diffusion in soft heterogeneous biomaterials · carrageen gel lijkt daarentegen wel veelbelovend en...

DIFFUSION IN SOFT HETEROGENEOUS

BIOMATERIALS

Hannes DEVELTER Student number: 01203622

Promoter: Prof. Dr. Kevin Braeckmans

Co-promoter: Prof. Dr. Niklas Lorén and Prof. Dr. Anette Larsson

RISE: Research Institutes of Sweden - Unit of Bioscience and materials - Agrifood and

Bioscience - Product Design and Perception

Commissioners: Dr. Juan Fraire and Dr. Evelien Wynendaele

A Master dissertation for the study programme Master in Pharmaceutical Care

Academic year: 2016 - 2017

COPYRIGHT

“The author and promotors give the authorization to consult and to copy parts of this thesis for personal use only. Any

other use is limited by the laws of copyright, especially concerning the obligation to refer to the source whenever

results from this thesis are cited.”

May 24, 2017

Promoter

Prof. Dr. K. Braeckmans

Co-promoter

Prof. Dr. N. Lorén

Author

Hannes Develter

ABSTRACT

The knowledge of mass transport is very important in pharmaceutical and food preparations. Many

techniques are established to determine the local mass transport of a substance. However, while most diffusion

experiments have been done in homogeneous materials, soft matter systems, like hydrogels, often have very

complex microstructures. They can be heterogeneous with many interfaces, which can have a large impact on

the local diffusion properties of the substance. It is therefore interesting to investigate the diffusion near these

interfaces. There is moreover a need for stable and flexible model systems to develop new quantitative

microscopy techniques.

In this thesis, the diffusion in hydrogels is investigated, starting with relatively homogeneous systems

and gradually introducing more heterogeneous models with interfaces. The diffusion in 1% κ-carrageenan and

4% alginate gels is investigated. Polystyrene spheres and alginate beads are introduced in the carrageenan gel,

evaluated and the diffusion near the alginate and carrageenan interface is studied. Sodium fluorescein and

70kDa FITC-dextran are tested as fluorescent diffusion probes. For each system, Fluorescence Recovery After

Photobleaching (FRAP) and Raster Image Correlation Spectroscopy (RICS) are applied for measuring the

diffusion. The applied techniques are investigated and tested near interfaces and inside the bulk of the gels.

It appears that 70kDa FITC-dextran is better suited as a probe than sodium fluorescein in the used model

systems regarding the extent of photobleaching and possible interaction with the gel network. Furthermore, PS

spheres inside a 1% κ-carrageenan hydrogel does not seem to be the optimal model for this thesis. The alginate

gel inside the carrageenan gel seems to be a more promising model and the internal method for producing the

alginate gel is superior regarding the homogeneity.

In general for both techniques, the measured diffusion coefficients of 70kDa FITC-dextran inside the

alginate gel are in the range of 3µm2/s to 7µm2/s and inside the carrageenan gel in the range of approximately

15µm2/s to 18µm2/s by RICS and 20µm2/s to 25µm2/s by FRAP. The diffusion coefficient of the probe in water is

found to be in the range of 27µm2/s to 30µm2/s using RICS. The measured diffusion coefficients of RICS are slightly

higher inside the alginate gel, but lower inside the carrageenan gel in comparison with the FRAP experiments.

The results of the FRAP experiments near the interface show a slightly increasing trend in diffusion of the probe

inside the carrageenan gel when moving the ROI further away from the interface. This is in contrast to the RICS

experiments near the interface inside the alginate gel, where the diffusion coefficient remains relatively

consistent at each investigated distance from the interface.

The mass transport in heterogeneous biomaterials and their interfaces remains an interesting but

difficult subject. The results in this thesis are promising but more experiments are necessary to receive more

closing and reliable results.

SAMENVATTING

De kennis van massatransport is zeer belangrijk in farmaceutische en voedsel preparaten. Vele

technieken zijn beschikbaar om de lokale massatransport van een stof te bepalen. Hoewel de meeste

experimenten rond diffusie uitgevoerd zijn in homogene materialen, zijn systemen van zachte materie, zoals

hydrogels, echter vaak complexe microstructuren. Deze kunnen heterogeen zijn met veel interfaces, die een

grote impact kunnen hebben op de lokale diffusie eigenschappen van een stof. Het is daarom interessant om de

diffusie in de buurt van deze interfaces te onderzoeken. Er is daarnaast ook nood aan stabiele en flexibele

modelsystemen om nieuwe kwantitatieve microscopie technieken te ontwikkelen.

In deze thesis is de diffusie in hydrogels onderzocht, startende met relatief homogene systemen,

waarbij geleidelijk meer heterogene modellen worden gepresenteerd. De diffusie in 1% carrageen en 4% alginaat

gels is bestudeerd. Polystyreen sferen en alginaat druppels zijn geintroduceerd in de carrageen gel, geëvalueerd

en de diffusie in de buurt van de alginaat en carrageen interface is bestudeerd. Natrium fluoresceïne en 70kDa

FITC-dextraan zijn getest als fluorescente diffusie probes. Voor elk systeem werden Fluorescence Recovery After

Photobleaching (FRAP) en Raster Image Correlation Spectroscopy (RICS) toegepast om de diffusie te meten. De

toegepaste technieken zijn bestudeerd en getest dicht bij de interfaces en in de bulk van de gels.

Het blijkt dat 70kDa FITC-dextraan beter geschikt is als probe dan natrium fluoresceïne in de gebruikte

model systemen met betrekking tot de mate van fotobleking en mogelijke interactie met het gelnetwerk. Ook

lijkt het model met de PS sferen in de 1% carrageen gel niet optimaal voor deze thesis. De alginaat gel in de

carrageen gel lijkt daarentegen wel veelbelovend en de interne methode voor de productie van de alginaat gel

is superieur inzake de homogeniciteit.

In het algemeen zijn voor beide technieken diffusiecoëfficiënten van 70kDa FITC-dextraan gemeten

tussen 3µm2/s en 7µm2/s in de alginaat gel en ongeveer van 15µm2/s tot 18µm2/s door RICS en 20µm2/s tot 25µm2/s

door FRAP in de carrageen gel. De gemeten diffusiecoëfficiënten bij RICS zijn iets hoger in de alginaat gel, maar

lager in de carrageen gel, in vergelijking met de FRAP experimenten. De resultaten van de FRAP experimenten

dicht bij de interface vertonen een licht stijgende trend in diffusie van de probe in de carrageen gel wanneer de

ROI verder weg van de interface wordt geplaatst. Dit staat in contrast met de RICS experimenten aan de interface

in de alginaat gel, waar de diffusiecoëfficiënt relatief consistent blijft op elke onderzochte afstand van de

interface.

De massatransport in heterogene materialen en hun interfaces blijft een interessant maar moeilijk

onderwerp. De resultaten in deze thesis zijn veelbelovend, maar meer experimenten zijn noodzakelijk om tot

meer sluitende en betrouwbare resultaten te bekomen.

ACKNOWLEDGEMENTS

Above all, I would like to express my sincere gratitude to my supervisor Niklas Lorén for the endless support

and enthusiasm in my project, the help with my experiments and for answering all of my questions and coming

up with great ideas during my work. I had a very interesting and educational experience during my stay, mostly

thanks to him. I could not have wished for a better supervisor. I would also like to thank my promotor Prof.

Kevin Braeckmans of Ghent University for all the help. I am most grateful for Niklas Lorén, Prof. Kevin

Braeckmans and Prof. Anette Larsson for giving me the opportunity to establish my master thesis at RISE.

Furthermore, I would like to thank Magnus for the big help with the analysis of my data and with the RICS

experiments and also Annika Altskär and Annika Krona for teaching me the basics of the CLSM. I would like to

thank all the people in general at RISE for the amazing time during my stay and for the fun and entertaining

fika and lunch breaks every day, especially the other diploma workers and interns for the support and talks

every day.

I would like thank all my new friends in Göteborg for the amazing time I had during my Erasmus and my

friends in Belgium as well for the support and help.

Finally, I am very grateful for my parents, brother, sister and girlfriend for giving me the opportunity and

support during my stay in Sweden and for everything they have done for me.

TABLE OF CONTENTS

1 INTRODUCTION ............................................................................................................................................................................................................. 1

1.1 DIFFUSION ................................................................................................................................................................................................................ 2

1.2 HETEROGENEOUS MATERIALS ....................................................................................................................................................................... 2

1.3 HYDROGEL ............................................................................................................................................................................................................... 3

1.3.1 Carrageenan ................................................................................................................................................................................................ 4

1.3.2 Alginate .......................................................................................................................................................................................................... 5

1.4 POLYSTYRENE MICROSPHERES ..................................................................................................................................................................... 5

1.5 CONFOCAL LASER SCANNING MICROSCOPY (CLSM) ............................................................................................................................6

1.6 FLUORESCENCE...................................................................................................................................................................................................... 7

1.6.1 Fluorescent diffusion probes .............................................................................................................................................................8

1.6.1.1 Fluorescein ............................................................................................................................................................................................ 8

1.6.1.2 FITC-dextran ..........................................................................................................................................................................................9

1.7 OPTICAL TECHNIQUES TO MEASURE DIFFUSION .................................................................................................................................10

1.7.1 Fluorescence Recovery After Photobleaching (FRAP) ........................................................................................................ 10

1.7.2 Correlation spectroscopy .................................................................................................................................................................... 11

2 OBJECTIVES .................................................................................................................................................................................................................. 13

3 MATERIALS AND METHODS .................................................................................................................................................................................. 15

3.1 HYDROGEL ............................................................................................................................................................................................................. 15

3.1.1 Carrageenan gel ...................................................................................................................................................................................... 15

3.1.2 Alginate gel ................................................................................................................................................................................................ 16

3.1.2.1 Droplet method ................................................................................................................................................................................. 16

3.1.2.2 Internal method ................................................................................................................................................................................ 16

3.1.3 Mixture of Carrageenan and Alginate ......................................................................................................................................... 17

3.2 POLYSTYRENE MICROSPHERES .............................................................................................................................................................. 17

3.3 FLUORESCENT DIFFUSION PROBES...................................................................................................................................................... 18

3.3.1 Fluorescein ................................................................................................................................................................................................. 18

3.3.2 70kDa FITC-dextran ............................................................................................................................................................................... 18

3.4 CLSM..................................................................................................................................................................................................................... 19

3.5 FRAP..................................................................................................................................................................................................................... 19

3.5.1 Analysis ....................................................................................................................................................................................................... 20

3.6 RICS ..................................................................................................................................................................................................................... 20

4 RESULTS ....................................................................................................................................................................................................................... 22

4.1 POLYSTYRENE SPHERES INSIDE CARRAGEENAN GEL ..................................................................................................................... 22

4.2 FRAP EXPERIMENTS ................................................................................................................................................................................... 23

4.2.1 Concentration effect of sodium fluorescein........................................................................................................................... 23

4.2.2 Carrageenan bulk .................................................................................................................................................................................. 24

4.2.3 Alginate bulk ............................................................................................................................................................................................ 25

4.2.3.1 Droplet method ................................................................................................................................................................................ 25

4.2.3.2 Internal method ............................................................................................................................................................................... 27

4.2.4 Interface ..................................................................................................................................................................................................... 28

4.2.5 Mixture of carrageenan and alginate gel ................................................................................................................................ 33

4.3 RICS EXPERIMENTS ..................................................................................................................................................................................... 34

4.3.1 Optimal scanning rate ........................................................................................................................................................................ 34

4.3.2 H2O ................................................................................................................................................................................................................. 35


4.3.4 Alginate bulk ............................................................................................................................................................................................ 37

4.3.5 Interface ..................................................................................................................................................................................................... 38

5 DISCUSSION ................................................................................................................................................................................................................40

5.1 POLYSTYRENE SPHERES INSIDE CARRAGEENAN GEL .................................................................................................................... 40

5.2 FRAP EXPERIMENTS .................................................................................................................................................................................. 40

5.2.1 Concentration effect of sodium fluorescein...........................................................................................................................40

5.2.2 Carrageenan bulk ................................................................................................................................................................................... 41

5.2.3 Alginate bulk ............................................................................................................................................................................................. 41

5.2.3.1 Droplet method ................................................................................................................................................................................ 42

5.2.3.2 Internal method ............................................................................................................................................................................... 42

5.2.4 Interface ..................................................................................................................................................................................................... 42

5.2.5 Mixture of carrageenan and alginate ........................................................................................................................................ 44

5.3 RICS EXPERIMENTS ..................................................................................................................................................................................... 44

5.3.1 Optimal scanning rate ........................................................................................................................................................................ 44

5.3.2 H2O ................................................................................................................................................................................................................. 45


5.3.4 Alginate bulk ............................................................................................................................................................................................ 45

5.3.5 Interface ..................................................................................................................................................................................................... 45

5.4 FURTHER RESEARCH .................................................................................................................................................................................. 46

6 CONCLUSION .............................................................................................................................................................................................................. 47

7 REFERENCES .............................................................................................................................................................................................................. 49

ABBREVIATIONS

CLSM Confocal Laser Scanning Microscopy

FITC Fluorescein-5-isothiocyanate

FRAP Fluorescence Recovery After Photobleaching

GDL Glucono-δ-Lactone

HPC Hybrid Photon Counting

NA Numerical Aperture

PMT Photomultiplier Tube

PS Polystyrene

PSF Point Spread Function

RICS Raster Image Correlation Spectroscopy

ROI Region Of Interest

1

1 INTRODUCTION

Many pharmaceutical and food formulations are dependent on the control of diffusion properties for

their performance, for example the water or fat migration in respectively bread and chocolate or controlled

release formulations of numerous drugs. It is often desirable to control both release and uptake of molecules

inside soft materials as well. In addition, the free diffusion in liquid will change when mechanisms such as

obstruction or interactions with the medium are active. In a pharmaceutical context, it is also important to

determine the release of drugs out of their formulations and to measure the uptake in the human body. This is

why knowledge of mass transport dynamics and its measurement is very important.

