Karam Patzak Is A

NOVEL SPECTROSCOPY
TECHNIQUES FOR FUNCTIONAL

IMAGING OF BACTERIAL BIOFILMS
AT MICROSCOPIC LEVEL
ANDREAS KARAMPATZAKIS
(M. Sc.)
A THESIS SUBMITTED FOR THE

JOINT DEGREE OF DOCTOR OF PHILOSOPHY
NUS GRADUATE SCHOOL FOR INTEGRATIVE

SCIENCES AND ENGINEERING
NATIONAL UNIVERSITY OF SINGAPORE
DEPARTMENT OF PHYSICS
IMPERIAL COLLEGE LONDON
2017
Supervisors:
Professor Thorsten Wohland, National University of Singapore
Professor Peter Török, Imperial College London
Examiners:
Associate Professor Sanjay Swarup, National University of Singapore
Dr Darryl Overby, Imperial College London
Associate Professor Kimberly Kline, Nanyang Technological University
Declaration
I hereby declare that this thesis is my original work and it has been written by
me in its entirety. I have duly acknowledged all the sources of information which
have been used in the thesis.
This thesis has also not been submitted for any degree in any university previ-
ously.
Andreas Karampatzakis
August 2017
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Copyright Declaration
The copyright of this thesis rests with the author and is made available under
a Creative Commons Attribution Non-Commercial No Derivatives licence. Re-
searchers are free to copy, distribute or transmit the thesis on the condition that
they attribute it, that they do not use it for commercial purposes and that they do
not alter, transform or build upon it. For any reuse or redistribution, researchers
must make clear to others the licence terms of this work.
Results of this thesis have been partially published in:
- A. Karampatzakis, J. Sankaran, K. Kandaswamy, S. A. Rice, Y. Cohen, T. Wohland “Mea-
surement of oxygen concentrations in bacterial biofilms using transient state monitor-
ing by single plane illumination microscopy” Biomed. Phys. Eng. Express 3 035020
(2017).
- A. Karampatzakis, C. Z. Song, L. P. Allsopp, A. Filloux, S. A. Rice, Y. Cohen, T. Wohland,
P. Török “Probing the internal micromechanical properties of Pseudomonas aeruginosa
biofilms by Brillouin imaging” npj Biofilms and Microbiomes, 3, 1–7, (2017).
v
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Acknowledgements
The long journey that has led to the writing of this thesis would not have been
possible without the help of many persons that I was fortunate enough to meet
along the way.
First and foremost, I would like to express my sincere thanks to my super-
visors Thorsten Wohland (NUS) and Peter Török (ICL) for giving me the oppor-
tunity to work on this project, and for their continuous support throughout this
project. Most importantly, I would like to thank them for their trust in me and
my work despite the difficulties along the way.
Many thanks to my thesis advisor committee members Nanguang Chen (NUS)
and Shu Sin Chng (NUS) for bringing their expertise to the discussions of my re-
sults. I would also like to thank professors Yehuda Cohen (SCELSE, NTU), Scott
Rice (SCELSE, NTU) and Alain Filloux (ICL) for accepting me in their labs where
most of the biological work for this thesis was done.
My appreciation goes to all my colleagues at CBIS, SCELSE, and ICL, and es-
pecially to Jagadish Sankaran, Cheng–Ze Song, Luke Allsop, Xue–Wen Ng, Emilio
Sanchez, Anand Singh, Kumaravel Kandaswamy, Sarala Neomi Tantirimudalige,
Pei–Ju Wung, Giuseppe Antonacci and Guillaume Lepert.
I would also like to express my gratitude to NUS Graduate School for Inte-
grative Sciences and Engineering for funding my studies and expenses via the
NGS Scholarship, and for being extremely helpful and accommodating.
Last but not least, I would like to thank my mother, and my wife, Charis Sim,
for their love, patience, understanding and support throughout these years.
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Contents
Summary xiii
List of Tables xv
List of Figures xviii
List of Abbreviations and Symbols xix
1 General Introduction 1
1.1 Historical perspective . . . . . . . . . . . . . . . . . . . . . . . . . . 1
1.2 Scope . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
2 Photophysics and Photochemistry 8
2.1 Light scattering . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
2.1.1 Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
2.1.2 Brillouin scattering . . . . . . . . . . . . . . . . . . . . . . . . 10
2.1.3 Brillouin modulus and mechanical properties . . . . . . . . 12
2.2 Luminescence phenomena . . . . . . . . . . . . . . . . . . . . . . . 15
2.2.1 Energy states . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
2.2.2 Singlet vs Triplet states . . . . . . . . . . . . . . . . . . . . . 18
2.2.3 Spectra . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
2.2.4 Quenching by Oxygen . . . . . . . . . . . . . . . . . . . . . . 21
ix
CONTENTS
3 Biofilms 25
3.1 The biofilm mode of life . . . . . . . . . . . . . . . . . . . . . . . . . 25
3.2 EPS and mechanical properties of biofilms . . . . . . . . . . . . . . 29
3.3 The role of oxygen in biofilms . . . . . . . . . . . . . . . . . . . . . . 32
4 Brillouin Microscopy for Mechanical Measurements 35
4.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35
4.1.1 Confocal microscopy . . . . . . . . . . . . . . . . . . . . . . . 35
4.1.2 Brillouin spectroscopy . . . . . . . . . . . . . . . . . . . . . . 37
4.1.3 The conventional use of term ’stiffness’ . . . . . . . . . . . . 40
4.2 Material and Methods . . . . . . . . . . . . . . . . . . . . . . . . . . 42
4.2.1 Experimental setup . . . . . . . . . . . . . . . . . . . . . . . . 42
4.2.2 Sample preparation . . . . . . . . . . . . . . . . . . . . . . . 45
4.2.3 Acquisition and analysis . . . . . . . . . . . . . . . . . . . . . 47
4.3 Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51
4.3.1 Measurements in hydrogel beads . . . . . . . . . . . . . . . 51
4.3.2 Characterisation of the stiffness of P. aeruginosa biofilms . 51
4.3.3 Brillouin shift measured at increasing depths . . . . . . . . 53
4.3.4 Temporal changes of stiffness . . . . . . . . . . . . . . . . . . 55
4.3.5 Effect of flow rate . . . . . . . . . . . . . . . . . . . . . . . . . 58
4.4 Discussions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 58
4.5 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63
5 SPIM – TRAST for O2 Measurements 65
5.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 65
5.1.1 Single Plane Illumination Microscopy (SPIM) . . . . . . . . 65
5.1.2 Transient States (TRAST) spectroscopy . . . . . . . . . . . . 67
5.2 Material and Methods . . . . . . . . . . . . . . . . . . . . . . . . . . 70
5.2.1 Simplified energy system . . . . . . . . . . . . . . . . . . . . 70
x
CONTENTS
5.2.2 TRAST monitoring theory . . . . . . . . . . . . . . . . . . . . 71
5.2.3 Derivation of TRAST contrast . . . . . . . . . . . . . . . . . . 73
5.2.4 TRAST contrast as a measure of [O2 ] . . . . . . . . . . . . . . 75
5.2.5 Experimental setup . . . . . . . . . . . . . . . . . . . . . . . . 76
5.2.6 Sample preparation . . . . . . . . . . . . . . . . . . . . . . . 78
5.2.7 Acquisition and analysis . . . . . . . . . . . . . . . . . . . . . 81
5.3 Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 83
5.3.1 Measurements in bulk solutions . . . . . . . . . . . . . . . . 83
5.3.2 Measurements in heterogeneous samples . . . . . . . . . . 85
5.3.3 Measurements in bacterial biofilms . . . . . . . . . . . . . . 86
5.4 Discussions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 89
5.5 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 94
6 Afterword 96
6.1 Overall conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . 96
6.2 Limitations and outlook . . . . . . . . . . . . . . . . . . . . . . . . . 98
Appendices
A Typical TRAST illumination scheme A-1
B Protocol for TRAST Contrast vs Oxygen calibration B-1
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xii
Summary
This thesis deals with the development and application of spectroscopy meth-
ods in the study of bacterial biofilms at the microscopic level. More specifically,
with a combination of Brillouin microscopy with fluorescence imaging, we were
able to probe the stiffness in the interior of growing biofilms in a non–contact
manner, where we found distinct profiles indicating colonies in different stages
of their lifecycle. With the implementation of Transient State (TRAST) imaging
on a Single Plane Illumination Microscope (SPIM) we were able to measure the
triplet state populations and develop a protocol that can quantify oxygen con-
centrations. When biofilms were measured, steep oxygen gradients were found.
The report is structured as follows: Chapter 1 provides a historical overview
of microscopy and spectroscopy methods, and sets the scope of the thesis. Chap-
ter 2 presents the theoretical background on light scattering mechanisms that is
essential for interpreting Brillouin imaging results, as well as on fluorescence
and quenching phenomena which are key concepts for understanding TRAST
imaging. Chapter 3 introduces and discusses the concept of biofilms as a mode
of bacterial life. In particular, the distinct structural morphology of biofilms is
highlighted, together with the importance of oxygen in their lifecycle. Chap-
ters 4 and 5 contain all the information for the development of the experimental
methods, together with the results and discussions for Confocal Brillouin mi-
croscopy and SPIM – TRAST, respectively. Lastly, Chapter 6 contains the overall
conclusions of this project and an outlook.
xiii
xiv
List of Tables
2.1 Types of scattering . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
2.2 Typical lifetimes of electronic transitions . . . . . . . . . . . . . . . 18
5.1 Characteristics of Eosin Y . . . . . . . . . . . . . . . . . . . . . . . . 78
5.2 Triplet relaxation and intersystem crossing times for Eosin Y in air–
saturated and deaerated solutions . . . . . . . . . . . . . . . . . . . 83
A.1 Lookup table for TRAST image acquisition settings . . . . . . . . . A-2
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xvi
List of Figures
2.1 Typical scattering spectrum. . . . . . . . . . . . . . . . . . . . . . . 10
2.2 Schematic of momentum conservation in Brillouin scattering . . 11
2.3 Types of material deformations based on the distribution of forces. 13
2.4 A typical Jablonski diagram. . . . . . . . . . . . . . . . . . . . . . . . 16
2.5 Absorption and emission spectra of Eosin Y. . . . . . . . . . . . . . 20
3.1 P. aeruginosa bacterial cells forming a colony . . . . . . . . . . . . 27
3.2 Schematic illustrating the late stages of a biofilm life cycle. . . . . 31
4.1 Diagram of a confocal microscope. . . . . . . . . . . . . . . . . . . . 36
4.2 Schematic of the confocal Brillouin microscope. . . . . . . . . . . . 42
4.3 Schematic of our continuous–culture biofilm flow cell setup . . . 46
4.4 Brillouin spectrum of water, used for calibration. . . . . . . . . . . 48
4.5 A typical Brillouin spectrum from a point inside a biofilm . . . . . 50
4.6 Brillouin imaging in hydrogel beads. . . . . . . . . . . . . . . . . . . 52
4.7 Brillouin, widefield and fluorescence images of typical colonies. . 53
4.8 Brillouin shift (average and standard deviations within ROIs) mea-
sured in colonies of various sizes. . . . . . . . . . . . . . . . . . . . 54
4.9 Brillouin shift measured in increasing depths in a biofilm colony. 54
4.10 Brillouin shift measured along a cross-section of a large colony at
various depths. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 56
xvii
LIST OF FIGURES
4.11 Brillouin shift (average and standard deviations within ROIs) mea-
sured in colonies at various times post–inoculation. . . . . . . . . 57
4.12 Brillouin shift measured along a cross–section of a colony at two
consecutive days. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 57
5.1 SPIM – TRAST schematic. . . . . . . . . . . . . . . . . . . . . . . . . 67
5.2 Simplified, three–state Jablonski diagram. . . . . . . . . . . . . . . 70
5.3 The effect of triplet relaxation and intersystem crossing time on
the appearance of TRAST curves. . . . . . . . . . . . . . . . . . . . . 73
5.4 TRAST contrast for a number of triplet relaxation times. . . . . . . 75
5.5 TRAST contrast plotted against oxygen concentrations. . . . . . . 76
5.6 Mounting of samples and optical sectioning schematics. . . . . . . 81
5.7 Assessment of bleaching recovery . . . . . . . . . . . . . . . . . . . 82
5.8 TRAST measurements in bulk solutions of Eosin Y. . . . . . . . . . 84
5.9 TRAST contrast measured in samples of varying oxygen content. . 84
5.10 TRAST measurements in hydrogel beads. . . . . . . . . . . . . . . . 86
5.11 TRAST measurements in P. aeruginosa biofilms. . . . . . . . . . . . 88
5.12 TRAST contrast image and oxygen concentration contours inside
a colony. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 89
B.1 Parallel measurements with an optical sensor and SPIM. . . . . . . B-2
xviii
List of Abbreviations and Symbols
List of Abbreviations
A. A. Alginate Acid
AFM Atomic Force Microscopy
BE Beam Expanders
BS Beam Splitter
CARS Coherent anti–Stokes Raman spectroscopy
C Cylindrical lens
CCD Charge–Coupled Device
CW Continuous Wave
DB Doublet lens
DICM Differential–Interference Contrast Microscopy
DM Dichroic Mirror
EPS Extracellular Polymeric Substances
e–DNA extracellular DNA
FCS Fluorescence Correlation Spectroscopy
FCCS Fluorescence Cross–Correlation Spectroscopy
FEP Fluorinated Ethylene Propylene
xix
ABBREVIATIONS AND SYMBOLS
FLIM Fluorescence Lifetime Imaging Microscopy
FRAP Fluorescence Recovery After Photobleaching
FRET Förster (or Fluorescence) Resonance Energy Transfer
FSR Free Spectral Range
FxP Frequency per Pixel
HOMO Highest Occupied Molecular Orbital
LB Luria–Bertani medium (Lysogeny Broth)
LUMO Lowest Unoccupied Molecular Orbital
M Mirror
NA Numerical Aperture
OPFOS Orthogonal–Plane Fluorescence Optical Sectioning
PCS Photon Correlation Spectroscopy
RLS Raman correlation spectroscopy
ROI Region Of Interest
S Spherical lens
SNR Signal–to–Noise Ratio
SPIM Selective (or Single) Plane Illumination Microscopy
TRAST imaging Transient State imaging
TIRF Total Internal Reflection Fluorescence
VIPA Virtually Imaged Phase Array
WD Working Distance
xx
List of Symbols
[Q] Concentration of quencher Q
α Duty cycle of pulse train
A Surface cross–section
γ Scaling factor that depends on the concentration of fluorophores and
the detection efficiency of the camera
Γ Concentration of fluorophores
δ Optical path difference
∆v B Spectral linewidth of Brillouin peaks
η Viscosity
θ Scattering angle
θi Angle between the prism and the beam at the common path interfer-
ometer
λ0 Wavelength of incident light
ξ Conversion factor between the emitted intensity and the digital read-
out of the camera
ρ Density
σexc Excitation cross–section
σ(λ) Wavelength–dependent molecular absorption cross–section
τ01 Excitation lifetime
τ10 Relaxation time of the first excited state
τT Triplet relaxation time
τT 0 Triplet relaxation time in absence of quencher
xxi
τf Fluorescence lifetime
τf 0 Fluorescence lifetime in absence of quencher
τi sc Intersystem crossing time
Bn Position of Brillouin peaks on the dispersion axis (n = 1, 2)
c Speed of light in vacuum
d Particle size
dg Length of glass prism of the common path interferometer
D Diffusion coefficient
E Young’s modulus
f Focal length
F Force
fQ Quenching efficiency
G Shear modulus
h Planck’s constant (≈ 6.63 × 10−34 J s)
I exc Excitation intensity
If Fluorescence intensity
If 0 Fluorescence intensity in absence of quencher
ICW Fluorescence intensity taken with 100% duty cycle
I shor t Fluorescence intensity taken with 1% duty cycle
K Bulk modulus
k0 Diffusion–controlled bimolecular quenching constant
k 0n Fluorescence excitation rate (n = 1, 2...)
k2 Triplet state population rate after the onset of excitation
xxii
kB Boltzmann’s constant (≈ 8.6 × 10−5 eV/K)
kf Fluorescence rate
ki Wave vector of incident photons
ki c Internal conversion rate
k i sc Intersystem crossing rate
k nr Rate of non–radiative processes
kp Wave vector of phonons
k ph Phosphorescence rate
kq Bimolecular quenching constant
ks Wave vector of scattered photons
kT Triplet, non–radiative, relaxation rate
M Longitudinal modulus
M0 Storage (or Brillouin) modulus
M 00 Loss modulus
NA Avogadro’s number (6.022 × 1023 mol−1 )
n Refractive index
P0 Population of ground electronic state
P1 Population of first excited electronic state
P l aser Excitation power
PT Population of triplet state

