autocorrelation for each slice using Fourier transform average all of the autocorrelation curves for all the slices to ge t the final autocorrelation curve. The final autocorrelation curve has just particular number of points to be chosen by the programmer in order to get smooth curve because plotting all the points would result in unwanted fluctuations within the final repre sentation. The code also has a feature that allows the user to remove any unwanted autocorrelation curve to enhance the final autocorrelation result. Measurements for fluorescence correlation spectroscopy were accomplished using a custom sof tware (MAZ 2015) written in Python programming language. Equation 1 was used to measure the fluorescence fluctuations for the system, whereas, equation 2 used to calculate the cross correlation function. To avoid the computational complexity involved in th e autocorrelation computation, the autocorrelation was calculated by means of the Wiener Khinchin theorem. This theorem uses the Fourier transform to compute the autocorrelation, resulting in a more convenient method, because it has several efficient algor ithms, such as the Fast Fourier Transform. An exponential axis is generated for the time delay, with a fixed number of points to avoid the unnecessary fluctuations in the resulted autocorrelation and cross correlation curves. A correction on the values of this array when the difference between each subsequent point is less than the sampling time is also

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30 accounted for. Normalized autocorrelation of a 3D freely diffusing sample can be expressed as: (Schwille & Haustein, 2004) (3) Where, = delay time. D = characteristic diffusion time N = average number of particle s within the light focal volume. w xy = beam waist of the laser focal volume in the XY direction w z = beam waist in the Z direction These last two parameters can be obtai ned from calibration measurements. Equation (3) can be used as model that can be fitted to the autocorrelation computed from equation (1). Once the normalized autocorrelation function has been computed, it can be used to find the parameters N and in e quation (3) using an appropriate fitting procedure. MAZ 2015 uses the SciPy Python library and its optimization package, which provides the least function that allows to minimize the square error between the values from the computed autocorrelation and a p arametric model given by equation (3), whose parameters are G (0) and where (Lakowicz, 2006) Once the Go and N parameters have been found via least squares minimization, the diffusion

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31 (4) Flowchart in Figure (6) summarizes the flow of the software: Figure (6): Flow chart represents the steps for the custom software (MAZ 2015).

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32 Figure 7: Graphic user interface for the custom software (MAZ 2015) Compute and fit autocorrelation buttons for file 2 Com pute and fit autocorrelati on buttons for file 1 Compute and fit cross correlation buttons for selected files

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33 Figure 8 : The working software when computing the autocorrelation for the fluctuations of the signal for the colloidal gold nanosphere solution.

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34 Figure 9: shows the selective feature to remove unwanted fluctuations using the colloidal gold nanoball solution with unwanted fluctuations, the yellow line at the left side, and the a) Before removing the unwanted trace b) After removing the unwanted trace result after removing it appearing at the right side. Figure 10: Cross correlation for the d ata from two different solutions ( colloidal gold nanoballs and colloidal gold nanorods ), it shows that there is no correlation between them.

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35 The quantification of the results done by calculation the root mean square error (RMSE) values for each result using the following equation, the lower RMSE is the best the result, (Kaggle, 2015). RMSE (5) Where n refers to the number of elements e refers to the error i.e. the difference between the fitted values and the measured ones refers to the calculated correlation at i. refers to the fitted values according to equation (3)

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36 CHAPTER III RESULTS No 1 Colloidal gold nanoballs RB solution 543 100 nM in PBS 7.8 10 377.67 52.8 2 Colloidal gold nanoballs RB solution 543 100 nM in PBS 7.8 20 230.49 86.56 3 Colloidal gold nanoballs RB solution 543 100 nM in PBS 7.8 10 409.49 48.7 4 Colloida l gold nanoballs RB solution 543 100 nM in culture media 7.8 10 392.24 50.8 5 Colloidal gold nanoballs RB solution 543 100 nM in culture media 2.3 10 552.8 36.09 6 Colloidal gold nanoballs RB solution 543 100 nM in PBS 1.6 10 24533.12 0.813 7 Colloidal gold nanoballs RB solution 543 100nM in nanopure water 2.3 10 1181.56 16.88 8 Colloidal gold nanoballs RB solution 543 Full strength 7.8 10 770920.7 0.030