Soft matter systems often have very complex microstructures. They can be heterogeneous, hierarchical,

and multiphase, which can have a large impact on the diffusion properties depending on variations within

temporal and spatial scales. A lot of interfaces are present in multiphase systems, thus making it pertinent to

investigate diffusion near them.

Many techniques have been established to determine the local mass transport of a substance, such as

fluorescence recovery after photobleaching (FRAP), single particle tracking (SPT), fluorescence correlation

spectroscopy (FCS), (raster) image correlation spectroscopy ((R)ICS) and many more. On the other hand, the

global mass transport inside a material can be measured as well by numerous techniques, like nuclear magnetic

resonance diffusometry (NMRd). Since the microscopy and mathematical models for analyzing of the data are

developing further as time passes, these techniques also become gradually more accurate. However, while most

diffusion experiments have been carried out in homogeneous materials, most materials have a rather

heterogeneous and complex structure. This makes it harder to measure the diffusion properties in these systems

and provides a need for stable model systems to develop new quantitative microscopy techniques.

In this thesis, the diffusion in hydrogels is investigated, starting with relative homogeneous systems

such as carrageenan and alginate gels and slowly introducing more heterogeneous models with interfaces.

Different techniques for measuring diffusion are applied to each system, more specifically RICS and FRAP. The

applied techniques are investigated and tested near interfaces to find out if there is any difference between the

diffusion inside the bulk and at these interfaces and if there is a relation to the difference in diffusion in function

of the distance from the interface. All systems are studied under the CLSM with the use of sodium fluorescein or

70kDa FITC-dextran as a fluorescent probe.

Chapter 1 of this thesis describes the theoretical aspects of the used materials and techniques. Chapter

2 provides an overview of the main objectives, while the used materials and methods are listed in chapter 3.

Finally, the results of all the experiments, the discussion of these results and the conclusion of this thesis are

described respectively in chapters 4, 5 and 6.

2

1.1 DIFFUSION

Diffusion is a passive transport mechanism (i.e. only thermal energy is needed), where the total mass

of a substance (solvents or solutes) moves in a medium, randomly or due to a concentration gradient (from

regions with higher concentration to regions with lower concentration) until an equilibrium is reached. Self-

diffusion is a process where the molecules collide with other molecules from their surroundings, causing a

movement (1). Fick’s second law of diffusion describes the diffusion process mathematically (1, 2) (1.1):

𝛿𝐶

𝛿𝑡= 𝐷

𝛿2𝐶

𝛿𝑥2 (1.1)

Where C: concentration (mole/m3)

t: time (s)

D: diffusion coefficient (m2/s)

x: position parameter (m)

The Stokes-Einstein equation describes the diffusion constant of a particle in an infinitely diluted solution (1.2)

(1, 2).

𝐷= 𝑘𝐵𝑇

6𝜋𝜂𝑟𝐻 (1.2)

Where D: diffusion coefficient (m2/s)

kB: Boltzmann’s constant (1.38e-23 m2.kg/(K.s))

T: temperature (K)

η: macroscopic dynamic viscosity (kg/m)

rH: hydrodynamic radius of a spherical particle (m)

1.2 HETEROGENEOUS MATERIALS

Many biomaterials are heterogeneous, as shown in the microscopic images of a carrageenan gel, gelatin

and maltodextrin, emulsion and chocolate as examples in FIG. 1.1. It is clear that many interfaces are present

inside these heterogeneous materials. In general, soft materials and cells are composed of various elements and

structures at different length scales that influence the mobility of diffusing particles. Microstructures can be

organized for example in a fractal, hierarchical or periodical manner (1), as illustrated in FIG. 1.2.

3

Hydrogels in particular can be classified as homogeneous and heterogeneous. Homogeneous hydrogels

show a random dispersion of mobile chains and pores in the gel network, for example PEG (polyethylene glycol)

and PVA (polyvinyl alcohol). Heterogeneous hydrogels on the other hand, show a gel network with a high polymer

interaction, with different properties in different directions. Examples of heterogeneous hydrogels are κ-

carrageenan, calcium alginate and agarose (3).

FIG. 1.1: Microscopic images as illustration of heterogeneous biomaterials at different length scales: a) 1% κ-carrageenan gel with 200 mM NaCl and 20 mM KCl; b) 4% gelatine and 7.3% maltodextrin labelled with RITC, where the bright phase shows the maltodextrin; c) bicontinuous emulsion with the oil phase visible; d) chocolate, where the bright phase represents the fat. The pictures are reproduced, with permission, from reference (2).

FIG. 1.2: Illustrations of possible organizations of heterogeneous microstructures: (a) fractal, (b) hierarchical or (c) periodical. The different length scales are shown by δ (2).

1.3 HYDROGEL

Hydrogels consist of cross-linked hydrophilic polymeric networks, swollen by the presence of water (4).

They are classified as “soft matter” and are an important medium in numerous food and pharmaceutical

applications to restrict and control the release of active substances, the texture and viscosity or to stabilize

products. The gel strand network acts as a sieve in which the molecules can move. It can cause a decrease in

diffusion transport of the particles, depending on the size of the particles and the size of the pores of the gel

strand network (2, 5). Gels consist mostly of at least two components: the solvent and a polymer that forms the

gel-network, giving the gel solid-like mechanical properties (6).

The two polysaccharide hydrogels used in this thesis are sodium alginate and κ-carrageenan. Due to

their biocompatibility, biodegradability, immunogenicity and nontoxicity, they are regularly applied in drug

delivery systems (7).

4

1.3.1 Carrageenan

Carrageenan is an anionic hydrocolloid seaweed gum collected from red algae (Rhodophyta). It is mainly

used as a stabilizer and gelling agent in foods and pharmaceuticals. Carrageenan is a linear polysaccharide as it

consists of many sulfated D-galactose residues. There exist many different types of carrageenan with various

solubility and gelation properties, depending on the manufacturing and chemical composition. The two most

common carrageenan types are known as κ-carrageenan (the gelling fraction) and λ-carrageenan (the non-

gelling fraction), both with a slightly different chemical composition. κ-carrageenan, which is used in this thesis,

consists of alternating α-(1-3)-D-galactose-4-sulphate and β-(1-4)-3,6-anhydro-D-galactose (8, 9) as illustrated

in FIG. 1.3a.

Through heating followed by cooling of the aqueous solutions of κ-carrageenan together with the

required cations (Na+, K+ or Ca2+, etc.) thermo-reversible cross-linked gels can be formed. In this thesis potassium

ions (K+) are used, which can bind on specific binding sites on the polymer (10). The mechanism involves a

conformational coil-helix transition: in solution-state and when heating, κ-carrageenan has a random coil

formation. However, by decreasing the temperature of the heated solution a conformational change occurs from

a random coil to a (double) helix formation as shown in FIG. 1.3b. These helices can aggregate in the presence of

cations (in adequate concentrations) by decreasing the repulsion of the negative sulfonic groups of κ-

carrageenan. The gel-network is now formed (9, 11, 12) (FIG. 1.3c).

The formation of the gel depends on the chemical structure and concentration of the carrageenan, the

nature of the cations and on the temperature (9). The mean pore size of the gel decreases with increasing

carrageenan concentration (13). Κ-carrageenan gels are interesting for determining the diffusion because the

gel structure can be fitted to meet the requirements (10).

FIG. 1.3: (a) Repetitive disaccharide in a κ-carrageenan chain; Formation and destruction of the gel-network by cooling and heating: (b) (double) helix formation and (c) further aggregation of helices in presence of cations (14).

(b)

(a)

(c)

5

1.3.2 Alginate

Alginate is generally collected from brown algae (Phaeophyceae), but it can also be produced by

bacteria. Like carrageenan, alginate is commonly used as a gelling agent and thickener in food and

pharmaceutical products, along with being suitable for biomedical applications. Alginate is a charged linear

copolymer polysaccharide composed of a variable ratio of (1–4) linked β-D-mannuronic acid (M) and α-L-

guluronic acid (G) (15), as illustrated in FIG. 1.4a.

It can form a gel in presence of most di- and trivalent cations (such as Ca2+) in low concentration. In

presence of calcium ions, as used in this thesis, an enhanced chelation between the hydroxyl groups of the poly-

G segments can occur via a two-step network formation mechanism. During this formation, a dimerization

process takes place, followed by a dimer–dimer aggregation of Ca2+ and G units, resulting in the formation of a

three-dimensional ionically cross-linked gel network (6), as illustrated in FIG. 1.4b. This process is also called the

egg-box model. Usually, if the calcium concentration increases at fixed alginate concentration, the gel network

becomes thicker (15). Alginate gels are typically nanoporous with an average pore size of 5nm (16).

In contrast to carrageenan, sodium alginate is a cold gelling agent that does not require heat to form a

gel network (6). Moreso, alginate gels are thermostable over the range of 0-100°C (17) and are for therefore

suitable to use in combination with carrageenan gels, which require a heating process up to 90°C (18).

FIG. 1.4: (a) Repetitive structure of G- and M-units in an alginate chain; (b) Network formation of the alginate gel in presence of calcium-ions (19).

1.4 POLYSTYRENE MICROSPHERES

Polystyrene is a synthetic aromatic polymer, composed of linked styrene monomers (20). Polymeric

micro-particles are suitable as a drug delivery system thanks to their controlled-release properties, limited size

and biocompatibility with body cells and tissue. Drugs can be coupled at the surface of the sphere or

encapsulated within the sphere. Although nanoparticles are more beneficial than microparticles, the latter have

been chosen in this thesis in order to improve the investigation of the diffusion at the interface.

(a)

(b)

6

Generally, negatively charged PS spheres, as used in this thesis, show a moderate gastrointestinal

uptake due to (low) affinity to intestinal tissues (21). The structure and dynamics of the matrix determine the

mobility of the spheres (22). In a 1% κ-carrageenan hydrogel for example, the PS spheres get completely

immobilized because of the high crosslinking of the polymers.

FIG. 1.5: Chemical structure of polystyrene (6).

1.5 CONFOCAL LASER SCANNING MICROSCOPY (CLSM)

The confocal laser scanning microscope (CLSM) is based on fluorescence and is used in this thesis to

form an image of the sample and to perform FRAP and RICS. The basic principle is visualized in FIG. 1.6a. The laser

can scan the sample pixel by pixel in the x- and y-direction and at different depths in the z-direction, which is

directed by the scanning mirrors. This can also result in three-dimensional images if the images from adjacent

focal planes (so called z-stack images) are added. It works by optical sectioning of the sample: it generates clear

images of thin sections in thick samples, without any need for physical sectioning (non-invasive method). Only

a bare minimum of sample preparation is required (23) thanks to this way of functioning. The CLSM can also be

used to detect dynamic changes in the microstructure such as phase separation or coalescence (24).