eq
PT Relative triplet state population at equilibrium
qf Quantum efficiency of a fluorophore
qT Triplet quantum yield
R Molecular radius
xxiii
Sn Singlet electronic energy levels (n = 0, 1, 2...)
T Period of pulse train
T ac q Total acquisition time
Ti l l Total illumination time
Tn Triplet electronic energy levels (n = 0, 1, 2...)
v Light frequency
va Frequency of absorbed photons
vi Frequency of incident photons
vf Frequency of fluorescence photons
vp Frequency of phonons
v ph Frequency of phosphorescence photons
vs Frequency of scattered photons
V Acoustic velocity
Vn Vibrational energy levels (n = 0, 1, 2...)
vB Brillouin frequency shift
w Pulse width
xxiv
Chapter 1
General Introduction
1.1 Historical perspective
It is believed that the first compound microscopes were conceived and pro-
duced in the 1590s by two Dutch spectacle makers, Zacharias Jansen and his
father, Hans, who experimented with placing multiple lenses in a tube. The first
documented scientific observations on microscopy, however, came from Robert
Hooke and his book Micrographia, published in January 1665 [1]. Amongst oth-
ers, in this book Hooke coined the term cell to describe the basic unit of life,
inspired by the resemblance of the microstructures he saw in plant samples to
the arrangement of cells in a monastery. Micrographia went on to become one
of the first scientific best sellers and is today recognised as a milestone in the
evolution of microscopy [2].
Another name associated with the birth of microscopy is that of Antonie
van Leeuwenhoek, who is also considered the father of microbiology [3]. van
Leeuwenhoek was not a scientist by training, and it is believed that his interest in
microscopy was sparked from reading Hooke’s Micrographia. He built “single–
lens microscopes” which would today be classified as magnifying glasses. His
microscopes were simpler in design compared to other instruments of his time
1
CHAPTER 1. GENERAL INTRODUCTION
(compound microscopes were already in use). However, due to his great skill
in lens grinding and his attention to lighting conditions, he was able to achieve
magnification levels exceeding 200 times and he produced clearer and brighter
images than any of his colleagues. This allowed him to make one of his most
important observations, what he called animacules (little animals), that were
moving in water. His letter to the Royal Society, date 9 October 1676, where he
reports these findings is believed to be one of the first observations on living
bacteria ever recorded [4]. His findings were later reproduced and confirmed by
Hooke.
In one of his most famous experiments, van Leeuwenhoek looked at the
plaque formed in his own teeth and compared his findings with samples taken
from his wife, daughter as well as from two older men who reportedly never
brushed their teeth. What he saw “with great wonder” was “very prettily a-
moving” [...] “animacules” of different sizes and shapes. More specifically, in
the case of the older men, he reports “an unbelievably great company of liv-
ing animalcules, a–swimming more nimbly than any I had ever seen up to this
time. The biggest sort ... bent their body into curves in going forwards ... More-
over, the other animalcules were in such enormous numbers, that all the water
... seemed to be alive”. With this, van Leeuwenhoek had effectively made the
first observation of living bacterial biofilms1 in the human body.
On the theoretical front, the physical limits in discernible objects were in-
vestigated in the 19th century. In 1835, George Airy studied the diffraction pat-
tern created by light passing through an illuminated circular aperture [5], which
is now referred to as “Airy disc” and considered a fundamental concept in mi-
croscopy. A few years later, in 1873, Ernst Abbe in a landmark paper reported
that “the smallest resolvable distance between two points using a conventional
microscope may never be smaller than half the wavelength of the imaging light”
1
An introduction and historical perspective of biofilms can be found in Chapter 3.
2
[6], which has become the well known Abbe’s resolution criterion.
Around the same period, the first observations of fluorescence are docu-
mented. In 1845, Sir Frederik William Herschel noted that a quinine solution,
exhibits a “vivid and beautiful celestial blue color” when illuminated and ob-
served under certain incidences of sunlight [7]. In 1852, George Stokes described
Herschel’s observation in greater detail and set the theoretical foundation for
fluorescence [8].
It did not take long till the first fluorescent dye, fluorescein, was synthesized
by Adolf von Bayer in 1871 [9], and the first fluorescence microscopes were de-
veloped. In 1904, August Köhler used ultraviolet light to observe luminescence
in a microscope built at Zeiss Optical Works in Jena, Germany, and in the coming
years researchers started using fluorescent dyes to enhance autofluorescence of
cells and tissues [10].
A number of techniques that are heavily used today were introduced in the
first half of the twentieth century. Most notably, Frits Zernike’s work on Phase
Contrast Microscopy in the 1930s [11], Wilhelm Josef Schmidt and Shinya In-
oué’s work on Polarizing Microscopy [12, 13], and Francis Smith and Georges
Nomarski’s work on Differential–Interference Contrast Microscopy (DICM) in
the 1950s [14, 15] have all revolutionised the way we look at cells. Around the
same time came the invention of the Confocal Microscope by Marvin Minsky
which led to an improvement of resolving power of fluorescence [16, 17]. The
first confocal microscopes were produced in late 1980s [18], however, scanning
confocal microscopes as those routinely used today only appeared in the ’90s,
after advances in laser systems, electronics etc. allowed their production [19].
Other developments in the last decades of the twentieth century include
the application of the phenomenon of Total Internal Reflection in Fluorescence
(TIRF) microscopy, as demonstrated by Axelrod in 1981 who visualized cellu-
3
lar focal adhesions on a glass slide [20]. Additionally, the concept of illumina-
tion perpendicular to detection – which was introduced by Siedentopf and Zsig-
mondy in 1902 as ultramicroscopy [21] – was revisited by Voie et al. who built
an Orthogonal–Plane Fluorescence Optical Sectioning (OPFOS) microscope in
1993 [22]. The latter has led to the development of what is commonly known
today as Selective (or Single) Plane Illumination Microscopy (SPIM) – a term
introduced by Stelzer and colleagues in 2004 [23] – a valuable technique that
allows high spatio–temporal resolution, minimal photo–toxicity and thus more
measurements per sample [24].
Certainly, the use of microscopes goes beyond imaging; first, a number of
fluorescence spectroscopy methods have been developed in the last decades,
often referred to as F–Techniques [25]. For instance, Fluorescence Correlation
Spectroscopy (FCS), which was developed independently by Watt Webb and
Rudolf Rigler during the early 1970s [26, 27], measures fluctuations in the flu-
orescence signal to quantify chemical and biological reactions, diffusion and
flow. The breakthrough of the technique was its implementation in a confo-
cal arrangement by Rigler and coworkers in 1990s, which allowed tracking at
single molecule level [28]. An extension of this method is Fluorescence Cross–
Correlation Spectroscopy (FCCS) that uses two distinct fluorophores. The method
was introduced by Ricka and Binkert in 1989 [29] and first applied in biological
systems by Schwille in 1997 [30]. Other techniques include Fluorescence Re-
covery After Photobleaching (FRAP), developed to study dynamics of proteins
within and between cellular membranes [31], Förster (or Fluorescence) Reso-
nance Energy Transfer (FRET) imaging, which can measure distance and detect
molecular interactions in a number of dynamic systems, such as during protein
conformational changes [32] and Fluorescence Lifetime Imaging Microscopy
4
(FLIM) that can be used to detect physicochemical changes in the environment
of the fluorophore [33].
Many non–fluorescence spectroscopic techniques have also been implemen-
ted in microscopic setups allowing the extraction of functional, chemical and
structural information at optical spatial resolutions. For example, Raman cor-
relation spectroscopy (RLS) [34] and coherent anti–Stokes Raman spectroscopy
(CARS) [35] are used to identify molecules and study chemical bonding, and
Photon Correlation Spectroscopy (PCS) is used to determine particle sizes in
suspensions [36].
1.2 Scope
Biofilms are structured bacterial communities in which cells are held together
within a matrix formed by self–secreted polymeric compounds, termed the Ex-
tracellular Polymeric Substances (EPS) [37]. Through cell–to–cell signalling, known
as quorum sensing, bacterial cells are known to coordinate the biofilm forma-
tion, and collaborate in a way that benefits the entire population [38, 39]. This
unique characteristic enables bacteria to survive unpredictable environmental
stress, such as temperature or pH changes, desiccation or pressure, thus mak-
ing them collectively very resistant to attacks that would otherwise destroy the
same cells in their planktonic state. Biofilms have a significant impact on hu-
man health being the leading cause of bacterial infections [40]. They are re-
sponsible for contaminations in the food and dairy industry [41, 42] and on sur-
faces of catheters [43] and medical devices [44], as well as for the degradation of
drinking water quality [45] and biocorrosion [46].
Biofilm colonies are still largely treated as uniform populations similar to
planktonic cells. However, this approach is incorrect, as studies have shown
that a number of chemical and physical gradients exist within the colonies [37].
5
The formation of gradients is stabilized by the immobilization of bacterial cells
within the matrix, and leads to the establishment of sub–populations of cells
that exist in distinct physiological states. As a consequence of this heterogene-
ity, studies of the genetic and physiological pathways that control biofilm devel-
opment, e.g., transcriptomics, metabolomics and proteomics, are almost inde-
cipherable due to the effect of averaging changes across the biofilm for cells in
different physiological states.
It is therefore essential to be able to identify and measure the aforemen-
tioned gradients so that the status of the cells can be defined along the differ-
ent stages of biofilm development, as well as in different structural parts of the
biofilm such as near the outer surface versus deep in the micro–colony core. In
this thesis, two novel spectroscopic techniques, namely Brillouin Imaging and
Transient State monitoring (TRAST) are presented, further developed and ap-
plied on the study of bacterial biofilms.
Brillouin spectroscopy measures the frequency shift of light scattered by in-
herent thermal acoustic waves, which is associated with the stiffness of the me-
dium. Recently, Brillouin spectroscopy has been combined with optical mi-
croscopy, allowing one to perform high–resolution mechanical imaging in a non–
invasive manner [47]. Here, we apply confocal Brillouin microscopy to study
the stiffness of living Pseudomonas aeruginosa biofilms along different stages of
their life cycle, as they grow under constant flow inside a flow cell.
TRAST monitoring was introduced by Tor Sandén, Gustav Persson and Jerker
Widengren [48, 49] as a method to measure kinetics of transient photo–induced
states without the need of fast–scale time–resolved measurements. In TRAST
imaging, the kinetic rates of transient states can be calculated by fitting on a
electronic state model the time–averaged fluorescence intensity of samples illu-
minated with pulse trains of different characteristics. The extracted triplet relax-
6
ation time can then be related to concentrations of quenching agents according
to the Stern–Volmer equation. Here, we implement TRAST imaging on a SPIM
set–up and present an experimental protocol that can measure concentrations
of molecular oxygen inside biofilm colonies.
Precise knowledge of the molecular concentration of oxygen and the me-
chanical properties of biofilms can improve our understanding of the processes
that take place at microscopic levels. For example, oxygen is a key metabolite
that is believed to be responsible for the cueing of dispersal, during which, dras-
tic changes of the structural properties of colonies are expected to take place.
Additionally, oxygen becomes a limiting factor in deeper parts the biofilm, from
where dispersal has also been found to originate. Therefore, we hypothesise that
the depletion of oxygen and the onset of dispersal could be correlated and col-
localised, and we propose the development of methods that can provide us with
quantitative information at optical resolutions. Findings can potentially be of
help in the assessment of drug treatments as well as for strategising methods to
inhibit the growth or promote the removal of biofilms.
7
Chapter 2
Photophysics and Photochemistry
This chapter provides a theoretical background of light–matter interactions that
is necessary for the understanding of the spectroscopy techniques presented in
Chapters 4 and 5, and for the interpretation of the results.
2.1 Light scattering
2.1.1 Overview
Scattering is termed the change of direction of light propagation, resulting from
its incidence and interaction with matter. It can be classified as inelastic or elas-
tic, depending on whether there is energy transfer during the light–matter inter-
action or not.
Other classification methods are based on the nature, size and geometry of
the scatterer: Rayleigh scattering refers to scattering by particles whose size (d )
is much smaller than the wavelength of the incident light (λ0 ), Mie scattering
refers to scattering by spherical particles of any size, and Tyndall scattering is
an extended model of Mie scattering that includes particles of any shape. Bril-
louin scattering – which is of main interest in this thesis and will be described in
8
CHAPTER 2. PHOTOPHYSICS AND PHOTOCHEMISTRY
detail later on – occurs from the interaction of photons with acoustic phonons
that exist in matter. Lastly, Raman scattering refers to the interaction of light
with optical phonons that arise from intra–molecular vibrations and rotations
with energies higher than acoustic phonons. Table 2.1 summarises the above; a
comprehensive review of the different scattering types can be found at ref. [50],
and an in–depth discussion about Brillouin scattering and its connection with
mechanical properties of material can be found at ref. [51].
Type Scatterer Spectral shift