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37 9 Colloidal gold nanoballs RB solution 488 100 nM in PBS 10.2 10 5869.81 4.014 10 Colloidal gold n anoballs RB solution 488 100 nM in PBS 33.1 10 631.98 37.28 11 Colloidal gold nanoballs RB solution 543 100 nM in PBS 1.6 10 5719.536 4.119 12 Colloidal gold nanoballs RB solution 543 100 nM in PBS 4.2 10 618.74 38.08 13 Colloidal gold nanorod solution 488 Full strength 10.2 10 24.936 860.67 14 Colloidal gold nanorod solution 543 2uL in 998uL PBS 1.6 10 33020190.13 0.0006 15 Colloidal gold nanoballsPhotosensitize r solution 543 Full strength 7.8 10 29275421.52 0.00068 16 Colloidal gold nanoballs solut ion 543 Full strength 7.8 10 74982283.2 0.00026 Table 1: Results from autocorrelation for different colloidal gold nanoparticles solutions (nanoshpere, nanorod, nanosphere conjugated with Rhodmine B in water, nanorod conjugated with Rhodmine B in water) the correlation analysis shows that the nanosphere conjugated with Rhodmine B in water with laser wavelength of 543 nm and power of 7.8 micro watt.

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38 According to the RMSE observations we got the following: Figure 11: Varying concentrations of colloidal gol d nanosphere conjugated with Rhodamine B in water solution. Undiluted sample Figure 12: Varying laser powers of colloidal gold nano balls conjugated with Rhodamine B in water solution

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39 is Figure 14: Diluting the colloidal gold soluti on in PBS nanopure water and culture media it is obvious that the PBS shows the best RMSE value over the other two solvents. Figure 13: Using different colloidal gold nanoparticles (nanosphere and nanorod ) under the same experimental conditions, the colloidal gold nano sphere RB solution shows the best results.

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40 C HAPTER IV DISCUSSION In this thesis, a software called MAZ 2015 was successfully implemented which is com parable to the commercial software used for the fluorescence correlation spectroscopy calculations, with this software one can do the correlation and cross correlation calculations with a special feature to remove the unwanted fluctuations caused by impuri ties or other sample fluctuations in order to get more accurate results, the user could specify the frequency, the focal spot size, and the size of division over which we are computing the correlation or the cross correlation. The tiny differences in the r esults between the custom software and the MAZ 2015 may relate to the approximations for the two different programming languages because the commercial software FV1000 uses Pascal whereas MAZ 2015 uses Python programming language. The colloidal gold nanopa rticles samples have been synthetized by Dr. Zheng's group at Fujian normal university in China in which three samples are manufactured with ball and rod shapes. Some of them were conjugated with photosensitizers, some with Rhodamine B, while the others we re not conjugated with neither molecules. All these were studied using FCS while the correlation curves were obtained to track the behaviour of each of these samples to determine which of them would be of the best choice as well as which are the best exper imental conditions to implement such experiment to make use of these results in the Photodynamic Therapy. The correlation results for the colloidal gold nanorod solutions by changing the concentrations and excitation wavelengths at each time were inconclus ive which gives astronomical diffusion coefficients values that cannot be considered as real

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41 values and this maybe because they are too heavy to diffuse so what we got is just a noise. The correlation results for the colloidal gold nanoball solution were a lso unreliable because there were problems with their synthesizing. The same problem occurred with the colloidal gold nanoballs solution and the gold nanoballs conjugated with photosensitizer solution. The best result was obtained from the gold nanoballs c onjugated with Rhodamine B and 2 /sec and standard deviation of 71.32. Whereas, using lower laser powers did not provide reliable results given that the concentration is in the nanomolar range. The RMSE was used to quantify our observations, in accordance with the RMSE values we found that it is better to use low concentrations for the fluorescence correlation spectroscopy experiments. High concentration of the sample means high number of particles in the measurement volume reliable results, given that the number of particles within the observation volume should not be too few or too large. The ot her comparison we did with the aid of RMSE values was between different samples under the same experimental conditions it shows that the gold nanoparticles conjugated with Rhodamine B was of the best behaviour and that may return to the effects of size. Th is also proves that the conjugation with the Rhodamine B is better than conjugation with the photosensitizer and both of them are better than using the gold nanoparticles without any kind of conjugation. Our results showed that diluting the colloidal gold solution in PBS shows the best behaviour for these particles over diluting them in nanopure water or culture media. Using different solutions under the same experimental conditions showed that the RMSE value colloidal gold nanosphere solution was the lowes t which indicates that this sample is of the best behaviour. This may be related to the different sizes and shapes but unfortunately we were not able to do the size and shape characterization