Different lasers with specific wavelength and intensity can be employed and controlled by the acousto-

optical tunable filter (AOTF). These wavelength and intensity settings can precisely and instantaneously be

modified at a high scan rate on a linked computer. The laser illuminates the sample and can be focused onto one

spot in the sample, the focal plane. The fluorophores in the sample will re-emit fluorescent light picked up by a

detector, for each pixel separately. The beam splitter separates the laser light and emitted fluorescent light from

the sample and sends it to respectively the sample and the detector. The detector can consist of a

Photomultiplier Tube (PMT) or a Hybrid Photon Counting (HPC) detector that amplifies its signal (with the gain

setting) and records the fluorescence intensity. The HPC also counts the photons pixel by pixel (24). It creates a

digital signal that can be processed by a computer, which generates the image. The scanning laser beam can be

used to apply a ROI of any size and shape for FRAP (25).

7

A considerable advantage is that the CLSM only allows the emitted fluorescent light from the focal point

(i.e. in-focus light) in the sample to be detected. Most of the out-of-focus light (i.e. from above or below the focal

point that receive much lower laser intensity) can be eliminated with the confocal pinhole so that it doesn’t

reach the detector. The majority of the out-of-focus light is reflected and not included in the final image as

illustrated in FIG. 1.6b. This increases the optical resolution (26). Since CLSM is a fluorescence microscope, the

samples will have to be fluorescent or contain a fluorophore to be visible (24).

FIG. 1.6: (a) Basic principle of the CLSM (24); (b) The confocal pinhole eliminates the out-of-focus light (26).

1.6 FLUORESCENCE

Fluorescence is a particular case of luminescence (27). Fluorescent molecules or fluorophores can

absorb light energy (photons, for example from laser light) with a specific wavelength and re-emit it typically

at a longer wavelength (lower energy) within nanoseconds (24, 28). Only when photons with sufficient and

correct energy are absorbed can a transition happen from the ground singlet energy state (S0) to a higher excited

energy state (usually S1 or S2). The fluorophore receives all the energy that the photon originally had, described

in the Planck-Einstein equation (1.3):

𝐸 = ℎ𝑐

𝜆 (1.3)

Where: E: energy (J)

h: constant of Planck (J.s)

c: speed of light (m/s)

λ: wavelength (1/m)

When the electron falls back to its original ground energy state, the fluorophore emits a photon on its

own as a means to lose its excess energy, as displayed in FIG. 1.7. Because the electron loses some energy by

(a) (b)

8

vibration, rotation and heat, the energy of the emission light is lower than the original absorbed light, which

corresponds to a longer wavelength (1, 24, 28). This difference between the absorption and emission

wavelengths is known as the Stokes Shift (28). The transition takes place swiftly. By emitting fluorescent light

the electrons fall back to the ground state (28).

FIG. 1.7: Example of a simplified energy (Jablonski) diagram illustrating the process of fluorescence (29).

1.6.1 Fluorescent diffusion probes

Fluorescent probes are fluorescent chemical components or fluorophores that can be used in

fluorescent optical techniques and experiments. They can directly be used or attached to molecules that are to

be studied under a fluorescent microscope (1). When determining the mobility properties, it is of great

importance that it does not greatly alter the diffusion and interaction of the molecule of interest (1, 30). For

FRAP experiments it is also necessary to have a fluorescent probe with a good balance between photostability

(stable against bleaching) and photoinstability (easy bleachable at low laser intensity) (2, 31).

1.6.1.1 Fluorescein

Fluorescein and its derivate Fluorescein-5-isothiocyanate (FITC) (FIG. 1.8a and FIG. 1.8b) are commonly

used hydrophilic fluorophores that emit green-yellow light (29, 31). Both fluorescein and FITC are evenly

photostable as instable (31) which is necessary in a FRAP experiment. They possess an opportune long absorption

maximum of approximately 494 nm and an emission maximum of approximately 521nm (29, 32), as described

in FIG. 1.8c.. The molecular weight of fluorescein and FITC is 376Da and 389Da (1), respectively.

Fluorescein is negatively charged and used in many FRAP experiments on its own as it can be bleached

relatively easily. The process of bleaching, however, is not a normal first order reaction, which complicates the

9

measurement of the diffusion coefficient (33). FITC on the other hand is often coupled with dextrans or proteins

(1, 31).

FIG. 1.8: (a) Chemical structure of Fluorescein (b) and its derivate Fluorescein-5-isothiocyanate (FITC); (c) Excitation and emission spectrum of FITC (29).

1.6.1.2 FITC-dextran

Dextrans are hydrophilic polysaccharides of anhydroglucose, typically defined by their high water

solubility and molecular weight (ranging mostly from 3kDa to 2,000kDa), inertness and low toxicity. They are

produced by Leuconostoc bacteria and commonly used as effective carriers for many fluorescent dyes, such as

FITC. They possess α-1,6-polyglucose linkages, which are resistant to cleavage by most glycosidases and are ideal

to use as live cell tracers for this very reason. Their net charge can vary, depending on the method of preparation

and the coupled fluorophore, but dextrans are mostly weakly anionic (32, 34). Due to the large range of molecular

weight, they can be used as a model for drugs, such as peptides and proteins (4).

As mentioned before, FITC is often coupled with dextran-molecules (FIG. 1.9), which gives an ideal

fluorescent probe for FRAP and RICS experiments. 70kDa FITC-dextrans are typically labeled with three to eight

dyes per dextran and are only weakly anionic (32, 33). The free diffusion of FITC-dextran depends on the ionic

conditions and temperature of the sample. 500kDa FITC-dextran shows phase-separation when used in a 1% κ-

carrageenan hydrogel, rendering it unusable in this thesis (5).

FIG. 1.9: Chemical structure of FITC-dextran. It is assumed that the attachment site of FITC (represented by *) is randomly associated with any free hydroxyl group of the dextran molecule (34).

(a) (b) (c)

10

1.7 OPTICAL TECHNIQUES TO MEASURE DIFFUSION

1.7.1 Fluorescence Recovery After Photobleaching (FRAP)

In a FRAP or photolysis experiment, a fraction of the fluorescent labels or fluorophores are

photobleached for a short period of time by irradiation with one or several high-intensity lasers (bleaching, t=0).

Photobleaching induces an irreversible loss of a molecule’s fluorescence ability due to the chemical interaction

of the fluorophore in the excited state with free oxygen (i.e. oxidation). This will cause an immediate decrease

of the fluorescence intensity in the bleached region of interest (ROI), as illustrated in FIG 1.10a. The fluorescence

intensity will however directly recover due to the diffusion of the bleached molecules out of the photobleached

region and the diffusion of the fluorescent molecules from surrounding unbleached areas into the ROI (t>0), as

shown in FIG. 1.10b. Typically a ROI in the range of 5 to 50µm in diameter or length is chosen for FRAP experiments

(1). The time evolution of this recovery can be monitored with the CLSM or another fluorescent microscope, from

which the diffusion rate can be measured on a micrometer scale (0.01 to 100µm2/s) (1).

This technique is very interesting as it is a non-invasive and very specific method to determine the

diffusion coefficient and interaction properties (molecular dynamics in general) of samples. The immobile

fraction of bleached molecules can also be determined by the difference between the fluorescence intensity

before the photobleaching and after the experiment (t∞) (1, 4). It is assumed that the photobleaching is

irreversible so that the fluorescent recovery is stated to nothing but the diffusion of the fluorophores (31).

FIG. 1.10: Simple presentation of a FRAP experiment: on t<0 a pre-bleach image is taken without any bleaching performed. On t=0 the circular ROI is fully bleached by the laser, causing the fluorescence intensity of the fluorophores to drop inside this ROI. The bleached fluorophores that lost their fluorescent ability are presented by the black spots in (b). Over time (t>0 and t∞) the bleached fluorophores will diffuse out of the ROI and un-bleached fluorophores from outside the ROI will diffuse inside the ROI, causing the fluorescence intensity to rise again. (a) Recovery curve of a FRAP experiment with the mean fluorescence intensity of the fluorophores inside the ROI in function of the time; (b) Illustration of the mechanism of the FRAP technique in relation to CLSM images over time (35).

(a) (b)

11

In order to carry out a FRAP experiment correctly, certain requirements must be met: All the fluorescent

probes (sodium fluorescein or FITC-dextran as used in this thesis) must be distributed evenly inside the samples.

The laser beam must also be able to pass through the sample and the used FRAP evaluation model and

experimental settings must be appropriate for the FRAP experiment (2). It is also important to use an objective

with a relative low numerical aperture (NA) to acquire a cylindrical bleaching profile, which is assumed in the

FRAP model (see 3.1.5), and gives a better bleaching as well (10). This way, only two dimensional lateral diffusion

has to be considered, as the bleaching generates no significant gradient in the z-direction (22). Another very

important criterion of a FRAP experiment is that a bleaching of 30% of the pre-bleach fluorescence intensity

should be achieved to have a good FRAP measurement (35).

1.7.2 Correlation spectroscopy

Fluorescence correlation spectroscopy (FCS) and imaging correlation spectroscopy (ICS) are important

correlation spectroscopy techniques for measuring molecular mobility. In FCS the fluctuations in the fluorescence

intensity of the observed diffusing fluorophores are analyzed, which correlates with the diffusion rate. On the

other hand, ICS is a type of extension (imaging analog) of FCS that provides better spatial coverage as it uses an

entire microscope image but is limited to rather slow diffusion (1, 36).

The fluorescent raster imaging correlation spectroscopy (RICS) combines the advantages of both

methods: spatial information from ICS and temporal information from FCS. It uses the raster-scan images of the

CLSM, where the scanning laser moves sequentially in the x-direction for a much shorter time than the adjoining

pixels in the y-direction, as illustrated in FIG.1.11. The fluorescence intensity is measured one pixel at a time,

where ‘pixel’ stands for a localized intensity measurement. The laser starts measuring at the top left pixel from

left to right. When the top row of pixels is collected, the laser starts collecting the second row from the left to

the right. This process goes on until the entire image is obtained. It generates temporal information (since each

pixel is collected at a different time) on the diffusion of the molecules of every single image in a range of seconds

(images), milliseconds (scan lines) and microseconds (pixels) (1, 36, 37).

Using RICS analysis, the diffusion coefficient and concentration is determined from the images, starting

with background subtraction followed by image correlation. The image autocorrelation is estimated in all

frames, averaged and fitted relating the correlation to the particle concentration and the diffusion coefficient

using the equation described in literature (38, 39). Typically a diffusion coefficient on a micrometer scale can be

measured, like FRAP (1).

The biggest difference with FRAP is that in RICS no photobleaching is performed and that, besides the

diffusion coefficient, RICS can also measure the concentration of fluorescent probes in mediums (39) It is also

possible to generate a two-dimensional diffusion coefficient map with this technique (1, 36, 37).

12

However, RICS is sensitive to experimental settings. For example, if the scanning rate is too high, but

the diffusion is slow the estimate of the diffusion coefficient will most likely be less accurate (38). It is therefore

interesting to investigate the best scanning rates for the sample, as carried out in this thesis. A normal RICS

method only accepts squared ROI. Another recently developed method exists that allows the use of ROIs with an

arbitrarily shape (of any shape), known as ARICS or Arbitrary-Region RICS (33).

FIG. 1.11: Movement of the scanning laser in a raster scan by CLSM, where τp and τl are respectively the scanning

time between pixels in x- and y-direction (37).

13

2 OBJECTIVES

Numerous types of solutions and gels with different fluorescent probes have been used as a model to

study the mobility of molecules in the past, but there is a need for a stable and heterogeneous model. In this

project the point of focus is the development of stable and flexible models for heterogeneous structures that

are easy to control. The second purpose of this thesis is to increase the understanding of the mass transport near

interfaces and in the bulk of two different phases in a heterogeneous model. In order to control, design and

optimize the diffusion properties of a substance, it is important to investigate the mass transport in

heterogeneous materials and its restrictions by the structure and the heterogeneity of the system on different

length scales.

Following tasks were formulated to accomplish the goals of this project:

Evaluation of a 1% κ-carrageenan hydrogel with 50µm PS spheres as a model system.

Determination of the effect of different concentrations (from 20ppm to 200ppm) of sodium fluorescein

on FRAP experiments in a 1% κ-carrageenan gel.

Evaluation of the use of sodium fluorescein and 70kDa FITC-dextran as a fluorescent diffusion probe in

a 1% κ-carrageenan gel.

Evaluation of a 1% κ-carrageenan hydrogel with a 2% or 4% alginate gel as a model system.

Evaluation of different production techniques for alginate gels.

Measurement of the diffusion coefficient of the fluorescent diffusion probe inside a carrageenan gel

and alginate gel separately using both FRAP and RICS techniques. Measurements of the probe inside

distilled water can be used as a reference.

Evaluating the use of different sizes (length of 50µm to 5µm) of rectangle ROIs in FRAP experiments

together with different zoom functions.