Rayleigh Particles (d << λ0 ) N/A
Mie Spherical particles (any d ) N/A
Tyndall Particles of any shape (any d ) N/A
Brillouin Acoustic phonons (density fluctuations) 5 – 20 GHz

Raman Optical phonons (vibrational or rotational modes) > 1 THz
Table 2.1: Types of scattering. d is the particle size and λ0 the wavelength of
incident light. Spectral shift corresponds to the amount of energy ex-
change during the inelastic scattering processes.
Information about the structure and dynamics of a material being probed by
light can be extracted by studying its scattering spectra. A typical spectrum of
the scattered light, where the frequency of scattered light is plotted on x–axis,
is shown in Figure 2.1. The strongest component corresponds to the elastic
(Rayleigh) scattering, giving rise to the peak located at v i that is the frequency of
the incident light. Additionally, peaks in higher (anti–Stokes) and lower (Stokes)
frequencies are typically observed. These correspond to the energy gain or loss,
respectively, of the photons as a result of scattering. Such scattering phenom-
ena where energy exchange takes place are referred to as inelastic scattering,
and include Brillouin and Raman scattering.
9
Rayleigh
Stokes anti-Stokes
Brillouin Brillouin
Raman Raman
vi frequency
Figure 2.1: Typical scattering spectrum showing the central, elastic, Rayleigh peak to-
gether with a Brillouin and a Raman peak on each side. The two inelastic
side bands are referred to as the Stokes and the anti–Stokes peaks associ-
ated with a lower and higher frequency shift, respectively.
2.1.2 Brillouin scattering
Brillouin scattering is an inelastic scattering process arising from the interaction
of light with density fluctuations – termed acoustic phonons or acoustic lattice
vibrations – that propagate inside a medium. The scattered photons experience
frequency shift by an amount equal to the frequency of the scattering sound
wave [52]. The phenomenon was first reported in 1922 by Lèon Brillouin [53].
For low photon densities, Brillouin scattering is termed as spontaneous and the
scattered light is proportional to the intensity of the incident beam. When in-
cident powers exceed certain thresholds, optical fields substantially contribute
to the phonon population leading to a stimulated excitation of the medium.
Stimulated Brillouin scattering is a non–linear effect commonly studied in the
context of optical fibres. It becomes more prominent for longer optical fibres,
and poses limitations in optical fibre communications since it creates signifi-
cant back–scattering of the beam [54]. This thesis only deals with spontaneous
Brillouin scattering.
10
ks
(a) (b)
θ
kp
kp ki
ks
ki
kp
ki
ks
(c)
Figure 2.2: Schematic of momentum conservation in Brillouin scattering. (a) The gen-
eral case. (b, c) Stokes and anti–Stokes scattering, respectively, from a back–
scattering geometry such as the one used in the experiments of this thesis.
From a classical physics point of view, a travelling acoustic phonon creates
a periodic change of density and hence of the local dielectric constant and re-
fractive index in a medium. To an incident beam of light, the medium acts as
a scattering lattice, which is moving at the speed of sound. For that matter, the
scattered light experiences a positive or negative shift in frequency, depending
on the direction of the phonon movement, due to the Doppler effect.
From a quantum mechanical point of view, an incident photon (wave vector
k i , frequency v i , Figure 2.2) interacts with a phonon and is annihilated. There is
instantaneous generation of a scattered photon (wave vector k s and frequency
v s , travelling at an angle θ) and an annihilation or creation of a phonon (wave
vector k p , frequency v p ). The scattered photon will have either gained or lost
energy equal to the energy of the phonon that was annihilated or created, re-
spectively, referred to as Stokes and anti–Stokes processes, which are responsi-
ble for the symmetry of the spectra (Figure 2.1).
Equations (2.1–2.2) show the energy and momentum conservation in Bril-
louin scattering, respectively. The minus symbol corresponds to the Stokes scat-
11
tering, where the material absorbs the photon, creates a phonon and inelasti-
cally emits a photon with a lower energy than that of the absorbed photon. The
plus symbol corresponds to the anti–Stokes scattering, in which the incoming
photon absorbs a phonon (by phonon annihilation), and a photon with a higher
energy than that of absorbed photon is emitted.
hv s = h(v i ± v p ) (2.1)
k s = ki ± k p (2.2)
The absolute frequency shift of the Brillouin peaks can be calculated by:
2n
vB = V sin(θ/2) (2.3)
λ0
where V is the acoustic velocity, n the refractive index, λ0 the wavelength of the
incident light and θ the scattering angle. It can readily be seen that for θ = 0◦
there is no shift in frequency, whilst, for θ = 180◦ , which is the case in back–
scattering set–ups such as the one used in this thesis, the shift is maximised so
that:
2n
v B max = V (2.4)
λ0
2.1.3 Brillouin modulus and mechanical properties
In material sciences, mechanical properties of materials refer to their response
to applied forces. While a number of measures can be used to quantify the be-
haviour or a sample under stress (force per unit area) depending on the distri-
bution of forces, all of them are based on the same principle, that is, expressing
the ratio of stress over strain (ratio of deformation over initial length).
12
F
A
(a) (b) (c)
Figure 2.3: Types of material deformations based on the distribution of forces. (a) Axial
stress (F /A), applied perpendicularly to the area cross–section (A), resulting
in deformation (∆L). (b) Shear stress (F /A) applied coplanarly to the area
cross–section (A), resulting in shear deformation (∆x). (c) Force (F ) applied
uniformly to the material leading to a change in volume.
In an isotropic material, Young’s modulus, E , is defined as the ratio between
a stress that acts to change the length of a body and the fractional change in
length caused by this force, as shown in Figure 2.3a. The shear modulus, G, is
defined as the ratio between shear stress (stress applied coplanarly to the area
cross section) and shear strain, and describes a situation where all the strains
perpendicular to the stress direction are zero, that is, the volume of the mate-
rial remains unchanged while alterations in the shape occur (Figure 2.3b). The
bulk modulus, K , refers to the situation where force is applied uniformly to the
material and all the strains normal to the stress direction are equal; it is com-
monly used for liquids, and is defined as the ratio of applied stress, or pressure,
to volume change (Figure 2.3c).
A parameter more closely related to our study is the longitudinal modulus,
M . It is defined as the ratio of axial stress to axial strain, that is stress and strain
applied only along the longitudinal axis of the material. It differs from Young’s
modulus only in that there is no shear strain produced. The stress that a sam-
ple experiences under longitudinal acoustic waves in a backscattering Brillouin
microscope, such as the one used in our study, fall under this category.
Biological samples exhibit a viscoelastic behaviour, that is they respond to
stress by a fast deformation followed by a return to the original state – unless
strained beyond their breaking point – with a time delay which corresponds to
13
the energy that is lost during the process. The mathematical formulation of the
complex longitudinal modulus of such a medium can be derived from the dif-
ferential equation describing the propagation of a longitudinal acoustic wave in
a medium [55] and is expressed as:
∆v B
µ ¶
M = M 0 + i M 00 = ρV 2 + i ρV 2 (2.5)
vB
where ρ is the density and ∆v B is the spectral linewidth of the Brillouin peaks.
Further, M 0 and M 00 are termed storage – or Brillouin – and loss moduli, respec-
tively.
Recalling equation (2.3), it can be seen that the Brillouin modulus is con-
nected to the experimentally measured Brillouin frequency shift, u B , via:
¶2
vB λ
µ
0
M =ρ . (2.6)
2n
It should be noted, however, that this relation is valid in general for isotropic me-
dia; in anisotropic matter such as biological samples where direction–dependent
hypersound velocities exist and moduli are expressed as tensors, it can only be
specifically applied to a particular directions. Therefore, even though the lon-
gitudinal modulus is generally related to the bulk and shear moduli by M =
K +4/3G, a direct translation of the measured quantity to commonly–used mod-
uli is not possible. Caution is thus needed when interpreting Brillouin spectra,
as it is later discussed in section 4.4.
14
2.2 Luminescence phenomena
Luminescence is generally termed the phenomenon of light emission by a sub-
stance that has not been heated. There are a number of different types of lu-
minescence; for example, chemiluminescence and electrochemiluminescence
is the emission of light resulting from a chemical or biochemical reaction, re-
spectively, bioluminescence results from biochemical reactions in a living or-
ganism, crystalloluminescence is emitted during crystallization, tribolumines-
cence is the result of breaking bonds inside a material when it is scratched or
rubbed, etc.
In this chapter we will be focusing on photoluminescence that is light emis-
sion from a material after absorption of photons. Photoluminescence can fur-
ther be separated into fluorescence and phosphorescence; in both processes,
light is produced during the electronic transition from higher to lower electronic
states, and the energy of the emitted light is characteristic of the transition en-
ergy. The main way that fluorescence differs from phosphorescence, however,
is that the electronic energy transition that is responsible for fluorescence does
not involve a change in electron spin. This is not a trivial difference, and, as will
be discussed later in detail, has a great impact in the transition dynamics. The
theory presented in this chapter is largely based on the textbook of J. R. Lakowicz
[56], which is an excellent reference point for fluorescence.
2.2.1 Energy states
As quantum mechanical systems, molecules of fluorophores can only take on
certain discrete values of energy. These electronic states and the transitions be-
tween them are usually depicted using Jablonski diagrams, which are line dia-
grams that group the states vertically by energy and horizontally by spin multi-
15
plicity. A typical Jablonski diagram is shown in Figure 2.4 where radiative transi-
tions are shown by solid straight arrows and non–radiative transitions by dashed
arrows.
vibrational relaxation, kic