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42 experiment. We also found that as the excitation power increases the RMSE value decreases; this is because as the power increases the number of observed photons from each fluorophore increases due to the Brownian motion and this result in increasing the strength of the recorded intensity signal given that the concentra tion in the nanomolar range. We also found that when we used 100 nM and full strength concentrations of colloidal gold nanoparticles RB solution with the same laser power, excitation wavelength of 543 nm, and sampling time of 10 microsecond per pixel. We m ay notice that the RMSE value is much lower for the 100 nM concentration value. This can be connected with the number of fluorophores in the observation volume, as the number of fluorophores increases the value eases. Note that Go = (1/N) where N is the number of fluorophores in the observation volume and hence the amplitude of the correlation function will be too small for reliable measurements.

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43 C HAPTER V CONCLUSIONS In light of the aforementioned results, the following conclusions could be reached: the customized software (MAZ 2015) works well and we can use it for autocorrelation and cross correlation analysis. Moreover, its selective feature enables the users to remove unwante d fluctuations for more accurate calculations, increases the laser power also enhances the accuracy of the correlation results. This occurs because increasing the laser power increases the fluorescence intensity of the sample which, in turn, increases the number of photons obtained by the detector. Diluting the colloidal gold nanoparticles in water solution with the Phosphate Buffered Saline (PBS) yields good signals for analysis, compared to diluting them in nanopure water and culture media to obtain the d iluted colloidal gold nanoparticles solution. The concentration of 100 nM of colloidal gold nanoparticles solution also yielded better results, compared to the tests conducted using higher concentrations. The green He Ne laser (543 nm) was the most suitabl e wavelength for this experiment, as was duly demonstrated using the absorption spectra. The colloidal gold nanoparticles solution diluted in PBS of nanospheres conjugated with RB yielded the most promising results.

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44 C HAPTER VI FUTURE WORK The optical behavior of colloidal nanoparticles can be tuned by changing their dimensions, shape, and surrounding environment. Moreover, different sizes of nanoparticles can be used, since the gold nanoparticles are plasmonic particles. When the light propagates near the colloidal nanoparticles, it interacts with the free electrons at the surface of the metal and induces them to vibrate collectively creating an electric field. When the electric field of the vibrated free electrons resonate wit h the electric field of the incident light, we call this phenomena surface plasmon resonance. The oscillations of the free electrons on the surface of the metal affect the absorption of light. For instance, the absorption of light for small gold nanopartic les occurs in the blue green portion of the spectrum, but as their size increase the light absorption of the surface plasmon resonance wavelength shifts to the red portion of the spectrum. Regarding nanorods, as the aspect ratio (length/width) increases th e fluorescence bands also drastically increase, given that the nanorods have two plasmon resonance bands that correspond to the length and width of the rod shaped nanoparticle. It has been showed that increasing the length of these particles leads to impro vements of the fluorescence quantum efficiency (Li et al., 2005). We may also use different shapes of nanoparticles like the nano urchins to further improve the results in which the absorption and emission from rough surfaces of nano urchins are better than from smooth ones. In order to reduce the aggregation to the lowest level, specific materials must be added to the colloidal nanoparticles solution. These characteristics help in tailoring the optical properties for nanoparticles to meet the requireme nts of different

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45 applications. For photodynamic therapy, it is important to have the absorption in the therapeutic window (around 650 1000 nm) where light can penetrate deeper layer of the skin, compared to other portions of the spectra (Scholz and Dedic, 2012). Probes other than the Rhodamine B and silicon phthalocyanine (Pc4) photosensitizer can be conjugated to the nanoparticles when doing the FCS experiment to determine which one is the efficient to enhance the fluorescence of the observed particles. Fu rthermore, nanoparticles made of different materials can be tested in the future. Silicon nanoparticles could yield positive results given that it has been found that they are able to generate cytotoxic singlet oxygen upon illumination when used in photody namic therapy. We may also try to use concentrations lower than 100 nM and determine if it would yield better results because the FCS works well for solutions concentrations in the nanomolar range.

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