Determining the optimal scanning rate for the measurement of the diffusion coefficient of the probe in

a carrageenan gel and evaluating different zoom functions using RICS.

Measurement of the diffusion coefficient of the probe near the interface of a carrageenan and alginate

gel using both FRAP and RICS techniques.

Because the PS spheres have a solid and impenetrable surface, the boundaries will be extremely sharp,

resulting in a well-defined and limited interface. This will be the more homogeneous model. In the model with

the alginate gel inside the carrageenan, the interface will be less well defined and not limited because of the

penetrable and diffusible alginate gel. This will be the more heterogeneous model. FRAP and RICS will be

14

performed on each model to determine the diffusion and to test each technique at the interfaces and bulk. For

the FRAP and RICS experiments near the interface, each observed ROI will always be placed at the interface of

the two gels and gradually further away from the interface in each hydrogel phase. Measurements of the

diffusion coefficient will also be made in the bulk-phase of the gels to investigate any possible difference in

mobility between the experiments near the interface.

The purpose of this project is to increase the understanding of diffusion properties inside

heterogeneous biomaterials. The structure designs of the systems used in this thesis can be employed as a model

for real food or pharmaceutical products in order to control the diffusion properties of these preparations, like

controlled release preparations.

15

3 MATERIALS AND METHODS

All gels and solutions were made with distilled and ultrafiltrated water from a NANOpure system

(Barnstead/Thermolyne, Dubuque, IA, USA). All gels were made and stored in glass vials of 5 mL with a plastic

snap-cap (Hecht-Assistent, Sondheim, Germany). The observed samples mostly contained 7 to 8µL of the

hydrogel or solution in a Secure-Seal™ adhesive spacer (Molecular Probes, Invitrogen, Eugene, OR, USA) that is

absorbed in a sandwich manner onto two cover-glass slides giving perfectly defined dimensions of the sample:

120mm in depth and 9mm in diameter. It also avoids evaporation and convection (33) and restricts possible flow

(5). Some samples were observed in a metallic cup instead, with a cover glass placed on top. The carrageenan

hydrogel was poured into the metallic cup and cooled down, giving a flat surface. The alginate bulk gel, produced

by the internal method was instead cut and placed inside the metallic cup, as the gelling process is different

from the carrageenan gel. Every sample was prepared twice to establish reproducible experiments. Generally,

the samples were analyzed a few hours after the preparation, to ensure the obtaining of the final

microstructures after the gelation process.

3.1 HYDROGEL

3.1.1 Carrageenan gel

First a stock solution of 500mM (0.5M) KCl (Merck KGaA, Darmstadt, Germany) was made by dissolving

1.86g KCl in 50mL distilled water. 5mL of a 1% w/v κ-carrageenan hydrogel was made by adding 0.05g κ-

carrageenan (Danisco Cultor, Grindsted, Denmark) in a closable vial of 5mL with 5mL of total solution. The total

solution of 5mL consists of 100mM (0.1M) KCl, the required volume of the desired concentration of the

fluorescent diffusion probe and distilled water. The mixture was then heated under stirring at 90ᵒC in a warm

water bad for 15 minutes, while closing the vial to prevent evaporation. It was then cooled down to room

temperature (20ᵒC) to initiate the gelling-process (18). The final sample must always be covered by aluminum

foil when not in use. This prevents possible bleaching of the fluorescent probe by stray light (40, 41).

Because of the inability to pipet the final hydrogel into the secure seal spacer on the cover glass, the

hydrogel must be pipetted right after the heating of the mixture, when it is still in liquid phase. The pipet points

were put into an oven on 90ᵒC for a few minutes to be on the same temperature as the hydrogel. The sample

was then pipetted into the closed seal on the cover glass as quickly as possible to avoid clotting of the gel in the

pipet.

16

3.1.2 Alginate gel

A stock solution of 20mL 2% and 4% (w/v) sodium alginate solution was made, by gradual adding respectively

0.4g and 0.8g sodium alginate (Aldrich, Sigma-Aldrich, St. Louis, USA) to 20mL distilled water at room

temperature under vigorous stirring until it completely dissolved. If necessary the dispersion could also be

warmed up to 80ᵒC in a water bath under stirring while covering it to avoid evaporation (42). For the FRAP and

RICS experiments, a 4% alginate solution was made with respectively 100ppm and 80nM 70kDa FITC-dextran.

3.1.2.1 Droplet method

The alginate gel-beads were prepared by using the dripping technique, as shown in FIG. 3.1. the sodium

alginate solution was released dropwise from a stainless steel needle and collected in a 0.5M CaCl2 solution

(using calcium chloride dehydrate, CaCl2.2H2O, Merck KGaA, Darmstadt, Germany) (43). This way the calcium ions

diffuse from the outside of the drop towards the center of the bead,

causing the gelling of the outside of the drop to be faster than the inside.

The alginate particle size depends on the size of the initial drop. This way

the beads of 500µ to 200µm could be formed. To produce smaller drops,

a tweezer is used to take the small alginate drop from the needle and

place it in the CaCl2 solution. These beads were small enough to be placed

in a closed seal spacer. For the FRAP interface experiments 5 of the

smallest beads were placed in a closed seal spacer on a sample glass. The

heated carrageenan gel was pipetted onto the cover glass with the beads

and finally a cover glass was placed on top of the sample. Smaller microbeads could in theory be produced with

the modified emulsification method as reported in some articles (44). This is however a method to produce solid

core-microbeads instead of gel-beads and therefore not appropriate for this thesis.

3.1.2.2 Internal method

To produce a relatively homogeneous alginate gel, the so-called internal method was employed by

controlled release of calcium. In this method, insoluble calcium carbonate was dispersed in the alginate solution.

Next, a slowly hydrolyzed acid glucono-δ-lactone (GDL) is added to the system. GDL gets deprotonated over time

by slow hydrolysis of the lactone causing the calcium salt to be solubilized and the gelling process to start.

Because of the fast chelation of calcium alginate, it is necessary to slowly introduce the calcium ions in the

mixture (15).

FIG 3.1: Preparing the alginate gel-beads by using the dripping technique in the CaCl2 solution.

17

2mL of a 4% sodium alginate solution was prepared with the required volume of the stem solution of

70kDa FITC-dextran to obtain a final concentration of 100ppm in the mixture. CaCO3 (Acros Organics, Thermo

Fisher Scientific, New Jersey, US) and GDL (Jungbunzlauer S.A., Basel, Switzerland) were introduced to the

alginate and FITC-dextran mixture. A final concentration of 30mM CaCO3 and 60mM GDL was obtained. The vial

was sealed and covered with aluminum foil and stored at room temperature for 2 days prior to use. It is necessary

to always maintain a CaCO3 to GDL molar ratio of 0.5 to prevent changes in pH value (15, 45).

For the RICS experiments in the bulk of the alginate gel and at

the interface between the alginate and the carrageenan gel, the alginate

gel was cut into small pieces and placed in a metallic cup in random order

with a sample glass on top of it. For the interface experiments, the

heated carrageenan gel was quickly poured between the alginate gel

pieces, to create the interfaces between the two gels, as shown in FIG.

3.2. Both gels were prepared with the 70kDa FITC-dextran concentration

of 80nM.

3.1.3 Mixture of Carrageenan and Alginate

A mixture of 2mL 1% carrageenan gel and 1mL 4% alginate gel was made, both with a 70kDa FITC-dextran

concentration of 100ppm. The alginate mixture was prepared using the internal method protocol as described

in 3.1.2.2 on the same day as the carrageenan gel. The carrageenan mixture was prepared using the protocol

described in 3.1.1, without heating. The alginate mixture was then mixed with the carrageenan gel while heating

and stirring the entire solution. After heating the mixture was cooled off to form the carrageenan gel. The

mixture was kept at room temperature to complete the formation of the alginate gel.

3.2 POLYSTYRENE MICROSPHERES

The non-fluorescent PS microspheres with carboxylate surface modifications (Phosphorex, Inc.,

Hopkinton, MA) were selected in this thesis with a diameter of 50μm, to observe the diffusion at the boundary

inside a 1% carrageenan gel. The hydrophilic carboxylate modifications were chosen to distribute the spheres

easier in the hydrogel. The particular diameter of 50µm was selected to have a well-defined surface and to fit

inside the closed-seal samples. Smaller particles might lead to more undefined interfaces and an undesirable

curved surface. Another reason for this diameter is that the used ROIs in the FRAP experiments (50µm to 5µm in

length) should not be bigger than the bead itself.

The spheres must first be dispersed in some distillated water and vortexed for 5 minutes to ensure the

microspheres are fully distributed in the mixture. The PS spheres were then added in the 1% κ-carrageenan

FIG. 3.2: 4% alginate (rather opaque) and 1% carrageenan gel (transparant) with 80nM 70kDa FITC dextran in a metallic cup.

18

mixture with a concentration of 1% and 0.1%. Instead of heating up to 90ᵒC, the total mixture was now heated

under stirring at 80ᵒC for 15 minutes. Heating up the PS spheres above this temperature might lead to problems

as the glass transition temperature (Tg) is 94̊C, according to the manufacturer (20).

3.3 FLUORESCENT DIFFUSION PROBES

For FRAP experiments sodium fluorescein (Fluka, Sigma-Aldrich, St. Louis, USA) and 70kDa FITC-dextran

(Invitrogen, Eugene, Oregon, USA) were used as a diffusion probe. For RICS experiments only 70kDa FITC-dextran

is used. The probes must always be dissolved in distilled water first to ensure a homogeneous and unhindered

distribution in the water and afterwards in the sample (final hydrogel-solution mixture) as well. Otherwise the

diffusion in the gel can be affected by the presence of a possible concentration gradient of the probe. Initially a

more concentrated stem solution was made in distilled water (e.g. 500ppm) to fully allow a homogeneous

distribution. The probe solutions were then added to the vial of the mixture always in such volumes to reach the

desired final probe concentration (e.g. 100ppm) (5) together with the desired volume of the KCl stem solution

and the distilled water. Before adding the carrageenan, the mixture was always vortexed for a couple of minutes

for the same reason as above. To prevent the bleaching by stray light, the stock solution must always be covered

with aluminum foil as well (40, 41).

The chosen concentrations of the fluorescent probes are all well-within the concentration range in

which a linear ratio is shown between the fluorescence intensity (signal) and the concentration of the

fluorescent probe (41). This is required since the used analysis model assumes that the fluorescence intensity of

the used fluorescent probes is more or less linear depending on the concentration, see 3.5.1.

3.3.1 Fluorescein

A stock solution of 500ppm was made by dissolving 25mg sodium fluorescein in 50mL distilled water.

A concentration of 20ppm, 50ppm, 100ppm and 200ppm were obtained from this solution. These concentrations

were used to determine which concentration is the most optimal to use in a FRAP experiment.

3.3.2 70kDa FITC-dextran

A stock solution of 400ppm was made by dissolving 4mg 70kDa FITC-dextran in 10mL distilled water.

As mentioned by the manufacturer, the aqueous stock solution of FITC-dextran should be stored at 2-6ᵒC when

not in use (6). From this solution, a concentration of 50ppm and 100ppm were obtained for the FRAP

experiments. However, for the RICS experiments, lower concentrations of the probe were used, such as 20nM,

50nM and 80mM. The fluctuations in fluorescence intensity when the fluorescent probe enters or leaves the ROI

are registered with the RICS technique, which is only possible if the concentrations of the probe are low enough

19

(in the nanomolar concentration range). If higher concentrations are used, the fluorescence intensity would be

constantly high, resulting in no fluctuations to be seen. This concentration corresponds to one molecule per

detection volume (46). The concentrations of 70kDa FITC-dextran were chosen as found in some literature, where

sodium fluorescein was used to determine the concentration range with the lowest associated errors (38) and

as described in some articles with 500kDa FITC-dextran as a probe for RICS experiments (42).

3.4 CLSM

All FRAP experiments were performed on a Leica SP2 AOBS (Acousto-

Optical Beam Splitter) CLSM as showed in FIG. 3.3 and all RICS experiments on

a Leica SP5 CLSM (Heidelberg, Germany). For all experiments, the built-in

488nm emission argon (Ar-ion, Ar/ArKr) laser of the CLSM was operated for

imaging and bleaching. The PMT and HPC detector were both set at the

wavelength range of 500nm to 650nm (ideally for sodium fluorescein and

FITC dextran, see 1.7). All diffusion coefficients were calculated using Matlab

(MathWorks, Natick, MA).