LUMO S2
HOMO
internal
conversion, kic
inte
r
LUMO S1 cro system
ssin
g
HOMO
k
isc T1 LUMO
HOMO
fluorescence, kf
non-radiative
relaxation knr
triplet relaxation
excitation k0n
non-radiative
rescence, kph
phospho-
hva hvf
hvph
kT
V3
V2
LUMO S0 V1
HOMO V0
Figure 2.4: A typical Jablonski diagram showing the singlet ground state (S 0 ), two ex-
cited states (S 1 and S 2 ) and the lowest energetic triplet state (T1 ). For each
electronic state, four vibrational states (V0 – V3 ) are shown. Photon absorp-
tion of energy hv a can lead to excitation to state S n , at a rate of k 0n , where
n > 0 depends on v a as per equation (2.8). De–excitation mechanisms in-
clude vibrational relaxation and internal conversion (k i c ), fluorescence (k f ),
non–radiative processes (k nr ) and intersystem crossing (k i sc ), which leads
to build–up of T1 . T1 is subsequently depopulated by phosphorescence
(k ph ) or by non–radiative triplet relaxation processes (k T ). The arrows in
the boxes indicate the spin directions at each state.
Typically, fluorophores exist in the ground electronic state (S 0 ). Further, for
each discrete energetic state, the molecule may adapt several vibrational modes,
which are represented by the thin vertical lines in Figure 2.4. In room tem-
perature (T = 300 K) the thermal energy of molecules is E = k B T ≈ 0.026 eV,
where k B ≈ 8.6 × 10−5 eV/K is the Boltzmann constant. The energy separation
between vibrational modes is typically ∆E ≈ 0.19 eV, therefore, when consider-
ing the Boltzmann distribution that gives the probability of particles in a system
to be in a particular state:
E Vn − E Vn−1
µ ¶
P (Vn ) ∝ exp − . (2.7)
kB T
16
One can see that fewer than 1 in every 1,000 molecules will be at the first ex-
cited vibrational state V1 . Further, when considering the separation of electronic
states, which is in the order of a few eV, the probability of a molecule to be at S 1
in room temperature becomes vanishingly small. Therefore it is safe to assume
that all excitation events occur from S 0V0 .
The S 0 state is characterised by a fully occupied Highest Occupied Molecular
Orbital (HOMO) and an empty Lowest Unoccupied Molecular Orbital (LUMO).
When a system absorbs a photon, it gains energy and can enter an excited state,
S n , where n = 1, 2, 3... The exact state that the system will be excited to depends
on the wavelength (or energy) of the incident photon, hv a , which has to be equal
to the energy difference between the states (∆E ) according to:
∆E = hv = hc/λ (2.8)
where h is Planck’s constant, v the frequency, λ the wavelength and c the speed
of light in vacuum. The molecule can now be at different vibrational states of the
excited electronic state, however, fast vibrational relaxation (k i c ) quickly leads
them down to the lowest vibrational level of the S 1 state. From there, one way for
the system to relax to S 0 is by fluorescence, that is by emission of a photon (k f ).
Usually, the system returns to an excited vibrational state of S 0 , and internal
conversion events follow leading to complete de–excitation. The energy of the
emitted photon (hv f ) is lower than that of the absorbed one, and its exact value
can again be calculated using equation 2.8. Fluorescence is a random process
and can be described by a decaying exponential for the S 1 evolution:
µ ¶
−t
S 1 (t ) = S 0 exp (2.9)
τf
17
where τ f is the fluorescence lifetime. For commonly used fluorescent dyes, τ f
is in the order of ns (Table 2.2).
Other than fluorescence – which is a radiative transition – there exist a num-
ber of non–radiative transitions (k nr ). These include the relaxation of each ex-
cited state to its lowest vibrational level, which happens in the ps scale and is
termed vibrational relaxation, the internal conversion that occurs when a vibra-
tional state of an electronically excited state couples to a vibrational state of a
lower electronic state and intersystem crossing (k i sc ). The latter is the transition
to a state with a different spin multiplicity, for example from S 1 to T1 . T1 can
subsequently be depopulated by phosphorescence (k ph ) with the emission of a
photon (hv ph ), or by non–radiative triplet relaxation processes (k T ). Transitions
between states of different spin multiplicity are slower (Table 2.2) since they in-
volve a spin flip, as will be further discussed in the next section. It should also
be noted that it is always v a < v f < v ph as a result of the respective energy loses
before each emission.
Transition Time scale (s)

Absorption 10−15
Vibrational Relaxation 10−12 − 10−10
Internal Conversion 10−11 − 10−9
Fluorescence 10−10 − 10−7
Intersystem crossing 10−10 − 10−6
Phosphorescence 10−4 − 102
Table 2.2: Typical lifetimes of electronic transitions [56].
2.2.2 Singlet vs Triplet states
Singlet and triplet states differ in the electron configuration of their valence
bands – that is the highest energy levels where electrons can be found – as de-
picted by the rectangles beside each energy state in Fig 2.4. The multiplicity of
18
each state is defined by M = 2S +1, where S is the total spin number i.e. the sum
of all individual spins.
Singlet states (S n ) have the property that the electron spins are paired in such
a way that for each electron with an “up” spin (s = +1/2) there is an electron
with a “down” spin (s = −1/2); note that in the excited singlet states, the spin of
the excited electron remains paired with the ground state electron. These states
have therefore S = 0 and M = 1 (hence the name “singlets”). On the other hand,
triplet state molecules have an unpaired spin configuration that gives S = 1 and
M = 3.
The transitions between singlet and triplet states are said to be “quantum–
mechanically forbidden”, meaning that they have much lower probabilities to
occur compared to transitions within states of the same spin multiplicity. They
occur via intersystem crossing (k i sc ) which is a non–radiative process. The prob-
ability of intersystem crossing increases when the vibrational levels of the two
excited states overlap, since little or no energy must be gained or lost for the
transition to happen, and it is higher in molecules with large spin–orbit cou-
pling. Such molecules can experience high triple state build–up at relatively
lower irradiances, and are therefore suitable for TRAST monitoring.
Since another spin flip is required for the relaxation of triplet states, they are
much longer lived in comparison to the singlet excited states. Typical relaxation
times τT range from µs to s, versus singlet relaxation times which are in the ns
range. This stability is exploited in TRAST monitoring [48]. Fluorophores re-
laxing back to the ground state originating from a triplet state may emit weakly
(phosphorescence) or not at all, which is why triplet states are often called dark
states.
19
2.2.3 Spectra
Figure 2.5 shows the absorption and emission spectra of Eosin Y, a dye used
extensively in this thesis. It can be seen that the emission spectrum is shifted
to longer wavelengths than the absorption. This shift corresponds to the energy
dissipation due to internal conversion that occurs between the events of absorp-
tion and emission (Figure 2.4). The spectral shift between the maxima of ab-
sorption and emission is termed the Stokes shift – in honour of Sir George Stokes
who first observed this phenomenon – and is the key concept behind spec-
tral separation that is essential in most fluorescence–based techniques. From
a quantum mechanical perspective, each energy state can be described by a
wavefunction. The absorption maximum represents the most probable tran-
sition, that is the excitation from the lowest vibrational mode of the ground
electronic state, S 0V0 , to that vibrational level of S 1 , S 1Vn , whose wavefunction
overlaps the most significantly with S 0V0 . This is the so–called Franck–Condon
principle, a rule that states that during an electronic transition, the likelihood
for a change from one vibrational level to another will be proportional to the
square of the overlap integral of the initial and final vibrational wavefunctions.
Emission
COO- Absorption
Br Br
Intensity (AU)
O- O O
Br Br
200 300 400 500 600 700 800

Wavelength (nm)
Figure 2.5: Absorption and emission spectra of Eosin Y dissolved in basic ethanol (data
from ref. [57]). Inset: Chemical structure of Eosin Y.
20
It can also be seen that the spectra are continuous, which might appear to
be in disagreement with the image painted by the Jablonski diagrams that show
distinct, quantised energy levels. This apparent discrepancy, however, can read-
ily be explained when considering the fact that organic dyes are large molecules
and thus have plenty of vibrational modes between which transitions can occur
(Figure 2.5:inset). Additionally, the individual line spectra are usually not seen
because of thermal (or Doppler) broadening of the spectra at room tempera-
tures where experiments typically take place.
Another two interesting and important characteristics of fluorescent spectra
are that the emission spectra are typically independent of the excitation wave-
length, and that the fluorescence spectra are approximately the mirror image of
the absorption spectrum. The former results from the fact that all fluorescent
relaxation events originate from the lowest vibrational level of S 1 . This allows
us to acquire the full fluorescence spectrum even when a source of different
wavelength than that of maximum absorption is used. The latter reflects the
fact that the vibrational modes are approximately equally spaced at the differ-
ent energetic states, and that Franck–Condon principle is applied both for the
absorption and the emission.
2.2.4 Quenching by Oxygen
Fluorescence quenching refers to any process that decreases the fluorescence
intensity of a sample. Quenching phenomena can be classified as static or dy-
namic (also referred to as collisional). In static quenching, the fluorophore and
quencher form non–fluorescent complexes while the fluorophore is in the ground
state. In contrast, dynamic quenching refers to energy transfer that occurs while
the fluorophore is in the excited state, and thus creates a new, non–radiative
pathway for de–excitation. Therefore, dynamic quenching affects the lifetimes
21
whereas static does not. This thesis focuses on dynamic quenching events, for
which to take place, the fluorophore and the quencher must come in contact.
Therefore, the quencher must be able to diffuse to the fluorophore during the
lifetime of the excited state.
The relationship between the concentration of a quencher and the fluores-
cence intensity is described by the Stern–Volmer equation:
If 0
= 1 + k q τ f 0 [Q] (2.10)
If
where I f 0 is the intensity without and I f with a quencher, k q is the bimolecu-
lar quenching constant, τ f 0 the fluorescence lifetime of the fluorophore in the
absence of quencher, and [Q] is the concentration of the quencher.
The Stern–Volmer equation (2.10) can be derived as follows (after [56]). The
fluorescence intensity at absence and presence of the quencher (I f 0 and I f , re-
spectively) are proportional to the population of the excited state S 1 at absence
and presence of the quencher, respectively, that is I f 0 ∝ S 1abs and I f ∝ S 1pr s .
When considering continuous excitation, f (t ) ∝ k 01 , a population equilibrium
is established: d S 1 /d t = 0. Therefore, we have:
d S 1abs
= f (t ) − (k f + k nr )S 1abs = 0 (2.11)
dt
in the absence, and,
d S 1pr s
= f (t ) − (k f + k nr + k q [Q])S 1pr s = 0 (2.12)
dt
in the presence of the quencher. Combining equations (2.11–2.12), it follows
that:
S 1abs (k f + k nr + k q [Q]) k q [Q]
= = 1+ (2.13)
S 1pr s (k f + k nr ) k f + k nr
22
which can be rewritten in the form of (2.10), given that the rate of decay at
the absence of quencher is k f + k nr = τ−1

10 . The bimolecular quenching con-
stant is a measure of the accessibility of the fluorophores to the quencher and is
given by k q = f Q k 0 where f Q is the quenching efficiency and k 0 is the diffusion–
controlled bimolecular quenching constant.
One of the most potent quenchers in nature is molecular oxygen. It has a
unique property that its ground state is a triplet state while its lowest available
excited state is a singlet state. The exact mechanism of quenching by oxygen
remains a subject of debate. Reports have suggested that it occurs by mixed
mechanisms that include intersystem crossing, charge transfer, and electron
exchange [56]. The most probable explanation, however, is that when a fluo-
rophore which is excited in a singlet state encounters a triplet oxygen molecule,
a change of its excited singlet state to an excited triplet state occurs. Since the
triplet states are also quenched by oxygen, and are long–lived, they are likely to
be quenched again by the same agent, driving the molecules to the ground state
via a non–radiative path.
For oxygen, it is f Q ≈ 1 and we can relate the bimolecular quenching con-
stant to the diffusion coefficient via the Smoluchowski equation:
k q = 4π(R f + R q )(D f + D q )N A (2.14)
where the sum of R f and R q (the molecular radii of the fluorophore and quencher,
respectively) defines the collision radius, D f and D q are the diffusion coeffi-
cients of the fluorophore and quencher, respectively, and N A is Avogadro’s num-
ber. The diffusion coefficient can be calculated from Stokes–Einstein equation:
D = k B T /6πηR (2.15)
23
where k B is Boltzmann’s constant, η is the solution viscosity and R is the molec-
ular radius.
Dynamic quenching is an additional rate process that depopulates the ex-
cited state, therefore, the lifetime in the presence of quencher becomes τ =
(k f + k nr + k q [Q])−1 . It can readily be shown that an equivalent decrease in flu-
orescence intensity and lifetime is expected to take place. Considering the first
half of equation (2.13), we have:
S 1abs τ−1
= (2.16)
S 1pr s τ−1
0
where τ0 is the lifetime in the absence of quencher. Equation (2.16) can take the
final form of:

If 0 τf 0
= . (2.17)
If τf
Apart from excited singlet states, triplet states can also be quenched by oxy-
gen. In fact, when considering the diffusion coefficient of O2 in H2 O (≈ 2 ×
10−6 cm2 s−1 ) and the usual concentrations of dissolved O2 in air–saturated H2 O
(≈ 30 µM) one can see that, with a typical value of k q ≈ 1010 L mol−1 s−1 [58],
we have k q [O 2 ] ≈ 3 × 106 s−1 . This time scale is orders of magnitude slower than
the fluorescence lifetime (which is a few ns). We can therefore assume that oxy-
gen is a much more competitive quencher of the slower triplet states instead,
and it is possible to re–write the Stern–Volmer equation (2.10) in terms of triplet
lifetimes as:
τT 0
= 1 + k q τT 0 [O 2 ] (2.18)
τT
where τT 0 and τT are the triplet lifetimes in the absence and presence of oxygen,
respectively.
This relationship between the triplet relaxation time and molecular oxygen
is the key concept behind the development of our protocol for quantification of
oxygen concentrations, as will be discussed in Chapter 5.

24
Chapter 3
Biofilms
The present chapter aims to introduce the reader to the biofilm concept. A his-
toric overview is given, followed by discussions on the importance of two fac-
tors, namely oxygen concentration distribution and mechanical properties, on
the growth and lifecycle of the biofilms.
3.1 The biofilm mode of life
Biofilms are structured bacterial communities in which cells are held together
within a matrix formed by self–secreted polymeric compounds, termed the Ex-
tracellular Polymeric Substances (EPS). They are considered to be one of the old-
est, most successful and widespread form of life on Earth [37]. The first recorded
microscopic observation of such ensembles dates back to the 17th century when
van Leewenhoeck experimented with plaque samples taken from human teeth
[4], however, it was only in 1978 when the term biofilm was coined by Bill Coster-
ton [59], who is considered the founding father of the field [60].
Biofilms have a deleterious impact on many domains of life as they can affect
both human health and man–made systems. It is estimated that up to 80% of all
bacterial infections are biofilm related [40], including cystic fibrosis [61], endo-
25
CHAPTER 3. BIOFILMS
carditis [62], urinary tract infections [63], periodontitis [43], and other chronic
infections [64]. Additionally, many medical devices, especially catheters [63],
prosthetic heart valves, cardiac pacemakers, skull implants, cerebrospinal fluid
shunts and orthopedic devices, have been found to be easily colonized by biofilms,
leading to device–associated infections and urgent need for replacement [65, 66,
44]. Other sectors affected are the food and dairy industry [67], where biofilms
can cause food spoilage potentially leading to outbreaks of foodborne pathogens
[41], water distribution systems and plumbing [45, 68], metalworking, structural
and civil engineering where mechanical blockages, impedance of heat transfer
processes and increase in the corrosion rate of surfaces have been documented
[42]. On the other hand, biofilms can also serve beneficial purposes, and are
currently used for the treatment of drinking water, purification of wastewater
and detoxification of hazardous waste [69, 70].
Remarkably, it has been found that the genetic expression of bacteria which
are organised in a biofilm formation can differ from that of the same type bac-
teria that exist in a planktonic state [71, 72]. This is a key concept in under-
standing why remedies developed against planktonic bacteria often fail to work
effectively against biofilms [73, 74], and highlights the importance of studying
the dynamics of formed biofilms rather than individual bacteria.
Another unique feature of biofilms is the ability of their member cells to
communicate with each other and regulate the expression of specific genes once
thresholds in cell densities are reached to promote phenotypes that are the most
beneficial for the entire biofilm. This process is known as quorum sensing [38,
75], and it it because ot it that biofilms are able to survive environmental stress,
such as temperature or pH changes, desiccation or pressure which would oth-
erwise destroy these cells in their planktonic state [39].
26
CHAPTER 3. BIOFILMS
(a) (b)
Figure 3.1: Widefield images of Pseudomonas aeruginosa bacterial cells forming a

colony on a coverslip. (a) Image taken 24 hours and (b) 90 hours post–
inoculations. Scalebar: 2 µm.
Biofilm formation is a dynamic process during which cells change and adapt
the regulation of motility and the production of different EPS components that
affect the physico–chemical properties of the cell surfaces, hence determining
the adhesiveness of the cells [76, 77, 78]. In general, the life cycle of biofilms
is considered to begin when free–floating, or planktonic, bacteria encounter
and attach to a surface (Figure 3.1a). EPS production starts, and the emerg-
ing biofilm community develops a complex, three–dimensional structure [79].
During this stage of maturation, biofilms can take many forms, including pillar–
and mushroom–like microcolonies (Figure 3.1b, top–view), filamentous stream-
ers as well as undifferentiated layers of cells [80]. In the latest of stages of the
biofilm lifecycle, propagation is done through detachment of clumps of cells,
or by release of individual cells. This is known as dispersal, a process that in-
volves bacteria swimming away from the interior portions of colonies, leaving
behind hollow structures (Figure 3.2). The single or clusters of escaped cells are
then able to attach on a surface and create new colonies, or merge with existing
colonies downstream [75, 81].
The dispersal can be triggered in different ways by a number of metabolites.
For example, it has been found that biofilms undergo dispersal in response to a
27
CHAPTER 3. BIOFILMS
sudden increase in nitric oxide [82], oxygen depletion [83], as well as in response
to either a sudden decrease or increase in carbon substrate availability [84]. At
the dispersal stage, detachment of either single motile cells, or sloughing off of
large aggregates of cells happens [85, 81]; this leaves behind empty shell–like
colonies [86, 87] while at the same time, the single cells and multicellular aggre-
gates that dispersed can go on to initiate new biofilms downstream [81].
A number of factors affect the extend of biofilm formation. Biofilms can be
developed under diverse nutrient conditions, from abundant to almost non–
existent. They are more dense in nutrient–rich environments which promote
the transition of bacterial cells from planktonic to biofilm state [70]. Similarly,
pH plays a role in the formation of EPS. For the majority of bacteria, the optimal
pH value is around 7, yet it varies between species [88]. Lastly, the surface of at-
tachment greatly influences the proliferation of biofilms. For example, surface
roughness at microscale levels enhances the adhesion of bacteria by providing
more surface area for cell attachment in the initial phases, and reduces the shear
force experienced by bacterial cells that grow in the presence of liquids flowing
at high flow rates [88]. Other factors such as charge, hydrophobicity and elastic-
ity of the surface are also influential in microbial attachment [89].
In nature and pathology, biofilms can comprise cells from up to several hun-
dred different species [231, 90]. Undoubtedly, in such systems, the interspecies
interactions become important and are expected to affect the biofilm structure
and functionalities. However, given the complexity and the exponential increase
in possible combinations as the species number increases, most biofilm studies
are traditionally carried out in single–species models.
Likewise, in this thesis, biofilm colonies of Pseudomonas aeruginosa are cul-
tured and studied. P. aeruginosa is a common Gram–negative, opportunistic,
rod-shaped (length ≈ 1.5 µm), biofilm–forming pathogen bacterium that can
28
CHAPTER 3. BIOFILMS
thrive both in aerobic and hypoxic conditions. P. aeruginosa can grow as a biofilm
of considerable thickness (up to hundreds of µm) and can secrete large quanti-
ties of polysaccharides which surround the cells by forming a glycocalyx [91].
P. aeruginosa biofilms may exhibit up to 100 times greater resistance to biocides
than the corresponding planktonic cells [92]. It is one of the most widely studied
systems as it is considered a model organism for investigating biofilms [85].
3.2 EPS and mechanical properties of biofilms
Biofilms have been metaphorically called “cities of microbes” [93]. EPS, which
accounts for 50 to 90% of their total organic matter [44], is comprised of polysac-
charides (long polymeric chains that usually represent the largest component of
EPS [94]), various proteins, glycoproteins, glycolipids, and in some cases, extra-
cellular DNA (e–DNA) [95]. At the most basic level, EPS can be thought of as the
key component for maintaining the biofilm architecture and cellular organisa-
tion [96, 97, 98]. Getting back to the analogy of a city, it is not surprising that EPS
has already been termed the “house of the biofilm cells” [95].
The role of EPS is neither static, nor limited to providing structural support
to cells. It determines the porosity, density and water content [96, 37, 97]. The
matrix is also responsible for fortifying the biofilm against desiccation and en-
vironmental stresses [99, 100], providing antimicrobial resistance and tolerance
[39] and making biofilms robust towards chemical perturbations [101].
Another key function of EPS is to facilitate resource capturing by passive
sorption, which gives biofilms a phenotypic advantage over the planktonic mode
of life [39]. This process involves a continuous exchange of nutrients, gases and
other molecules between the environment and biofilms, leading to the estab-
lishment of chemical gradients that facilitate the transport of solutes, nutrients
and small molecules (e.g. oxygen) in and out, as well as within the biofilm [102,
29
CHAPTER 3. BIOFILMS
81, 39]. These gradients have been observed in biofilms regardless of their age
and size [103], and become more prominent in multilayered biofilms. For ex-
ample, in aerobic biofilms, depletion of oxygen in the lower layers of the biofilm
leads to stratification of organisms according to local oxygen availability. [37]
Similarly, nutrient availability gradients can lead to different growth states in
different parts of the biofilm, as starvation of organisms in the lower layers oc-
curs due to the nutrient consumption by organisms in the upper layers [37].
In EPS, different polysaccharides are responsible for different processes; for
instance, adhesion has been linked to the polysaccharides Pel and Psl [79], and
structural stability and cohesion to the polysaccharides of algae, because of their
binding affinity to Ca2+ or Sr2+ ions that form cross–linked polymer networks
[104]. These three exopolysaccharides are produced by P. aeruginosa bacte-
rial cells, and have been identified within the matrix of formed P. aeruginosa
biofilms [80].
The production rate of each polysaccharides can play an important role in
the morphology and the mechanical properties of the colonies [79], and, re-
markably, it has been found that the matrix changes its properties through-
out the lifecycle of the biofilms in ways that benefit the microbial population
[80, 85, 105]. For example, in the early stages of P. aeruginosa biofilm develop-
ment, Psl is anchored on the cell surface in a helical pattern promoting cell–cell
interactions to facilitate the assembly of a matrix that holds bacterial cells in the
biofilm and on the surface [85]. During the maturation stage, Psl has been ob-
served to localise in the periphery of the colonies, facilitating the formation of
matrix cavities that prepare the biofilm colony for seeding dispersal [85, 84] and
eventually leading to the formation of hollow colonies [86, 87].
Being linked with such a large number of functionalities, it is not surpris-
ing that the mechanical properties of the EPS have been a subject of interest for
30
CHAPTER 3. BIOFILMS
Cells EPS
i ii iii
Figure 3.2: Schematic illustrating the late stages of the lifecycle of a biofilm, starting
from a compact colony (i), evolving to a mature colony that facilitates seed-
ing dispersal (ii), and finally to a post–dispersal colony (iii).
many years. In general, biofilms are considered as viscoelastic materials, mean-
ing that they undergo both reversible elastic responses as well as irreversible
deformation, depending on the forces acting on the EPS matrix [106, 101]. The
environmental conditions under which biofilms grow also a role; more specifi-
cally, biofilms grown under higher shear stress are more strongly attached and
cohesively stronger than those grown under lower shear stress [106].
A number of techniques at different scales have been employed, such as
Atomic Force Microscopy (AFM) [107, 105], magnetic tweezers [108], tensile test-
ing using micro–cantilevers [109], microindentation [110], small–scale bulk rheom-
etry [98], microbeads AFM [111], single particle tracking [112], video particle
tracking [80] and others [113, 94, 100]. However, the aforementioned meth-
ods have some limitations: they either require direct interactions between the
sample and a probe; they are able to measure only in discreet positions and
only near surfaces; or they are suitable only for bulk measurements. There-
fore, their applicability in imaging thick, living, three–dimensional samples is
limited, even more so when specimens are grown under continuous flow [105].
Hence, there is a great need of developing a method that can assess stiffness in
living samples non–invasively, in real time and in three dimensions. With that in
mind, we have here applied confocal Brillouin microscopy, a technique capable
31
CHAPTER 3. BIOFILMS
of probing the mechanical properties of specimens by measuring the frequency
shift of light due to thermal fluctuations [47, 114], to investigate the stiffness of
P. aeruginosa biofilms along different stages of their lifecycle.
3.3 The role of oxygen in biofilms
Oxygen is required for respiration in aerobic organisms, such as P. aeruginosa,
and, being the major electron acceptor during cellular respiration, it plays an
essential role in the generation of energy that is necessary for cell maintenance
and growth [115, 116]. The distribution of oxygen within biofilms can be het-
erogenous and may result in bacterial cells exhibiting distinct physiological states
depending on their local oxygen availability [39].
During the initial stages of the biofilm life cycle, the base of the biofilm is
usually aerated. However, as the biofilm develops into larger aggregates, respi-
ratory activity rapidly consumes oxygen, which then becomes depleted on the
interior of the biofilm [117, 119, 118] as the consumption of oxygen by aerobic
organisms in the higher layers of the biofilm is faster than the rate of diffusion
[39]. This dynamic behaviour leads to the formation of oxygen gradients, which
can be relatively stable, or fluctuate and move vertically over time with time
scales of seconds, minutes or hours [115]. Oxygen limitation significantly affects
metabolic activities. For some bacteria, oxygen depletion has been identified as
an environmental cue for seeded dispersal [87, 120, 83], which often occurs from
the centre of the biofilm, in agreement with the understanding that oxygen be-
comes limiting in the interior of biofilms. Lack of oxygen has also been linked
with changed physiology, where some organisms may grow fermentatively, oth-
ers may switch to alternative electron acceptors and some bacteria may enter a
stationary phase and show little cellular activity [44].
32
CHAPTER 3. BIOFILMS
Traditionally, measurements of oxygen have been carried out using Clark
type electrodes [121]. The main limitation of using electrodes, however, is that
they consume oxygen while measuring [122]. Additionally, measurements with
microsensors are invasive; their tips – which can be up to 10 µm in diameter
[119] – can obstruct the local flow field and alter the micromechanical proper-
ties of the sample leading to artefacts [123].
Another approach is the use of optical methods, which do not consume oxy-
gen and are able to image oxygen concentrations over large areas [124]. Such
techniques are based on the principle of dynamic quenching of luminescence
of an indicator in the presence of oxygen [125, 56, 126]. The simplest applica-
tion comes in the form of planar optodes [119], which have been used to obtain
oxygen profiles of various types of biofilms, such as tap water biofilms [126],
biofilms formed by an activated sludge from a waste water treatment plant [119]
and biofilms of P. putida [128]. One of the shortcomings of optodes is that they
often lead to erroneous measurements due to the heterogeneity in the excitation
light field. To overcome this, ratiometric [129] or lifetime [130] measurements
often need to be employed. Another important limitation is that such devices
can only measure close to the surface of the sensor, limiting their applicability
in thick samples such as biofilms.
Hence, there is a need for developing tools to measure oxygen dynamics
in three–dimensions without depth limitations in order to map the profile of
an entire biofilm structure. In this study, we have used TRAST monitoring by
time–modulated illumination, which is a technique that can measure the rel-
ative populations of fluorophores in singlet and triplet (transient) states, and
their transition rates [48, 49]. This is done by measuring and integrating the
fluorescence emission intensity while exciting the sample with trains of pulses
of different characteristics. We have also developed a protocol that allows faster
33
CHAPTER 3. BIOFILMS
acquisition and can provide absolute values of local oxygen concentrations, which
was validated in different microenvironments.
The method was here combined with SPIM, an imaging approach commonly
used in thick biological samples as it allows for unparalleled optical slicing and
decreased phototoxicity [131]. Using this approach we were able to identify ar-
eas of different oxygen concentrations inside biofilm colonies of P. aeruginosa.
34
Chapter 4
Confocal Brillouin Microscopy for
Mechanical Measurements
This chapter introduces the application of Brillouin Spectroscopy on a Confocal
Microscope for measuring the stiffness of living biofilm colonies. Detailed in-
formation about the methods and techniques are presented, followed by a dis-
cussion of results from measurements on hydrogel gels and PA biofilms. Results
of this chapter have been partially published in ref. [132].
4.1 Introduction
4.1.1 Confocal microscopy
The concept of confocal microscopy was patented in 1957 by Minsky [16, 17],
who wanted to overcome the limitations of existing wide–field microscopes in
order to image dense, unstained neural networks by optical sectioning. The first
usage of the term “confocal”, however, came from Colin J. R. Sheppard and his
group, who published a theoretical analysis of confocal and laser–scanning mi-
35
CHAPTER 4. BRILLOUIN MICROSCOPY FOR MECHANICAL MEASUREMENTS
detector
pinhole
dichroic mirror
Source
objective lens
focal plane
sample
Figure 4.1: Schematic outlining the key components and operating principles of a con-
focal microscope.
croscopes in 1977, and later developed microscopes with epi–laser–illumination,
stage scanning and photomultiplier tubes as detectors [18].
In a confocal microscope, light is emitted by a point source and is directed to
the sample. A pinhole1 is placed in front of the detector at an optically conjugate
plane with the sample and rejects out–of–focus light rays, which would lead to
image degradation (Figure 4.1). Confocal microscopy is able to produce thin
(0.5 to 1.5 µm) optical sections through thick fluorescent specimens.
Confocal microscopy is one of the most popular techniques currently used
in life sciences and cell biology due to the achievable image quality and the rel-
ative ease of implementation and operation [133].
The main drawback of a confocal microscope is the sequential, point by
point (usually by a scanning galvanic mirror) image acquisition, which results
in rather slow image acquisition times. Another consideration is the possible
increased photobleaching due to the continuous scanning. Those issues have
however been addressed by the development of spinning disk confocal microsco-
py, which allows fast sample scanning that decreases the total acquisition time
1
In certain applications, the pinhole is replaced by a single mode fibre.
36
and hence photobleaching [134], and by multi–photon microscopy that reduces
overall photobleaching by limiting it to the focal plane, even though bleaching
and higher–order photon interactions within the focal volume remain an issue
[135].
4.1.2 Brillouin spectroscopy
The theoretical background of spontaneous Brillouin scattering has been intro-
duced in Section 2.1.2; in short, when light interacts with density fluctuations
–termed acoustic phonons– inside a medium, there is an energy exchange that
causes a frequency shift in the scattered light.
Brillouin spectra yield information about the phonon’s properties, which can
be associated with the viscoelastic characteristics of the medium [114]. First,
information about the acoustic velocity and the viscoelastic coefficients of the
material can be inferred from the magnitude of the frequency shifts. Second,
the spectral line width can be related to the lifetime of the photon–phonon in-
teraction, which gives information about the viscous properties, and, last, polar-
ization of the scattered light can reveal information about the local anisotropic
properties of the medium. This thesis deals with measuring the magnitude of
spectral shifts for probing the local stiffness of the samples.
Brillouin scattering was first observed experimentally by Gross [136]. The
first experiments had been limited to compressed gases and fluids, however,
with the advent of laser sources in the 1960s, Brillouin spectroscopy started to
find a wider range of applications in solids, liquids and gases [137]. Since then,
the technique has been extensively used in material physics and environmental
sensing to study the viscoelastic and high–frequency viscous behavior of crys-
talline and amorphous solids [138, 139, 140, 141], polymers [142], as well as to
investigate the behaviour of elastic constants as a function of pressure [143] and
temperature [144].
37
Despite its rapid adaptation in those fields, Brillouin spectroscopy was not
equally well–embraced by biomedical imaging for reasons that will be discussed
later. The first such application is dated to 1980, when Vaughan and Randall
measured the elastic properties of the lens and cornea of the eye [55]. Even
though this study showcased that Brillouin spectroscopy is compatible with bi-
ological matter, the technique suffered from very long acquisition times and low
Signal–to–Noise Ratio (SNR). The major challenge in Brillouin scattering has al-
ways been isolating the Brillouin signal, which is on the scale of a difference of
a few pm, and orders of magnitude weaker than the elastically scattered light,
which makes conventional spectrometers unable to resolve it. This is partic-
ularly problematic in biological samples where significant elastic scattering is
commonly expected. Due to these limiting factors, early research was focused
only on ex vivo measurements on fixed samples that can remain unchanged for
long periods of time, such as collagen fibres [145, 146, 147] and bone [148].
Traditionally, high–finesse scanning Fabry–Perrot interferometers or angle–
dispersive etalons were used. These are optical devices that consist of a trans-
parent plate with two reflecting surfaces, or two parallel highly reflecting mir-
rors. As light travels through the paired flats, it is multiply reflected and pro-
duces multiple transmitted rays which are collected and focused to produce an
interference pattern. However, due to their inherent limitation in transmission
efficiency, etalons are too slow to provide a satisfactory SNR in most biological
samples [149]. In 1970, Sandercock showed that this shortcoming could be sig-
nificantly improved by a multipass arrangement [150]. Such designs have since
been used to a number of studies with biological interest, such as to observe
Brillouin scattering from single muscle fibres [151], to investigate thermal de-
naturation of lysozyme [152], and to determine the complete elastic moduli of
spider silks [153].
38
Up to this point, Brillouin spectroscopy had been limited only to acquisition
of spectra in point measurements. In 2005, however, Koski and Yarger intro-
duced the concept of Brillouin microscopy, which refers to the pixel–wise ac-
quisition of spectra while the sample is being scanned. In their first paper, they
presented a Brillouin image of a solid–liquid interface at a spatial resolution of
20 µm [47]. It should be noted, however, that the acquisition time was still too
high (10 s per pixel) to be applicable in dynamic biological measurements.
An important advancement came about three years later, in 2008, with the
implementation of Virtually Imaged Phase Arrays (VIPAs) in Brillouin micro-
scopy [154]. A VIPA is a solid Fabry–Perot etalon with a highly reflective front
surface, except for a narrow window through which the beam enters, while the
back surface is partially reflective. These devices were originally developed in
1996 [155]. In VIPA arrangements, the input beam is focused by a cylindrical
lens and enters the etalon at an angle through the aforementioned window. The
beam makes multiple internal trips by reflecting on the surfaces. An array of
output beams with increasing phase delays is produced. These beams interfere
at the output and components of different frequencies are emitted at different
angles. VIPAs can achieve large angular dispersion and low light loss leading to
faster data acquisition speeds as compared to conventional Fabry–Perot etalons.
Indeed, by using a VIPA, imaging of a mouse eye in situ became feasible with ac-
quisition time of only 500 ms [154].
Nonetheless, because the signal strength of the Brillouin scattered light is
small compared to the elastic signal, a single VIPA often does not allow clear
separation of the Brillouin peak from the background. A solution to improve the
suppression of the non–specific background that in turn permits clearer differ-
entiation of the Brillouin peak is cascading a number of sequential spectrome-
ters. For example, cascading two and three VIPAs in a cross–axis configuration
has shown a 55 dB and 80 dB SNR improvement, respectively [156].
39
A highly efficient Charge–Coupled Device (CCD) camera is commonly used
in conjunction to facilitate data acquisition, however, even in two–stage VIPA
systems, discrimination against strong light scattering can be difficult in thick,
opaque samples [157]. Therefore, additional methods for background clean-
ing have been developed and employed. These methods include molecular ab-
sorption cells (that can offer up to 50 dB suppression) [157, 158], coupling of
a Michelson interferometer [160], multi–pass Fabry–Perot interferometer [159],
tunable etalon–based notch filters [161] and common path interferometric fil-
tering [162].
All these improvements, together with background studies that showed the
applicability of high Numerical Aperture (NA) objective lenses [163], have al-
lowed Brillouin spectroscopy to find a number of applications in the life sci-
ences and medicine, both in tissue–level, such as for in situ and in vivo biome-
chanical imaging of the eye tissues [154, 156, 52, 164, 162], screening for in-
creased total protein in cerebrospinal fluid (CSF) during meningitis [165], study-
ing the viscoelasticity of amyloid plaques in transgenic mouse brain [166], and
quantification of plaque stiffness in arteries [167], and in single–cell or subcel-
lular level [168, 169, 170, 171].
4.1.3 The conventional use of term ’stiffness’
As discussed earlier in Section 2.1.3, the measured frequency shifts are con-
nected to the speed of sound in the material, and, in turn to the longitudi-
nal modulus as well as to other mechanical moduli. However, it is currently
not possible to convert the measured frequency shifts to absolute values that
are traditionally used in biomechanical measurements (as for example Young’s
modulus) for a number of reasons: First, equation (2.3) only holds for isotropic
materials, while, in anisotropic media, M is a tensor, that is, there exists a sep-
40
arate modulus in each direction, corresponding to direction–dependent hyper-
sound velocities. Second, the refractive index and material density are not al-
ways known in a pixel–wise manner, and local differences might exist. Third,
the relationship between macro– and micro–mechanical quantities is, in most
cases, complicated and very much dependent upon the medium being inves-
tigated, and, last, Brillouin microscopy probes a material at ultrafast GHz fre-
quencies, such that the viscoelastic behaviour may drastically differ from the
behaviour exhibited when the same material is probed by other methods that
operate in much slower scales, from seconds to minutes.
Unsurprisingly, results acquired by Brillouin imaging studies differ by sev-
eral orders of magnitude from those acquired by rheology, AFM or other es-
tablished techniques [156]. Some researchers have tried to correlate Brillouin
modulus with Young’s modulus, based on empirical observations [156, 168],
something that has led a number of studies to report Brillouin microscopy re-
sults using Pa scale [172, 170]. A more recent study attempts to shed some light
on what exactly Brillouin microscopy measures [173]. By investigating the fre-
quency shifts in differently prepared hydrogels, measured at different degrees
of swelling, the authors of the latter study suggest that the Brillouin modulus
is not directly related to the Young’s modulus, but rather to the polymer solid
fraction that is intrinsically coupled to Young’s modulus due to the mechanics
of swelling.
It is evident that, to date, there is a lack of a proper theoretical model and
fundamental understanding of the connection between Brillouin shift and elas-
tic modulii in biological material, however, it is the consensus that the Brillouin
shift is indicative of the stiffness of the probed material. Therefore, in this the-
sis, the raw measured Brillouin shift values (in GHz) are used, and, for simplic-
ity, materials with a larger Brillouin frequency shift will be referred to as “stiffer”,
similar to other studies in the literature [167, 169, 162].
41
4.2 Material and Methods
4.2.1 Experimental setup
Confocal microscope for Brillouin and fluorescence imaging The Brillouin
fluorescence confocal microscope was constructed using a commercial Olym-
pus IX–71 microscope platform. The laser source for Brillouin imaging is a Con-
tinuous Wave (CW) diode–pumped solid state laser (Cobolt 05–01 Jive) operat-
ing at 561 nm with a maximum power of 300 mW. The fluorescence excitation
laser is a diode laser (Cobolt 06–01 488) operating at 488 nm with maximum
power of 60 mW.
water cuvette
M filter
sample DM2 BS to filter