3.5 FRAP

The used objectives were PL FLUOTAR 10x 0.30NA dry (Leica, Heidelberg, Germany) and HCX APO L 20x

0.50NA Water immersion U-V-I objective (Leica, Heidelberg, Germany). An image format of 256x256, gray scale

and depth of 12bit was chosen for every experiment without the use of a beam expander (it was kept at 1). The

pinhole was kept at 20µm. All other images apart from FRAP experiment and FRAP images were always in

1024x1024 format. For bleaching, 100% of the maximum power of the laser was used, with a zoom-in during

bleaching, to maximize the photobleaching (1). For most FRAP experiments 50 pre-bleach images were made,

with one bleaching frame and 100 to 150 post-bleach images. For the 10µm ROI however, it was generally

required to take 2 bleach images in order to achieve an adequate bleaching in relation to the pre-bleaching

fluorescence intensity. In general, the AOTF was set on 4% laser intensity, with the total laser power set on

approximately 40% for the used CLSM.

The optimal scanning speed of FRAP experiments depends on the diffusion coefficient of the analyzed

probe. But the higher the scanning speed, the lower the image quality will be, leading to more noise in each pixel

(35). The scanning speed of 800Hz was chosen in the unidirectional scanning mode for every experiment,

corresponding to a time of 0.5s per frame. When using the 20x objective, a zoom factor of 4 was used for the

50µm ROI, 8 for the 20µm and 16 for the 10µm ROI, yielding a pixel size of respectively 732.42nm, 336.21nm and

183.11nm. In every FRAP experiment the ROI was carefully placed in the center of the image (47).

FIG. 3.3: The Leica SP2 AOBS CLSM used in this thesis

20

The depth in the sample was mostly kept at 30µm under the cover glass for every experiment, unless

explicitly noted, to decrease the possible effect of inner filtering (1). Most FRAP experiments were repeated a

couple of times at different random positions in the same sample to receive reproducible results. In addition, for

every FRAP and RICS measurement in the bulk gels, the sample was always examined in three dimensions before

the start of the experiment to ensure that the diffusion in the ROI is not influenced by possible boundaries

nearby (25, 35).

3.5.1 Analysis

The model used for the analysis of the FRAP data was the rectangle FRAP (rFRAP) model, a newer

alternative pixel-based model, where a rectangular bleached area (ROI) can be used instead of the more usual

circular ROI. It is a very fast and practical method that reckon with the full temporal and spatial information of

the images and is not restricted by the ROI size, giving a maximum flexibility (25). This model can thus use ROI

with a length of 10µm or lower as it is valid for all rectangle sizes and aspect ratios. FRAP experiments can

therefore be performed closer to the interface, which is interesting in this thesis. It also not affected by diffusion

during the bleaching (1). In a pixel-based model every pixel in the images is used for extimating the parameters

(41).

One drawback is that it is only valid for a limited amount of photobleaching: as it assumes a linear

photobleaching process no more than 50% of bleaching should be carried out on the ROI (1, 25). It also assumes

that the fluorescence intensity of the used fluorescent probes is more or less linear depending on the

concentration. This is only valid if the concentration of the fluorescent probe (Na2Fluorescein or FITC-dextran) is

low enough (41). The calculations, recovery curves and residual plots of the FRAP experiments were analyzed

with an in-house developed Matlab script (48) based on (49).

3.6 RICS

For every RICS experiment 100 frames were taken, which is generally sufficient for a good S/N ratio (38).

The format of each frame is 512x512 and 8Bits and the time-mode xyt was used. A 1.2NA 63x water immersion

HCX PL APO objective (Leica, Heidelberg, Germany) was used. A zoom factor of 7, 12 and 16 was utilized for most

experiments, yielding a pixel size of respectively 68.78nm, 40.12nm and 30.09nm. The lights of the room where

the measurements were performed needed to be turned off, to protect the sample from tray light during RICS

experiments. A HPC detector was used instead of the PMT for the experiments. The PMT detector was however

used to find the desired area in the sample, both set in the wavelength range of 500nm to 650nm.

To make it practically achievable, 400 frames were used in most RICS experiments. By increasing the

laser intensity, a higher photon count was acquired. However, to prevent photobleaching, the AOTF was set on

21

4% laser intensity for all experiments, giving a photon count of approximately 10 for each frame. The total laser

power was set on 20% for the used CLSM. The HPC detector, which is more sensitive, compensates for the low

laser power (39).

The depth in the sample was kept at 10µm under the cover glass. A series of experiments with a scanning

rate of 10Hz to 1000Hz were performed. In the carrageenan sample a FITC-dextran concentration of 20nM, 50nM

and 80nM were used, in the alginate and water sample only the 80nM concentration. It became clear that the

20nM and 50nM concentration of the probe in the sample needed too much laser intensity (i.e. 10% to 20% laser

intensity) by the AOTF to achieve a sufficient photon count in the RICS experiments, which is not favorable

because of the higher risk of bleaching. The probe concentration of 80nM required less laser intensity and was

therefore chosen for all next experiments.

The whole image was always selected for data analysis. A Matlab script kindly provided by Prof. Marcel

Ameloot and Dr. Nick Smisdom from Hasselt University (UH RICS program, Belgium) was used to analyze the RICS

experiments and estimate a diffusion coefficient using the calculated correlation function and residual plot as

described in (50, 51).

22

4 RESULTS

At first, the model system of a 1% carrageenan hydrogel with polystyrene spheres is evaluated.

Preparations with sodium fluorescein or 70kDa FITC-dextran as a diffusion probe are investigated to define the

best fluorescent probe to use. Next, the diffusion in rather homogeneous preparations is studied at micrometer

scale: 1% κ-carrageenan gels and 2% to 4% alginate gels. Finally the diffusion in more heterogeneous

preparations are investigated, i.e. the carrageenan gel together with the alginate gel in the form of beads or

bulk gel. In particular, the diffusion near the interface is studied. In this chapter, the FRAP experiments are listed

first, followed by the RICS experiments. It is important to note that all diffusion coefficients are described as a

mean of the diffusion coefficient of the performed experiments together with the calculated standard deviations

as error bars in the graphs. All listed images are shown in self-chosen colors (not the real colors of the

preparations) and generated with the CLSM.

4.1 POLYSTYRENE SPHERES INSIDE CARRAGEENAN GEL

FIG. 4.1: CLSM images of 1% carrageenan gel with 1% 50µm PS-spheres and 100ppm Na2Fluorescein. A scale bar of 100µm (left) and 50µm (right) is specified.

FIG. 4.1 shows the CLSM images of the PS-spheres inside the 1% κ-carrageenan gel with sodium

fluorescein as a fluorescent diffusion probe. The green background represents the carrageenan gel. The black

and dark or very bright circles represent the PS-spheres. Light-scattering of the laser light of the CLSM on the

spheres causes the boundaries of the spheres to appear unclear. It seems that the chosen concentration of PS

spheres is too high, as it would be difficult to measure the diffusion coefficient at the boundaries with FRAP as

the spheres are so close to each other as seen in FIG. 4.1. In order to remove the possibility of the diffusion being

altered by other PS spheres near the bleached area, a lower concentration of PS spheres inside the carrageenan

gel is chosen, namely 0.1%. This seems to be a more optimal concentration because the spheres are much further

away from each other, as represented in FIG. 4.2.

23

FIG. 4.2: CLSM images of 1% carrageenan gel with 0.1% 50µm PS-spheres and 100ppm Na2Fluorescein. A scale bar of 100µm (left) and 50µm (right) is specified.

4.2 FRAP EXPERIMENTS

The CLSM and the Matlab script shows for each FRAP experiment the recovery curve with the mean

fluorescence intensity of all pixels in the ROI of each image (a.u.) in function of the time (s). Some recovery curves

are listed in this chapter. The green line in the recovery curve of the CLSM refers to this mean fluorescence

intensity of all pixels in the ROI of each pre-bleach, bleach or post bleach image taken at different times. The

first straight line at the beginning of the curve describes the calculated intensity of the images before the

bleaching (pre-bleach). The drop of the fluorescence intensity presents the bleaching and the recovery of the

curve is described by the fluorescence intensity of the images right after the bleaching (post-bleach) as

illustrated in FIG. 1.10a. The red circles in the recovery curve by the Matlab script represent the experimental data

of the post-bleach images only and the solid black line illustrates the corresponding fit of the rFRAP model. It is

interesting to note that the recovery curves of the CLSM illustrate the bleaching extend in relation to the pre-

bleaching intensity, while the recovery curves of the Matlab script show the fitting of the model with the real

data of the FRAP experiments.

4.2.1 Concentration effect of sodium fluorescein

All experiments are performed with a circular ROI of 50µm in diameter. The diffusion coefficients are

not calculated. For the concentration of 20ppm (FIG. 4.3A) of sodium fluorescein, the recovery graph of the FRAP

experiment shows a very weak fluorescence intensity and a few fluctuations appear to overshadow the signal.

The concentration of 50ppm (FIG. 4.3B) shows a slightly better bleaching than the 20ppm, but the bleaching

extend is less than 30% of the pre-bleach fluorescence intensity, as well. The concentrations 100ppm (FIG. 4.3C)

and 200ppm (FIG. 4.3D) show a better bleaching profile. It appears that only a concentration of 100ppm of

sodium fluorescein can achieve a bleaching extend of more than 30% of the pre-bleach fluorescence intensity.

24

FIG. 4.3: The recovery curves displayed by the CLSM. All investigated samples are a 1% κ-carrageenan gel with Na2fluorescein as a diffusion probe. A circular ROI of 50µm in diameter is used. The concentration of the probe is (A) 20ppm; (B) 50ppm; (C) 100ppm; (D) 200ppm.

4.2.2 Carrageenan bulk

The results of the FRAP experiments inside the 1% κ-carrageenan gel are listed in FIG. 4.4. The ROI with

a length of 10µm has a lower standard deviation because only 2 experiments had a good recovery curve and they

happened to be close. For the ROI with a length of 20µm, on the other hand, 7 good recovery curves were found,

which led to a higher standard deviation. The diffusion coefficients are relatively consistent for the two ROI sizes.

FIG. 4.4: Average diffusion coefficients of 100ppm 70kDa FITC-dextran in 1% κ-carrageenan gel with a rectangle ROI of 10µm and 20µm in length.

An example of the recovery curve of the CLSM of a FRAP experiment and the recovery curve of its analysis

by the Matlab script is listed in FIG. 4.5. In contrast to the bleaching of sodium fluorescein in previous

experiments, bleaching appears to be easier with 70kDa FITC-dextran as a fluorescent probe in the carrageenan

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gel. Even with just one bleaching frame and a lower ROI size (20µm instead of the 50µm with sodium fluorescein

in the first experiments described in 4.2.1), a bleaching of more than 30% of the pre-bleach fluorescence intensity

is achieved.

FIG 4.5: A FRAP experiment inside a 1% κ-carrageenan hydrogel using a rectangle ROI with the length of 20µm. (A) the recovery curve formed by the CLSM; (B) the recovery curve plotted by the Matlab script.

The analysis of these FRAP experiments of 70kDa FITC-dextran inside a carrageenan gel using a binding

and diffusion model shows relatively extreme values of kon and koff (generally in the order of respectively 10-4

and 101).

4.2.3 Alginate bulk


The first image in FIG. 4.6 is taken directly after inserting 2% alginate beads in a 50ppm 70kDa FITC-

dextran 1% carrageenan gel, without dissolution of the 70kDa FITC-dextran in the alginate bead beforehand.

After approximately 30 minutes, it is clear on the second image that the probe distributes from the carrageenan

hydrogel into the alginate bead, and this in a heterogeneous matter. Next, the alginate beads are kept in the

carrageenan gel for 2 days and the distribution of the fluorescent probe is evaluated again. It appears that the

fluorescent probe never fully distributes equally between the carrageenan gel and the alginate bead.

Because of this distribution process, it is assumed that a higher concentration of FITC-dextran is needed,

i.e. 100ppm instead of 50ppm. Also, in order to have a higher probability to have a difference in diffusion

coefficient between the alginate and the carrageenan gel, a higher concentrated alginate bead is made for the

FRAP and RICS experiments, i.e. 4% instead of 2%. Furthermore, to perform better FRAP experiments inside the

alginate beads, the beads are to be made with 100ppm ́70kDa FITC-dextran.

A B

26

FIG. 4.6: 2% alginate bead (represented as the dark circle on top of the image), without any FITC-dextran in 1% κ-carrageenan gel with 50ppm FITC-dextran, in a metallic cup. At first no probe is present inside the alginate bead (left). After approximately 30min the fluorescent probe starts to diffuse inside the alginate bead in a heterogeneous manner (right). Scale bar is 200µm left and 100µm right.