BK7
(b)
sCMOS
EF 2-VIPA spectrometer
DB
DB S2
Mask 2
BE
VIPA 2
Pinhole S1
Mask 1
Detector DM1 C2
488 nm VIPA 1
C1
(a) 561 nm (c)
Figure 4.2: (a) Confocal arrangement for Brillouin and fluorescence imaging. DM:
Dichroic mirror, BS: beam splitter cube, BE: beam expansion, M: mirror, EF:
emission filter, DB: achromatic doublet lens. (b) Schematic of the common–
path interferometric filter using BK7 glass slab. (c) Crossed-axis VIPA spec-
trometer. C1, C2: cylindrical lenses, S1, S2: spherical lenses, DB: achromatic
doublet pair.
Two laser beams were delivered to the microscope using optical fibres (kine-
FLEX), combined by a long pass dichroic mirror (DM1) (Chroma T550lpxr) and
then expanded by 6× beam expanders (BE) to fill the entrance pupil of the ob-
jective. The beams were split by a beam splitter (BS) (Thorlabs BS019) with a
power ratio of 30:70 (R:T). The transmitted beams were reflected by a mirror
42
(M) to a 10X microscope objective, which focuses the beams into the water cu-
vette that is used for calibration purposes. The reflected beams were directed to
the oil–immersion microscope objective lens (Nikon UPLFLN / 100X, NA = 1.3)
for the Brillouin and fluorescence imaging (Figure 4.2a).
Light collected from the sample was first spectrally separated by the dichroic
mirror DM2 (Chroma ZT488/561TPC), which transmits the Brillouin and Rayleigh
signals and reflects the fluorescence signal. Approximately seventy percent of
the Brillouin and Rayleigh signals were transmitted by the beam splitter (BS) and
coupled into the fibre to be delivered to the common path interferometric filter.
The fluorescence signal reflected by DM2 was further filtered by an emission
filter (Chroma ET525/50) (EM). An achromatic doublet lens ( f = 75 mm) (DB)
was used to focus light on a motorised pinhole (Thorlabs MPH16) that couples
the signal to a multi–mode fibre delivering the signal to the detector. A photon
counting detector (Hamamatsu H10682–110) together with a data acquisition
unit (NI USB–6351) were used to measure the fluorescence signal strength.
Common path interferometer A wavefront division interferometer was used
to suppress the elastically–scattered light, as presented in ref. [162]. More specif-
ically, light delivered from the microscope through the fibre was collimated to
achieve a beam diameter of 4 mm. A glass slab was aligned so that exactly half
of the beam was transmitted through the slab while the other half propagated
above the glass (Figure 4.2b). The optical path difference between the two parts
of the beam (δ) that depends on the angle between the prism and the beam (θi )
is given by:
θi2
Ã !
δ = d g (n − 1) 1 + , (4.1)
2n
where d g and n are the length and refractive index of the glass slab, respectively.
43
Destructive interference occurs when the two halves of the beam overlap
upon and couple into a single–mode fibre. The exact wavelength at which de-
structive interference occurs can be adjusted by titling the prism to vary the
angle θi , and therefore the interferometer can be tuned to suppress only the
Rayleigh scattered light. The transmission function is a cosine function with
a Free Spectral Range (FSR) of c/δ, where c is the speed of light in vacuum.
A d g = 30 mm long BK7 glass prism (n = 1.51) was used in our experiment to
achieve a FSR of 20 GHz. A maximum extinction of 40 dB can be achieved with
our 1 MHz linewidth laser source.
VIPA spectrometer A two stage, crossed–axis VIPA spectrometer, as shown in
Figure 4.2c, was used to extract the Brillouin frequency shifts in our experiments
[156, 169]. Both VIPA 1 and VIPA 2 were custom–built with a FSR of 39.5±0.5 GHz
(LightMachinery Inc, Ontario).
Light from the interferometric filter output fibre was collimated to produce
0.35 mm beam waist and focused by a 100 mm cylindrical lens (C1) onto the first
VIPA entrance window. A second 100 mm cylindrical lens (C2) was used to form
the spectrum at its focal plane. A mask (Mask 1) is used to shape the spectrum.
A 100 mm spherical lens (S1) was used to focus the light further onto the second
VIPA whose axis was orthogonal to the first VIPA. Another 100 mm spherical lens
(S2) forms the spectrum at its focal plane where a second mask (Mask 2) was
used to shape the spectrum. A pair of 100 mm achromatic doublets (AC508–
100–A–ML, Thorlabs) with separation of 10 mm between them (DB) was used to
magnify and image the spectrum onto a sCMOS camera (Andor Neo 5.5 sCMOS)
(sCMOS).
44
4.2.2 Sample preparation
Hydrogel beads
Calcium alginate hydrogels were prepared similar to the protocol described in
ref. [174]. In specific, sodium alginate (Acros Organics, U.K.) solutions (concen-
trations of 1% and 3%) in distilled water (dH2 O) were prepared and autoclaved.
Using a pipette, droplets of 10 – 20 µL of the prepared sodium alginate solution
were gently dropped into the wells of a 6 well plate (Nunclon Flat, Thermoscien-
tific) containing filter–sterilised CaCl2 (100 mM and 400 mM). The cross linking
was almost instantaneous. The drops were left to gelate and settle for 30 min
in CaCl2 , before they were washed twice with sterile dH2 O. All imaging exper-
iments were performed the same day. Beads were kept in sterile dH2 O before
imaging.
Biofilms
Bacterial strains and growth conditions P. aeruginosa strain PAO1 were grown
in Luria–Bertani (LB) medium at either room temperature or 37o C with the ad-
dition of agar, as required. GFP–tagged P. aeruginosa PAO1 was engineered fol-
lowing the procedure outlined in ref. [175, 176].
Flow cell Biofilm assay Biofilms formation under continuous flow was per-
formed as described in established protocols [177, 178], as shown in Figure 4.3a.
In specific, a flow cell system consisting of a 2 L medium bottle, Fluorinated
Ethylene Propylene (FEP) tubing (30 mm long, 2.1 mm ID and 2.3 mm OD) (Mas-
terflex, Germany), a bubble trap (Technical University of Denmark, Department
of Systems Biology), a flow cell (made by gluing – using Silicone glue (3M Super
Silicone Sealant Clear) – a 50 × 24 mm glass coverslip on a pre–fabricated poly-
carbonate base on which three channels were milled, each 1 mm deep × 4 mm
45
wide × 40 mm long) (Technical University of Denmark, Department of Systems
Biology) as shown in Figure 4.3b, and a 2 L waste bottle was assembled and used
in all experiments. Technical drawings and a protocol for constructing the flow
cell can be found at ref. [177]. A six roller peristaltic pump (Langer Instruments,
U.S.A.) was used to pump the medium through the biofilm flow cell at a flow
rate of 6 or 20 ml / h, for the experiments with standard and high flow, respec-
tively, giving rise to flow speeds of 0.042 and 0.14 cm s−1 , respectively. A stage
was specifically manufactured at Imperial College London for the flow cell to be
placed under the microscope (Figure 4.3c).