It appears that the alginate beads are slightly bigger than 120µm, which is the depth of the closed-seal.

When the beads are placed inside the closed-seal sandwiched sample glasses, they will be deformed because of

the pressure, leading to a more compact alginate bead as seen in FIG. 4.7.

FIG. 4.7: Deformation of an 4% alginate bead in the center of the image with 100ppm FITC-dextran inside, in a 1% κ-carrageenan closed seal sample (left). Right is a zoomed-in image to visualize the heterogeneity inside the bead. Scale bar is 200µm left and 50µm right.

The alginate drop is very heterogeneous and compact, which makes it very hard to perform FRAP

experiments inside this bead. The attempted FRAP-experiments were not successful and resulted in variable and

strange recovery curves. A different sample with a big alginate drop was made inside a metallic cup, giving better

recovery curves. The results of the experiments are listed in FIG. 4.8.

27

FIG. 4.8: Average diffusion coefficients of 100ppm 70kDa FITC-dextran in 4% Alginate gel produced by the droplet method with a rectangle ROI of 20µm and 50µm in length.


A more homogeneous alginate gel is obtained with the internal method. It appears that the gel made

with the internal method is less heterogeneous than the gel made with the droplet method. However, there is

still a certain degree of inhomogeneity visible inside the alginate gel as seen in FIG. 4.9, though less

heterogeneities are visible in contrast to the alginate drop as seen in FIG. 4.7.

FIG. 4.9: CLSM image a 4% alginate gel with 100ppm 70kDa FITC-dextran, made with the internal method. Scale bar is 200µm.

It is experienced that the bleaching of 70kDa FITC-dextran in the FRAP experiments is easier in contrast

to the bleaching of the probe inside the carrageenan gel. A relative low diffusion coefficient of the probe is

measured with FRAP in the alginate gel, produced by the internal method, as listed in FIG. 4.10.

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FIG. 4.10: Average diffusion coefficients of 100ppm 70kDa FITC-dextran in a 4% alginate gel produced by the internal method by FRAP using a rectangle ROI of 10µm and 20µm in length.

4.2.4 Interface

All the FRAP experiments at the interface are carried out in a 1% κ-carrageenan hydrogel with 100ppm

70kDa FITC-dextran, near the interface of 4% alginate beads without 70kDa FITC-dextran, as shown with two

CLSM images in FIG. 4.11. The sample is examined 24 hours after its production. The experiments started with the

placement of the ROI relative far away from the interface to gradually closer by.

FIG. 4.11: 4% alginate bead (black area, C) inside a 1% carrageenan gel (lighter (A) and slightly darker areas (B)) in a closed seal sample. The concentration of 70kDa FITC dextran in the carrageenan gel is 100ppm. Scale bar is 100µm (left) and 200µm (right).

It appears that the carrageenan show lighter and darker phases inside the closed seal samples, in

contrast to the samples in the metallic cups. All FRAP experiments are performed inside the lighter carrageenan

areas since these phases are bordering with the alginate beads.

FIG. 4.12 shows the results of the FRAP experiments executed with different sizes of ROI. The average

diffusion coefficients with their standard deviations are plotted in function of the distance of the chosen ROI

from the interface.

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29

FIG. 4.12: Average diffusion coefficients of 100ppm 70kDa FITC-dextran in 1% κ-carrageenan gel in function of the distance of the interface with the 4% alginate bead. A rectangle ROI of 10µm, 20µm and 50µm in length are used.

There appears to be a somewhat increasing trend in diffusion of the probe in the carrageenan bulk when

placing the ROI further away from the interface with the alginate bead. The measured diffusion coefficients at

the range of 30µ to 100µm from the interface are relatively consistent. It is interesting to notice that the ROI of

50µm in length shows less noise, but measures the average diffusion coefficient as the ROI covers a relative

large distance, also illustrated in FIG. 4.13. It gives the diffusion coefficient of the probe in a bigger fragment of

the structure. The ROI of 10µm in length, on the other hand, shows more noise but covers less distance and shows

the diffusion coefficient of the probe in a more localized region in the hydrogel.

FIG. 4.13 is an illustration of the performed FRAP experiments near the interface, with the sizes of the

ROI in function of the distance from the interface. The distance from the interface should be interpreted as the

the distance from the interface to the left border of the ROI for all experiments. The rectangles represent the

ROIs according to their sizes. All ROIs are placed inside the carrageenan gel. FIG. 4.14 is an illustration of the

experiments shown with the real CLSM images with the bleached ROI of 20µm in length.

As an example of the FRAP experiments near the interface of the 4% alginate bead inside the 1%

carrageenan gel, some grayscale images of the experiments are listed in FIG. 4.15 for each ROI size used, as well

as the recovery curves of the CLSM and the recovery curve formed with the Matlab script in FIG. 4.16.

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30

FIG. 4.13: Illustration of the FRAP experiment near the interface with the ROI size in function of the distance from the interface. Each rectangle ROI of 10µm, 20µm and 50µm in length is placed inside the carrageenan gel with a distance far away from the interface to closer to the alginate bead.

FIG. 4.14: An example of CLSM images of a general FRAP interface experiment with a rectangle ROI of 20µm inside the carrageenan gel (shown as red in the image, A) with a distance far away from the interface to closer (from left to right) to the alginate bead (shown as green in the image, B). The ROI is illustrated as a white rectangle, always placed in the center of the image. Scale bar is 25µm.

A zoom-in during bleaching is carried out for each FRAP experiment, which explains the bigger size of

the ROI in bleaching frame in FIG. 4.15. The gray background represents the carrageenan gel, the white (t=0) and

dark (t>0) rectangles display the bleaching of 70kDa FITC-dextran in the sample. The fluorescence intensity of

the probes in the ROI recovers by diffusion of the probes as shown by the dark pixels fading away in the next

images (t>>0). The bleaching of the sample using the ROI with size 50µmx50µm is much clearer and it takes a

longer time for the fluorescence intensity to recover in contrast to the smaller ROI sizes.

A

B

A

A

B

31

In the post-bleaching frame 10s after the bleaching, there are still some bleached probes present in the

ROI, in comparison with the smaller ROI sizes. The fluorescence intensity in this ROI will eventually recover as

well over time.

ROI

size

PRE-BLEACH

t<0

BLEACH

t=0

POST-BLEACH

t>0 (0.5s)

t>>0 (10s)

10µm

X

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20µm

X

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50µm

X

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FIG. 4.15: Typical pre-bleach (t<0), bleach (t=0) and post-bleach (t>0: 0.5s after bleaching; t>>0: 10s after bleaching) images from the FRAP experiments for different sizes of the chosen ROI. For the ROI with a length of 10µm, 20µm and 50µm the distance from the interface of the alginate gel was respectively 20µm, 40µm and 100µm. The image sizes of the pre- and post-bleaching images are respectively 45.88µmx45.88µm, 93.75µmx93.75µm and 187.50µmx187.50µm.

FIG. 4.16 shows the common recovery curves of the performed FRAP experiments. It is again clear that

the recovery is slower in the ROI of size 50µmx50µm in relation to the recovery in fluorescence intensity of the

ROI of size 20µmx20µm and 10µmx10µm, which is in agreement with FIG. 4.15. When looking at the recovery

graphs of the CLSM, the 20µm ROI shows the largest bleaching extend (drop in fluorescence intensity), in contrast

to the bleaching extend for the 50µm and 10µm ROI.

When analyzing the recovery curves of the Matlab script, it appears that the fit of the model is

appropriate for all experiments. However, it appears that the model fit is not perfect for the data of the

experiment using the ROI of size 50µmx50µm and 10µmx10µm, shown by the black arrows in FIG. 4.16. For the

ROI of size 50µ there appears to be an underestimation of the model, while for the ROI of size 10µm there seems

to be an overestimation of the model in the indicated areas.

32

ROI

size

Recovery curve by CLSM Recovery curve by Matlab

10µm

X

10µm

20µm

X

20µm

50µm

X

50µm

FIG. 4.16: The common recovery curves of the FRAP interface experiments for each chosen ROI. The curves shown left are the curves produced by the CLSM right after the experiments. The curves shown right are the curves calculated by the Matlab script in the analysis of data of the experiments. For the ROI with a length of 10µm, 20µ and 50µm, the distance from the interface of the alginate gel was respectively 20µm, 40µm and 100µm.

The residual plots of the FRAP experiments, produced by the Matlab script, can be used as a guideline

for analyzing the quality of the model fit. The common residual plot is shown in FIG. 4.17. It is the plot of a FRAP

experiment with an ROI with a length of 20µm, placed 40µm from the interface of the alginate gel. The residual

plot of the real experiment data is presented in the top half of the image with the bleached probes shown as

dark blue stripes. The lower half of the image shows the model of the Matlab script with the bleached probes as

dark blue stripes without the residuals. The fluorescence intensity recovers as the dark blue stripes become

33

smaller and ultimately fade away. Notice that the images with the post-bleach ROIs are compromised and placed

next to each other. The residuals should be as random as possible (random noise) and the model should be

similar to the real data. No structure should be seen on the residual plot. It appears that the model fit is

appropriate for the data.

FIG 4.17: A common residual plot of the data of the FRAP experiments in comparison with the model. For this plot a ROI with a length of 20µm is used, placed 40µm from the interface of the alginate gel. The residual plot is in relation to the recovery graphs in FIG for the same experiment. The residual plot of the real data is shown on top, with the model without residuals shown below.

Some FRAP interface experiments with alginate beads inside a carrageenan gel in a metallic cup are

also performed at lower depth in the sample, but they did not show good recovery graphs and are therefore not

analyzed.

4.2.5 Mixture of carrageenan and alginate gel

A mixture by stirring and heating of a 1% carrageenan gel with a 4% alginate gel is tried out to

investigate the use of this system for the experiments in this thesis. As shown in FIG. 4.18, one can see small

unclear indications of phase-separation due to the patterns.

FIG. 4.18: CLSM image of the mixture of 1% carrageenan and 4% alginate gel, both with 100ppm 70kDa FITC-dextran. Scale bar is 25µm.

34

4.3 RICS EXPERIMENTS

All fitted correlation functions and their corresponding residual plots are plotted by the Matlab script.

Gs(ξ,ψ) represents the autocorrelation function plotted in three dimensions and ΔGs(ξ,ψ) is the difference in

autocorrelation function (residual). ξ and ψ are the pixel coordinates of the image, where ξ is the horizontal

correlation shift and ψ the vertical correlation shift. The magnitude of the autocorrelation function is shown by

the colors in the graphs.

4.3.1 Optimal scanning rate

Different scanning rates are tested to find out what the optimal scanning rate is for the diffusion

coefficient of the probe in the systems. The common fitted correlation functions with their top view are listed in

FIG. 4.20 for the RICS experiments in 1% κ-carrageenan hydrogel with 80nM 70kDa FITC-dextran as a fluorescent

diffusion probe. A scanning rate of 10Hz, 400Hz, 600Hz and 1000Hz is employed. The correlation function graphs

show the correlated fluorescence fluctuations of the fluorescent probe over space and time in the stack of CLSM

images of one experiment.

The two most common residual plots are listed in FIG. 4.19. FIG. 4.19a shows an interesting pattern, while

FIG. 4.19b shows a more random residual plot. The scale bar of the first plot shows higher residuals in contrast

to the second plot.

It appears that no correlation is visible in the ψ direction for the 10Hz scanning rate, as described in FIG.

4.20a. One basically see a somewhat broaden width of the Point Spread Function (PSF) at this scanning rate. As

the scanning rate rises from 10Hz to 1000Hz, it can be seen that the plot shows more and more correlation in

the ψ direction. It can be seen that the diffusing molecules contribute to the correlation function for the higher

scanning rates (400Hz, 600Hz and 1000Hz), in contrast to the scanning rate of 10Hz. For this reason a higher

scanning rate (400Hz to 1000Hz) was chosen for the next experiments.

FIG. 4.19: The residual plots of the RICS experiments inside a 1% carrageenan gel with 80nM 70kDa FITC-dextran as a diffusion probe. A scanning rate of (a) 10Hz and (b) 1000Hz is used.

(a) (b)

35

FIG. 4.20: The fitted correlation spectrums in 3D (left) and top view (right) of 80nM 70kDa FITC-dextran in a 1% κ-carrageenan gel. The RICS experiments are performed with a zoom of 7 and a scanning rate of (a) 10Hz, (b) 400Hz, (c) 600Hz and (d) 100Hz. Gs(ξ,ψ) represents the auto correlation function and ξ and ψ are the pixel coordinates.

4.3.2 H2O

The diffusion coefficients of 70kDa FITC-dextran in distillated water measured by RICS are listed in FIG.

4.21. The results are consistent with the different zoom factor used (7 and 12) and a gradually higher diffusion

coefficient is measured when increasing the scanning rate from 400 to 1000Hz. There is no clear advantage

when using a zoom factor of 7 or 12.

(a)

(b)

(c)

(d)

36

FIG. 4.21: Average diffusion coefficients of 80nM 70kDa FITC-dextran in H2O measured by RICS. For each experiment a scanning rate of 400Hz, 600Hz and 1000Hz were employed with a zoom of 7 and 12.


The average diffusion coefficients of 70kDa FITC-dextran in a 1% κ-carrageenan gel measured by RICS

are listed in FIG. 4.22 with their standard deviations. Again, it appears that the diffusion rate rises when a higher

scanning rate (from 400Hz to 600Hz and 100Hz) is used, also giving better correlations. As in the previous

experiments, there is no clear advantage when using a zoom factor of 7, 12 or 16.

FIG. 4.22: Average diffusion coefficients of 80nM 70kDa FITC-dextran in 1% κ-carrageenan gel with RICS. For each experiment a scanning rate of 400Hz, 600Hz and 1000Hz is used with a zoom of 7 and 12. For the scanning rate of 1000Hz a zoom of 16 is also used.

In the first RICS experiments, some results are very inconsistent. For example, a diffusion coefficient of

44µm2/s is measured inside a brighter area of the carrageenan gel in a closed seal spacer. Therefore, it is decided

to only perform RICS experiments in the metallic cup samples for the next experiments.

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37

4.3.4 Alginate bulk

FIG. 4.23: Average diffusion coefficients of 80nM 70kDa FITC-dextran in 4% alginate gel with RICS. For each experiment a scanning rate of 400Hz, 600Hz, 800Hz and 1000Hz is used with a zoom of 7. Only one experiment is performed with a scanning rate of 400Hz to 800Hz, 4 experiments are performed with a scanning rate of 1000Hz.

The diffusion coefficients of 70kDa FITC-dextran in a 4% alginate gel, produced by the internal method,

are listed in FIG. 4.23. One can again observe an increasing trend in diffusion coefficient from a scanning rate of

400Hz to 1000Hz. The correlation spectrum and its residual plot is shown in FIG. 4.24 of a RICS experiment inside

the alginate gel. A scanning rate of 400Hz is used in this experiment. The residual plot shows some interesting

patterns, most likely caused by the heterogeneities in the alginate gel. The fitted correlation spectrum shows a

good correlation in all directions.

FIG. 4.24: The graphs of the analysis of the RICS experiment on the 4% alginate gel with 80nM 70kDa FITC-dextran. (a) The residual plot with (b) the fitted correlation spectrum of the experiment with a scanning rate of 400Hz and a zoom of 7.

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38

4.3.5 Interface

The RICS experiments are carried out near the interface of the 1% carrageenan gel and the 4% alginate

gel, both with 80nM 70kDa FITC-dextran as a diffusion probe. The results are listed in FIG. 4.25. The negative

distance refers to the experiments in the carrageenan gel, while the positive distance refers to the experiments

in the alginate gel. It appears that the diffusion of the probe decreases for measurements in the carrageenan

phase to further away from the interface in the alginate phase. The diffusion coefficient measured at the

interface is the average of the diffusion coefficients inside both gel phases, as shown in FIG. 4.26b. Apart from

the measurements done at the interface, only one experiment is done on each distance from the interface in

both gels. The results of the measurements performed in 40µm, 70 and 100µm from the interface inside the

alginate gel are relatively similar.

FIG. 4.25: Average diffusion coefficients of 80nM 70kDa FITC-dextran in 4% alginate gel and 1% carrageenan gel with RICS. For each experiment a zoom of 7 is used with a scanning rate of 600Hz. The positive distance from the interface refers to measurements inside the alginate gel, zero is at the interface and the negative distance is inside the carrageenan gel. The image size of the experiments is always 35.14µmx35.14µm. The CLSM image of the interface between the carrageenan and alginate gel is shown in FIG. 4.26a. The

carrageenan gel is shown on top of the image, the alginate gel is shown at the bottom. Notice that the interface

of the two gels shows an interesting transition. FIG. 4.26b shows an illustration of the RICS experiments near the

interface in relation to the graph in FIG. 4.25. The distance from the interface should be interpreted as the

distance from the interface to the closest border of the CLSM image.

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FIG. 4.26: (a) A CLSM image of the carrageenan and alginate interface. The 1% carrageenan gel with 80nM 70kDa FITC-dextran is shown on the top half of the image and the 4% alginate gel with the same concentration of the probe is shown on the down half of the image. The size of this grayscale image is 35.14µmx35.14µm. (b) Illustration of the RICS experiments performed near the same interface as the CLSM image. The rectangles represent the images with a size of 35.14µmx35.14µm. Image A is placed inside the carrageenan gel, image B at the interface and image C in the alginate gel.

(a) (b)

40

5 DISCUSSION

5.1 POLYSTYRENE SPHERES INSIDE CARRAGEENAN GEL

It was expected that the PS beads would have clear, sharp boundaries when observed under the CLSM.

However, the boundaries are not sharp but rather unclear as shown in FIG. 4.2. This is most likely due to the

excessive reflection of laser light at the boundaries of the spheres since the refractive index of the polystyrene

beads (n is 1.61 for a wavelength of 488nm (52)) is much higher than the refractive index of water (n is 1.34 for

a wavelength of 488nm (53)). For this reason, it appears that this model system is not the optimal model to use

for investigating the diffusion near the interface of the spheres and the carrageenan gel. The model system is

therefore replaced by the carrageenan gel with the alginate gel as beads or bulk.

5.2 FRAP EXPERIMENTS

5.2.1 Concentration effect of sodium fluorescein

A concentration of 20ppm of sodium fluorescein as a fluorescent probe gives an undesirable ratio

between signal and background noise (signal-to-noise ratio, SNR) and a low fluorescence intensity, which is also

described in literature as an effect of too low concentration of the fluorescent probe (1). On the other hand, inner

filtering effects may occur when using a concentration that is too high (41). Inner filtering is the absorption of

the emitted light of fluorophores in the focal plane by nearby probe molecules so the light does not reach the

detector, causing the observed fluorescence intensity to drop. This can cause a change in the linearity of

fluorescence at high fluorophore concentration (1). This might be the case for the 200ppm concentration. The

bleaching extend seems to be greater as well for a concentration of 100ppm (and 200ppm). For these reasons

it can be concluded that a 100ppm concentration of sodium fluorescein is the optimal concentration to use for

FRAP experiments in a 1% κ-carrageenan hydrogel.

When looking at the recovery curves of 70kDa FITC-dextran in FIG. 4.5 in contrast to the recovery curves

of sodium fluorescein in FIG. 4.3, the latter is more difficult to bleach inside the carrageenan gel than the 70kDa

FITC-dextran. A bleaching profile of 30% of the pre-bleach fluorescence intensity inside the ROI or more (which

is necessary for a good FRAP measurement, as stated in (35)) is very hard to obtain in contrast to the FITC-

dextran, even with 5 bleaching images and an ROI of 50µm in diameter. The reason for this difference in

bleaching extend is most likely because of the difference in molecular mass between the two probes. As 70kDa

FITC-dextran has a higher molecular weight then fluorescein, it will diffuse slower, as stated in (33), resulting

in a slower fluorescence recovery and higher visible bleaching depth.

In addition, and in contrast to 70kDa FITC-dextran, there is a possibility of sodium fluorescein to be

influenced by interaction with the carrageenan gel-network, as this interaction is also present in a study of the

41

same probe in a β-lactoglobulin gel (BLG) (33). Because of the repulsion of the negatively charged sodium

fluorescein and κ-carrageenan polymer network, it is possible that a depletion area occurs close to the polymer

strings causing the sodium fluorescein to move in a more crowded environment. If this is indeed the case for the

carrageenan gel as well, it is necessary to describe the recorded recovery with a model that takes binding into

account for determining the diffusion coefficient of this fluorescent probe (33).

The relatively extreme values of kon and koff when analyzing the diffusion of 70kDa FITC-dextran using

the binding and diffusion model in experiments in the carrageenan bulk (as described in 4.2.2) can also be found

in literature (33). This indicates that the diffusion of the 70kDa FITC-dextran probe is most likely not influenced

by binding events in the κ-carrageenan gel. It can be assumed that the rFRAP model without implementing

binding, can be safely used for analyzing the diffusion of this probe. The difference in polymer interaction

between the two probes could be explained by the difference in surface-charge density (33). These two problems

result in the choice of 70kDa FITC-dextran as fluorescent diffusion probe in the following experiments.


The diffusion coefficients are relatively consistent in FIG. 4.4 for both ROI sizes. The standard deviation

of the experiments are acceptable when the results of other FRAP experiments are observed in literature (33).

The results should be compared with the diffusion coefficient of the same probe in a 2% (w/v) agarose hydrogel

measured by FRAP and RICS, which is described to be approximately 28µm2/s for both techniques (46). The free

diffusion rate (D0) of 70kDa FITC-dextran in water at room temperature is described to be 29.9±3.1µm2/s (33).

The measured diffusion coefficients of the probe in a 1% carrageenan gel are thus lower than the diffusion of the

probe in an agarose hydrogel and in water, which is plausible. The recovery graph listed in FIG. 4.5 shows an

appropriate fit of the model.

5.2.3 Alginate bulk

The measured diffusion coefficient for both production methods are very different. It is however not

clear if the FRAP experiments are made in the bead or in the liquid layer on top of the bead. The liquid layer may

give rise to a flow or sample drift, which can lead to an apparent increase in fluorescence recovery rate as stated

in literature, leading to higher measured diffusion coefficients (1). It appears that the results of the internal

method are more trustworthy. However, more experiments should be performed, with both RICS and FRAP to

assure the accuracy of the diffusion coefficients. There is another method to dissolute the probe inside the

alginate gel, as described in literature, called external-diffusive mechanism (15). This could be used in further

experiments and could possibly result in better FRAP and RICS experiments.

42


The beads are very heterogeneous as seen in FIG. 4.7 and it is difficult to control their properties because

of the almost instantaneous gelling process, which is also described in literature (19). It also appears that the

distribution of the fluorescent probe is not homogenous inside the alginate bead, which could possibly indicate

a certain internal structure of the alginate bead. This inhomogeneous distribution is however only a seen for the

alginate gel made by the droplet method. When an alginate gel is produced with the internal method, a more

homogeneous distribution of the probe can be reached. In addition, the lower concentration of the probe inside

the alginate bead after two days is a possibly due to a higher affinity of 70kDa FITC dextran for the carrageenan

gel in contrast to the alginate gel.


It is clear that the internal method is a method to obtain a more homogeneous alginate gel. This is

especially favorable if FRAP experiments are to be performed inside the gel. There are however still some clear

heterogeneities visible in the alginate gel as shown in FIG. 4.9. This may also give rise to less accurate results

with FRAP as these heterogeneities could influence the mobility of the fluorescent probes by obstruction or even

binding, as described in literature (1). It is however unknown what the effect of structural heterogeneity on FRAP

measurements is. The ROI for FRAP experiments should be placed in more homogeneous phases, as far away as

possible from the heterogeneities, as stated in (2). A more practical way to resolve this problem is the use of a

different model that can account for the heterogeneities in media or the use of line FRAP, where FRAP

experiments can be performed in smaller regions (2).

The measured diffusion coefficients of the probe in the alginate gel, produced with the internal method

are relative consistent and approximately between 3 and 4µm2/s. In literature it is found that the diffusion of

70kDa FITC-dextran is reduced by 80% inside a 2% alginate gel, produced by the internal gelation method (15).

If a free diffusion coefficient (D0) of approximately 30µm2/s (33) is assumed for the probe, a diffusion coefficient

of 6µm2/s is described in this article. It is likely that the diffusion of the probe is lower in a more concentrated

alginate gel as the gel network can interfere with the mobility of the probe. The results seem to be plausible.

5.2.4 Interface

The increasing trend in diffusion coefficient of the probe inside the carrageenan gel is observed. The

lower diffusion coefficient near the surface of the alginate bead is possible as the relative dens alginate bead

could obstruct the movement of the molecules near the interface in contrast to the molecules further away from

the interface. The standard deviations of the results are relatively large, which makes it hard to say with one

hundred percent certainty that the diffusion close to the interface is different. It is however a very interesting

observation. It would be interesting to do more experiments with higher resolution to better resolve the trend.