(a) Air filter
Microscope
Bubble
Peristaltic trap Flow cell Waste bottle
Medium bottle pump
40 mm
4 mm
(b) (c)
Figure 4.3: The continuous–culture biofilm flow cell setup used in the confocal Bril-
louin microscopy experiments of this thesis. (a) Schematic drawing (not to
scale). (b) Close–up photograph of flow cell. (c) The flow cell on the confocal
microscope.
The FEP tubings were autoclaved and primed with minimal medium ([15
mM (NH4 )2 SO4 , 34 mM Na2 HPO4 , 22 mM KH2 PO4 , 58 mM NaCl, 1 mM MgCl2 ,
0.1 mM CaCl2 and 0.001 mM FeCl3 ], supplemented with 2 g/L of Glucose plus
2 g/L of Casamino acids) before the start of every experiment. GFP–tagged P.
46
aeruginosa were grown overnight in 40 g/L LB medium (Difco) in a shaking in-
cubator at 37o C and 200 rpm. The culture was diluted to OD600 ≈ 2.0, in minimal
medium and 300 µL was injected into the FEP tubing near the entry port of each
of the channels of the flow cell. The inoculum was allowed to attach in the flow
cell for 1 h without flow, at room temperature. The flow of the minimal medium
was then resumed throughout imaging.
All protocols and risk assessments for developing biofilms in a flow cell were
approved by the Safety Department of Imperial College London.
4.2.3 Acquisition and analysis
Scanning and data acquisition was controlled by in–house developed Labview
(National Instruments, Austin TX, USA) software and data was analysed using
Matlab (The MathWorks Inc., Natick, MA, USA). Typical exposure times varied
between 500 – 900 ms for the Brillouin and 10 – 100 ms for the fluorescence
imaging. A 50 µm pinhole was used for the fluorescence imaging, which corre-
sponds to ≈ 2.25 Airy units2 .
Subsequently, as the sample was scanned, both the Stokes and anti–Stokes
Brillouin peaks were captured. Their positions (determined by fitting a Lorentzian
function on the spectrum) were used to calculate the frequency shift at every
point of the sample, as:
FSR − FxP × (B 2 − B 1 )
uB = (4.2)
2
where FxP is a coefficient that converts the spatial position along the dispersal
axis (in pixels) to frequency (in Hz), B 2 and B 1 are the spatial position of the
Stokes and anti–Stokes Brillouin peaks along the dispersion axis (Figure 4.4a).
2
d Airy = 1.22λ M/NA = 22.2 µm where λ ≈ 510 nm, M = 1 (magnification), NA = D/2 f = 0.28,
D is the 6× expanded beam (original radius 0.35 mm) and f = 75 mm the focal distance of the
achromatic doublet.
47
(a)
R
150
B1
Intensity (a.u.)
100
B2
50
R
0
10 20 30 40 50 60 70 80 90 100
(b) Number of Pixels
Figure 4.4: (a) Brillouin spectrum of water, measured by two-stage VIPA spectrome-
ter and interferometer. The Stokes and anti-Stokes Brillouin peaks (B) and
Rayleigh peaks (R) are shown. (b) The intensity profile along the dispersion
axis is fitted with Lorentzian curves to extract the positions of the Brillouin
peaks.
Brillouin microscope calibration At the start of every experiment the micro-
scope was calibrated using a distilled water sample placed in a cuvette. A typi-
cal acquired spectrum is shown in Figure 4.4, where the Brillouin and Rayleigh
peaks are marked with B and R, respectively. The Stokes and anti–Stokes Bril-
louin peaks are located with the software and their positions are used to define
the slope dispersion axis. The theoretical Brillouin frequency shift of water is
known to be 7.04 GHz for a 561 nm laser wavelength. Therefore, from the mea-
sured distance between the two peaks, a coefficient that relates frequency shift
and peak position in spatial domain FxP (Frequency per Pixel, in GHz) can be
calculated as:
FSR − 2 × 7.04
FxP = (4.3)
B2 − B1
where FSR is the free spectral range of the VIPA etalon, and B1 , B2 are the posi-
tions of the Stokes and anti-Stokes Brillouin peaks along the dispersion axis.
Zero–padding The Discrete Fourier Transform (DFT) converts a finite sequen-
ce of N samples of a recorded signal to an equally–sized sequence of samples in
the frequency domain. Zero–padding is commonly used in signal processing to
improve speed and precision of the results; it involves appending one or more
48
zeros in one domain and results in an increased sampling rate in the other do-
main [179].
A signal – here the spectrum image acquired by our camera – of N samples
can be expressed with an inverse DFT as:
NX
−1
f (m ? δx) = F (n ? δu) exp [i 2πmn/N ] (4.4)
n=0
where δx is the sampling distance in image space, δu is the sampling in Fourier
domain, f (m ? δx) is the spectrum image and F (n ? δu) is the discrete Fourier
transform of the spectrum image. It follows that:
1
δx = (4.5)
(N ? δu)
Padding M zeros at the borders of the transformed image in the Fourier do-
main allows us to obtain a denser sampling grid when applying the inverse DFT,
which improves the localisation of peaks when fitting the spectrum image with
Lorentzian curves to localise the Brillouin peaks (Figure 4.5).
This process does not improve the resolution as it does not produce new
information. However, it increases the number of points in the spectrum, as
they are directly proportional to the number of points used in the DFT. That is
essentially an interpolation in the image domain.
49
R 190 (b)
180
B1 170 Data points
Intensity (a.u.)
160 Lorentzian Fitting
150
140
130
B2 120
R 110
100
0 10 20 30 40 50 60 70 80 90 100
(a)
Pixel number along dispersion axis
20
(c)
19
18
Data points
17
Intensity (a.u.)
Lorentzian Fitting
16
15
14
13
12
11
0 50 100 150 200 250
Pixel number along dispersion axis
Figure 4.5: (a) Brillouin spectrum measured at a point inside a living biofilm. The Stokes
and anti–Stokes Brillouin peaks (B) and Rayleigh peaks (R) are shown. The
dispersion axis is marked by the red line. (b, c) Intensity profiles along the
dispersion axis, and Lorentzian fits, shown before (b) and after (c) zero-
padding.
50
4.3 Results
4.3.1 Measurements in hydrogel beads
In order to assess the applicability of the method in bacterial biofilms, we first
measured the Brillouin shift in cross–linked Alginate Acid (A. A.) – calcium chlo-
ride (CaCl2 ) hydrogel beads (Figure 4.6). Such hydrogels are commonly used as
model synthetic matrices, since alginates, which are abundant in biofilms, are
known to affect the mechanical properties of EPS in bacterial colonies [174, 180,
181].
Typical optical and Brillouin cross–sections of such a hydrogel bead (100
mM CaCl2 and 2% A. A.) can be seen in Figure 4.6c,d. The Brillouin shift mea-
sured in the region of interest (ROI) that is marked by the dashed line was 7.28 ±
0.03 GHz. Next, measurements were performed in beads prepared with a range
of biologically–relevant A. A. and CaCl2 concentrations. Figure 4.6e shows the
means of 10 technical replicate measurements that were performed at 3 differ-
ent points within each bead (total N = 30). The error bars represent the standard
deviations. It can be seen that the Brillouin shift was positively correlated to the
concentration of the A. A. and CaCl2 . An increase of A. A. concentration from
1% to 3% led to an increase in Brillouin shift from 7.02 ± 0.09 GHz to 7.51 ± 0.06
GHz when gelated in a 100 mM CaCl2 bath, and from 7.41 ± 0.07 GHz to 7.59 ±
0.05 GHz in 400 mM CaCl2 . All measurements were performed with the beads
immersed in sterile water.
4.3.2 Characterisation of the stiffness of P. aeruginosa biofilms
Previous studies have shown that P. aeruginosa biofilms form colonies that ex-
pand into pillar–like and mushroom–shaped colonies when grown and devel-
51
7.8
(a) (b) 7.4
7.0
6.6
GHz
7.8
7.6
Brillouin shift (GHz)

7.4
7.2
(c) (d) 7.0
6.8
6.6
1% A.A. 3% A.A. 1% A.A. 3% A.A.