43

A requirement for FRAP analysis is that the diffusion should be free and unaffected by boundaries in

the hydrogel (35). Some FRAP experiments are however performed near the border of the alginate bead, which

could lead to incorrect or less accurate diffusion rates. A new FRAP model could solve this problem, also stated

in 5.2.3.2, for instance the model described in (54) that can account for the heterogeneities in media. Some FRAP

experiments with a ROI of 5µm in diameter are also performed, but the signal to noise ratio is unfavorable. Next

experiments with a 63x objective could solve this problem.

The alginate beads employed in this experiment are produced without 70kDa FICT-dextran, in contrast

to the 100ppm concentration of the probe in the carrageenan gel. This may, however, create a variable diffusion

coefficient due to a concentration-dependent non-equilibrium phenomena, because of the different

concentrations in both phases. The experiments are however performed one day after the production of the

sample so the equilibrium could have been set already. Through, in literature it is described to avoid this

phenomena as much as possible (42). This should be kept in mind for further experiments in the future.

The reason for the strange recovery curves of the FRAP experiments at the interface of the alginate

beads inside the carrageenan gel in the metallic cup might be that the laser light experiences inner filtering

because of the greater depth of the FRAP experiments. The depth into the sample is 900µm for these

experiments, instead of the depth of 30µm for the usual experiments. This phenomena occurs if the

concentration of the probe is too high, as stated in 5.2.1. However, the effect of inner filtering can also grow if

the depth in the sample where the measurements are performed, increases (1, 35). For this reason, it is decided

that the depth should be kept at 30µm for all the other experiments.

The difference between the recovery graphs of the FRAP experiments in FIG. 4.16 using a ROI with a

length of 10µm and 20µm with the ROI with a length of 50µm is that it takes more time for the bleached probes

inside the center of the ROI to diffuse outside the bigger ROI in contrast to the smaller ROIs. As it takes more

time, the recovery of the fluorescence will be slower as well. As the average fluorescence of all pixels inside the

ROI is plotted on the recovery curve, there is a noticeable difference between the sizes of the ROI.

In literature, an enrichment of solvent in the interface between two coexisting polymer solutions is

described, because of the prevention of mutual contact of two incompatible polymers (55). The measured

diffusion coefficient at the interface should be approximately the same as the free diffusion rate of the probe in

water (D0) in that case. It seems that this is not the case at the FRAP and RICS experiments at the interface of

carrageenan and alginate gel. The reason for this could be that the polymers are compatible and do not phase

separate or that the enrichment of the solvent occurs at a much smaller length scale.

44

5.2.5 Mixture of carrageenan and alginate

It can be concluded that this method is not the desired method to investigate the diffusion at the

interface, because the interface of the carrageenan and alginate gel is not visible at the micrometer level. More

research is needed to form a conclusion on the phase-separation of the two gels.

5.3 RICS EXPERIMENTS

Generally, in all RICS experiments it is clear that the more frames are used, the lower the error in each

data point and the higher the precision of the fit. The correlation function shape does not differ much in relation

to the number of frames used, which is all in agreement with research literature (38). The measured diffusion

coefficient did not differ much in relation to a different zoom function (between 7 or 12) as stated in chapter 4.3.

This is in contrast to the FRAP experiments, where more accurate diffusion coefficients are found when using a

higher zoom function, in relation to the size of the ROI, while keeping in mind that the size of the ROI must never

be bigger than the image size (1).

5.3.1 Optimal scanning rate

For the evaluation of the analysis of the RICS experiments, it is important to check for the sharpness of

the fitted correlation function. A correlation function that is too sharp shows no correlation in the ψ-direction as

observed for the scanning rate of 10Hz in FIG. 4.20a. Here, the correlation function graph only shows the PSF

instead of the diffusion molecules. This is the case if the molecule of interest diffuses faster than the laser scans

the image in raster lines, as stated in (56). The diffusing molecules do not contribute to the correlation function

and the measured diffusion coefficient by the RICS analysis will be smaller than the true diffusion coefficient.

For the scanning rates of 400Hz to 1000Hz however, this is not the case. The scanning rates seems to be fast

enough (i.e. faster than the movement of the fluorophore) to show correlation between pixels in the ψ-direction.

It is important to note that the optimal scanning rate will depend on the diffusion rate of the molecule of interest

and should always be investigated to select the appropriate scanning rate. Only then accurate RICS

measurements can be performed (39). Because all images are scanned in the horizontal direction, all correlation

functions show an extension in the horizontal direction.

When observing the interesting pattern of the residual plot in FIG. 4.19a it is difficult to state that the

residual plot is not random enough. The same residual plot can also be found in literature where the diffusion

of gold nanoparticles in different glycerol:water mixtures is determined using photothermal RICS (36). On the

other hand, the residual plot in FIG. 4.19b appears close to random noise, which is desirable for a residual plot of

the RICS analysis (39).

45

5.3.2 H2O

In the literature the diffusion coefficient of 70kDa FITC-dextran in water at room temperature (D0) is

described as 29.9±3.1µm2/s (33). The results of the RICS experiments of the probe in water are similar to the

values of literature research.


The results listed in FIG. 4.22 are less consistent than the results of the RICS experiments in water. In

addition, the diffusion coefficients of 70kDa FITC-dextran measured in the bulk of the carrageenan gel is slightly

lower with RICS in comparison with FRAP. However, RICS and FRAP measurements cannot directly be compared,

because of the different size of ROI. Also, with FRAP the diffusion of fluorophores outside the ROI is indirectly

measured as well, while the diffusion coefficient with RICS is more locally determined with less power on the

sample (57). In some articles, approximately the same values are obtained for RICS and FRAP measurements,

while other results differ more between the two techniques (46).

The strange diffusion coefficient of 44µm2/s of 70kDa FITC-dextran inside a brighter area inside the

carrageenan gel is even higher than the normal diffusion coefficient of the probe in water. This should be

impossible, as the mobility is most cases decreased in a gel (1). It could perhaps be the reason of a too high

concentration of probes inside the area, which could lead to unreliable results. This is however just a hypothesis

and more research is needed to be able to explain the higher diffusion coefficient.

5.3.4 Alginate bulk

The measured diffusion coefficient inside the alginate gel by RICS is in the range of 4 to 6µm2/s, which

is slightly higher than the diffusion coefficient found by the FRAP experiments in the same system (3 to 4 µm2/s).

It is still slightly lower than the reported diffusion coefficient of the probe in a 2% alginate gel (15) as stated in

5.2.3.2 and seems therefore also plausible. The experiments with a scanning rate of 400Hz, 600Hz and 800Hz

are only performed once, because of shortage of time. More experiments should be performed with these

scanning rates to find out if the results are consistent.

5.3.5 Interface

The system used for these experiments is extremely interesting, as the diffusion could be measured in

both phases with ease. This is in contrast to the FRAP experiments performed at the interface with the alginate

beads, where the alginate bead is too compact for FRAP measurements. Unfortunately, there was not enough

time to perform more experiments by RICS in this model. If more time was available, more experiments would

be performed to find out if the results are consistent, as well with a higher zoom factor to measure the diffusion

46

closer to the interface in both phases. Arbitrary-Region RICS could also have been used for measuring the

diffusion closer to the interface using an arbitrary ROI (58).

The measured diffusion coefficients inside the carrageenan gel and alginate gel are approximately the

same as the measured diffusion coefficients inside the carrageenan bulk and alginate bulk separately by RICS.

This indicates that the results are consistent with the technique.

5.4 FURTHER RESEARCH

A promising technique for further research is to use quantum dots to investigate the diffusion at the

interface of the carrageenan and alginate gel. Quantum dots are very small and bright fluorescent nanoparticles.

They are more resistant to photobleaching than fluorescent proteins and organic dyes and show high

fluorescence quantum yields (59). They should be able to diffuse inside the alginate bead and inside the

carrageenan gel with ease, because there are so small, illustrated by FIG. 5.1. Instead of using the standard RICS

method to analyze RICS images, it would be interesting to evaluate a new method for measuring the diffusion

of single particles, namely Single Particle Raster Image Analysis (SPRIA), as described in (37).

FIG. 5.1: Illustration of quantum dots diffusing inside carrageenan gel and alginate bead.

Alginate bead

Carrageenan gel

47

6 CONCLUSION

To measure the diffusion coefficient near the interface in a hydrogel, the PS spheres inside a 1% κ-

carrageenan hydrogel does not seems to be the optimal model, regarding the unclear boundaries of the spheres

due to the excessive reflection of the laser light. The carrageenan gel with the alginate gel seems to be a more

promising model.

A concentration of 100ppm of sodium fluorescein seems to be optimal for FRAP experiments inside a

1% κ-carrageenan hydrogel. However, in terms of the extend of bleaching of the fluorescence intensity in relation

to the pre-bleaching fluorescence intensity and possible interaction with the gel-network it is clear that sodium

fluorescein is not the best choice as a fluorescent probe for the used hydrogels. 70kDa FITC-dextran seems better

suited as a probe in the used systems.

Of the two methods that are used to produce an alginate gel, the internal gelling method is superior in

terms of homogeneity in comparison with the alginate beads produced by the droplet method. The mixture of

carrageenan and alginate is not the optimal system to use for this thesis, regarding the unclear interfaces. The

1% κ-carrageenan hydrogel with a 4% internally set alginate gel seems a good model system for measuring the

diffusion near the interface by FRAP or RICS.

In general for both techniques, the measured diffusion coefficient of 70kDa FITC-dextran inside the

alginate gel is in the range of 3µm2/s to 7µm2/s and inside the carrageenan gel in the range of approximately

15µm2/s to 18µm2/s by RICS and 20µm2/s to 25µm2/s by FRAP. RICS measurements of the diffusion of the probe

inside the internally set alginate gel are slightly higher than the FRAP experiments. The RICS measurements of

70kDa FITC-dextran in water, results in diffusion coefficients in the range of 27μm2/s to 30μm2/s. When comparing

the RICS and FRAP results inside the carrageenan hydrogel, it is clear that the techniques slightly differ from

each other and that it is not easy to compare the results.

The bleaching of the probe in the sample using the ROI with size 50µmx50µm is much clearer and it

takes a longer time for the fluorescence intensity to recover in contrast to the smaller ROI sizes. It is interesting

to notice that the ROI of 50µm in length shows less noise, but measures the average diffusion coefficient as the

ROI covers a bigger fragment of the structure. The ROI of 10µm in length, on the other hand, shows more noise

but covers less distance and shows the diffusion coefficient of the probe in a more localized region in the

hydrogel. When analyzing the recovery curves of the Matlab script, it appears that the fit of the model is

appropriate for all FRAP performed experiments.

The diffusion coefficients in RICS experiments show a rising trend as the scanning rate is increased from

400Hz and 600Hz to 1000Hz, along with a better correlation of the fitted correlation spectrum in the ψ-

direction.

48

The fitted correlation function by a scanning rate of 10Hz shows no correlation in the ψ-direction. 600Hz

or 1000Hz seems to be a good scanning rate for measuring the diffusion of 70kDa FITC-dextran in the alginate

and carrageenan gels. An increased zoom factor does not seem to greatly alter the measurements of the

diffusion coefficient.

The results of the FRAP experiments near the interface of the 4% alginate bead inside the 1% κ-

carrageenan are interesting as there appears to be a slightly increasing trend in diffusion of the probe when

moving the ROI further away from the interface inside the carrageenan gel. However, more experiments with

higher resolution are necessary to better resolve the trend. A new FRAP model that accounts for heterogeneities

in the medium could be used for experiments near the interface in the future. This increasing trend in diffusion

of the probe is not seen in the RICS experiments near the interface in the alginate gel, but more research is

needed. The RICS and FRAP measurements of the diffusion of the probe far away from the interface in both the

carrageenan and alginate gel showes diffusion coefficients that are in agreement with the results in the separate

alginate and carrageenan bulk. It appears that the results are consistent for both techniques.

The mass transport in heterogeneous biomaterials and their interfaces remains an interesting but

difficult subject. The results in this thesis are promising, but it can be concluded that more experiments are

necessary to receive more closing and reliable results, especially for the RICS experiments near the interface.

Arbitrary-Region RICS could be used for measuring the diffusion closer to the interface using an arbitrary ROI.

Further research could also involve the measurement of the diffusion of quantum dots inside the model system

with the use of SPRIA analysis.

49

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