(e)
GHz
100 mM CaCl2 400 mM CaCl2
7.10 7.15 7.20 7.25 7.30 7.35
Figure 4.6: (a) Photograph of a typical Alginate Acid (A. A.) – calcium chloride (CaCl2 )
hydrogel bead. (b) Low–resolution Brillouin image of such a bead (pixel size:
30 µm). (c) Wide field image and (d) Brillouin image taken at the interface
of a hydrogel bead (prepared by cross–linking of 100 mM CaCl2 and 2% A.
A.) and water. (e) Brillouin shifts measured in hydrogel beads of different
compositions. The error bars denote the standard deviations of 30 measure-
ments: ten technical replicates at 3 different points within each bead. Scale
bar on (b – d): 200 µm. Note that all measurements were performed with
the beads immersed in dH2 O to avoid dehydration, however, for illustration
purposes only, panel (a) shows a bead resting on a petri dish outside water.
oped in flow cells similar to the ones used in our experiments [178, 105]. Here,
biofilm colonies (or colonies) are defined the visually observable convex struc-
tures that rise above the surrounding biomass, characterised by a circumference
and height. A total of 24 such colonies of different sizes (average diameters rang-
ing from 25 µm to 95 µm) were imaged at various depths and at different time
points along their life cycle.
In all images, gradients of Brillouin shifts within the biofilms were observed.
In particular, two different patterns were seen: 21 out of the 24 imaged colonies
tended to have increasing Brillouin shifts and biomass towards their centres, as
shown by the Brillouin and fluorescence images, respectively (Figure 4.7:Panel I,
example). In the remaining 3 colonies, the interior appeared to be less stiff than
the periphery, or possibly hollow (Figure 4.7:Panel II, example).
In order to investigate a possible relationship between colony size and stiff-
52
I GHz
7.6
7.4
7.2
7.0
10 μm (b) 10 μm (c) 10 μm
(a)
6.8
II GHz
7.6
7.4
7.2
7.0
(a) 20 μm (b) 20 μm (c) 20 μm
(c)
6.8
Figure 4.7: (a) Widefield, (b) Brillouin, and (c) Fluorescence images. Panel I: Compact
colony taken 60 h post inoculation at a depth of 24 µm inside a 42 µm thick
biofilm. Panel II: Dispersed colony taken 100 h post inoculation at a depth
of 15 µm inside a 35 µm thick biofilm.
ness, we calculated the mean Brillouin shift of each imaged colony by averaging
the results within a region of interest (ROI) that encloses the whole colony. The
results are plotted against the mean diameter in Figure 4.8, where no correlation
between colony size and average Brillouin shift was found (Pearson correlation
coefficient r = −0.0179 and p = 0.926).
4.3.3 Brillouin shift measured at increasing depths
Next, we investigated the possible correlation between depth and stiffness. The
thickness of the colonies imaged in our study varied from 16 µm to 55 µm. By
convention, we define Z = 0 at the vertex of each colony and therefore z increases
with increasing depth towards the substrate. Figure 4.9 shows such a z–stack
in a 32 µm colony taken in a biofilm 80 h post inoculation. The Brillouin shift
increased towards the centre of the cross–section at each of the different depths,
as well as overall with increasing depth (Figure 4.9b).
53
7.8
7.6
7.4
7.2
7.0
20 30 40 50 60 70 80 90 100
Mean diameter (μm)
Figure 4.8: Brillouin shift in colonies of various sizes. Data points denote the means,
and error bars the standard deviations from all pixels within the ROIs en-
closing the colony. ROIs drawn by visual inspection of the corresponding
widefield images. Biofilms grown under constant flow velocities of either
0.042 or 0.14 cm s−1 (circles and triangles, respectively).
Z = 4 μm Z = 9 μm
10 μm 10 μm 7.7
7.6
7.5
Z = 14 μm 10 μm
Z = 19 μm 10 μm
7.4
7.3 (b)
(a) GHz 5 10 15 20
7.0 7.2 7.4 7.6 7.8 Depth (μm)
Figure 4.9: (a) Brillouin image cross sections at different depths inside a single colony
of thickness = 32 µm, taken 80 h post insoculation. (b) Mean and standard
deviation of the Brillouin shift at different depths, measured within the ROIs
marked in (a). Data points connected by lines to aid visualisation. Z in-
creases with increasing depth towards the substrate from the vertex of the
colony.
54
To extend the study into larger colonies, we had to modify the imaging proto-
col in order to decrease the acquisition time. Therefore, in the following, instead
of acquiring complete two–dimensional Brillouin images at different depths,
we performed repeated measurements at points along a cross section running
through the middle of the colony, at various depths. Figure 4.10a shows the re-
sults from the technical replicates when examining such a large colony (thick-
ness = 38 µm, imaged 72 h post inoculation); it can be seen that there is decrease
in Brillouin shift towards the middle of the cross section when imaging at the
depths of 36 and 25 µm (as indicated by the black and green lines), while there
was an increase in Brillouin shift towards the center of the cross section when
imaging close to the top of the colony (depth = 10 µm, red line). Such a profile
indicates the existence of a shell of higher stiffness that includes a softer core,
or a void (Figure 4.10e). It is worth noting that the Brillouin shift measured at
the points outside of the colony boundaries was higher than that of the medium
around the colonies (≈ 7.05 GHz). This observation can be attributed to swim-
ming bacteria (as observed in previous studies [75]), which can move into the
focal volume during the exposure time of 900 ms, or due to the flocculent na-
ture of the colony walls. Widefield images at different depths of the colony are
shown in Figure 4.10b-d.
4.3.4 Temporal changes of stiffness
All measurements in this study were done between 36 and 120 h post flow cell
inoculation. Earlier than that, the biofilms were too thin to be imaged, as the
Rayleigh background scatter proximal to the coverslip is too strong to suppress.
Figure 4.11 shows the means and standard deviations of the Brillouin frequency
shifts of all imaged colonies, plotted against the time post inoculation. Each
datapoint corresponds to a ROI that encloses the imaged cross–section of the
55
7.5 10 μm 10 μm
7.4
0 0
7.3
(b) (c)
7.2
10 μm (e)
Depth: 10 μm
7.1 Depth:
36 μm 25 μm
25 μm 0
10 μm (a)
7.0 36 μm
-40 -20 0 20 40
(d) - 0 +
Cross section (μm)
Figure 4.10: (a) Brillouin shift measured along a cross-section of a colony (thickness =
38 µm, taken 72 h post inoculation), at various depths. Data points and
error bars represent the mean and standard deviations from 10 technical
replicates, respectively, at each point. The triangles on the top border de-
note the colony boundaries at each depth. (b-d) Widefield images of the
colony at different depths of 10, 25 and 36 µm, respectively. The white
dashed lines (drawn by visual inspection) enclose the in-focus area, which
is considered to define the boundaries of the colony at each depth. (e)
Schematic illustrating the imaged cross–sections. Darker colour indicates
increasing stiffness. Z increases with increasing depth towards the sub-
strate from the vertex of the colony.
colony. No correlation between the average Brillouin shifts within colonies and
time of imaging after flow cell inoculation was found (Pearson correlation coef-
ficient r = 0.234 and p = 0.212).
Next, we imaged a single colony at 48 h post inoculation, fixed the position of
the stage and repeated measurements of the same colony the following day (i.e.
at 72 h post inoculation). The Brillouin shifts were measured both days along a
line cross section at fixed depth at locations inside, near the border and outside
of the colony, and are shown in Figure 4.12a. It can be seen that the difference
in Brillouin shift between the periphery and the interior of the biofilm became
more pronounced at 72 h post inoculation. During this time, the colony had
grown in size, where the cross section increased from 55 to 74 µm (Figure 4.12b)
and the thickness from 33 to 38 µm.
56
7.8
7.6
7.4
7.2
7.0
40 60 80 100 120
Time post-inoculation (hours)
Figure 4.11: Brillouin shift in colonies at various times post–inoculation. Data points
denote the means, and error bars the standard deviations from all pixels
within the ROIs enclosing the colony. ROIs drawn by visual inspection of
the corresponding widefield images. Biofilms grown under constant flow
velocities of either 0.042 or 0.14 cm s−1 (circles and triangles, respectively).
7.5 (b)
7.4
7.3
7.2 0
7.1
Age:
48 h 10 μm
72 h (a)
7.0
-40 -20 0 20 40
Cross section (μm)
Figure 4.12: (a) Brillouin shifts measured along the cross–section of a colony at two con-
secutive days (at 48 and 72 h post inoculation). Data points and error bars
represent the mean and standard deviations from 10 technical replicates,
respectively, at each point. The triangles on top border denote the colony
boundaries at different days. (b) Widefield image of the colony taken 72 h
post inoculation. The white dashed lines enclose the in-focus area, which
is considered to define the boundaries of the colony at both days.
57
4.3.5 Effect of flow rate
During each experiment, the flow rate of the medium was kept constant through-
out the complete lifecycle of the biofilm. However, two different flow velocities
were tested. Most of the experiments (19 out of 24 imaged colonies) were per-
formed at 0.042 cm s−1 , while the remaining at 0.14 cm s−1 . We observed that
colonies which were grown under the faster flow setting were generally smaller
in diameter as well as flatter. However, in terms of stiffness, no effect of the flow
velocity on the average Brillouin shift of colonies was found, as can be seen by
comparison of datapoints that correspond to the two flow settings in Figures
4.8 and 4.11. The measurements done under the high flow rate were limited to
colonies imaged up to 70 h post inoculation, due to the high consumption rate
of the medium.
4.4 Discussions
In this chapter, we used Brillouin microscopy together with fluorescence imag-
ing to image P. aeruginosa biofilms and we investigated the effect of several fac-
tors including the size and thickness of the microcolony and flow rate. All mea-
surements were performed on living, active, samples of biofilms growing in a
flow cell of cross sectional area 4 mm2 under constant flow of medium.
Interpreting our results According to equation (2.3), the measured frequency
shift, u b , is linearly proportional to the average hypersound velocity, V , within
that volume and the refractive index, n. In turn, V is proportional to the ra-
tio of the Brillouin modulus, M , and inversely proportional to ρ, the material
density. Considering the above, it is evident that pixel–to–pixel variations of the
measured u b could arise from changes in either (or from any combination of)
58
n, M and ρ. First, let us address possible variations of n. Authors in a previous
study [182] have made the assumption that “since the biofilm has a high water
content, n can be assumed equal to 1.333”. In a later study, this was experimen-
tally confirmed as the refractive index of P. aeruginosa was measured n = 1.33 ±
0.17 and n = 1.348 ± 0.013 by optical density experiments and by using a refrac-
tometer, respectively [183]. Therefore, possible variations in the refractive index
(if any) would be within the detection limits of our spectrometer, as noted in a
previous Brillouin microscopy study [160].
Next, we discuss the dependence of Brillouin shift on local variations of den-
sity. To the best of our knowledge, no study on the density of P. Aeruginosa
biofilms with characteristics comparable to those of our study exists. However,
some estimations can be made on the basis of published results in similar sam-
ples. Using terminal velocity of particles, the density of a wastewater biofilm has
been calculated 1.14 g cm−3 [184]. In a later study, it was calculated that the wet
density of biofilms of mixed filamentous bacteria and bacillus ranges between
1 and 1.1 g cm−3 corresponding to dry densities between 0 and 0.05 g cm−3 , re-
spectively [185]. This is not very far from earlier studies that assummed that
biofilms have a wet density equal to that of water (1 g cm3 ), since they are well
hydrated [186] – it is now known that up to 90% of the wet weight of biofilms
is water[187]. So, even if we were to assume that the density of P. aeruginosa
is significantly higher than that of water, and that the measured changes in u b
result from variations in ρ alone, it would follow that the u b measured inside
biofilms would be smaller to that of water (u b water = 7.04 GHz), which is not
the case in our measurements. Therefore, the observed values of u b are propor-
tional to the Brillouin modulus M , which hence represent a measure of stiffness,
even though it does not seem to be possible to assign an absolute metric to M
without the exact knowledge of the local material density.
59
To further support our argument that local differences in u b do not reflect
density heterogeneities, we only found a single study that attempts to make spa-
tial differentiation of density within biofilms, in which the authors measured
1.001 – 1.003 g cm−3 at the top region of biofilms versus 1.01 – 1.02 g cm−3 at the
deeper regions [188]. That is a relative change of ≈1.3% that occurs over the spa-
tial extent of several hundred of micrometres to a few millimetres (their biofilms
were much thicker than those in our study). For reference, the z–stack shown in
our Figure 4.9 shows a 2.6% increase in Brillouin shift over only ≈ 20 µm of depth
gain, which, because it is twice the range of what was observed in ref. [188], is
unlikely to be due to density differences.
Therefore, as we have shown that the observed changes in u b cannot be ex-
plained by changes in density or refractive index, we infer that the profiles of
Brillouin shifts are indicative of the material stiffness; further, all our assump-
tions are based on using the raw Brillouin shifts without attempting to calculate
the hypersound velocity (which would require knowledge of n at each pixel) or
the Brillouin modulus (which would require knowledge of both n and ρ, at each
pixel).
Hydrogels as synthetic model material The applicability of Brillouin micro-
scopy on biofilms was first assessed by imaging cross–linked Alginate Acid (A.A.)
- calcium chloride (CaCl2 ) hydrogel beads, which are commonly used as model
synthetic matrices [174, 180, 181]. We were able to quantify and discriminate
between beads that were prepared by different concentrations of the reagents.
It should be noted, however, that biofilms are much more complex systems
than hydrogel beads, hence, the quantitative results obtained from the measure-
ments in the beads are not – and should not be – used as a calibration look–up
table to infer the composition of biofilms. Nevertheless, the increased degree of
heterogeneity that exists in biological samples does not invalidate our results,
60
since only the probed volume (confocal volume < 1 µm3 ) is considered uniform
in our measurements, and not the whole sample.
Characterisation of biofilms In this study, we captured biofilms during var-
ious stages of their development as they were imaged at different times post
inoculation. For most (≈ 88%) of the imaged biofilm colonies, the Brillouin
shift increased towards the centre of the colony, corresponding to growth on
the outer edges of the colonies, with a denser core [189]. These biofilms are
presumably in an earlier developmental stage. Contrastingly, we found some
colonies in which the Brillouin shift decreased towards their centres and within
such colonies, rotating bacteria were frequently observed. A possible explana-
tion is that these colonies are in transition to the dispersal phase [75, 85]. All
of the hollow colonies and colonies with interiors softer than their peripheries
were at least 45 µm in size (mean diameter). This is in agreement with previous
studies reporting that after the colony reaches ≈ 80 µm in diameter, the interior
disperses from a breakout point and leaves a nonmotile, empty colony behind
[190, 191].
Further, by investigated single colonies, we have shown that the Brillouin
shift of compact colonies increased with depth (Figure 4.9), however, no corre-
lation was found between the size of the colony and the measured Brillouin shift
when different colonies were analysed (Figure 4.8). This observation is different
from a previous study [105], however, measurements in our study were focused
on the interior of the colonies and include colonies of different thicknesses that
were imaged at different depths and at different time points after inoculation,
while the other study utilized AFM, which may primarily interrogate only the ex-
terior of the colonies. In agreement with a previous study [105], we did not find
a correlation between stiffness and flow velocity, as the mean Brillouin shifts of
colonies grown under constant flow at two different velocity settings were not
significantly different. Lastly, when we imaged a single colony for two consecu-
61
tive days we observed a profile was established where the interior of the colony
became less stiff than its periphery, possibly indicating a transmission to disper-
sal stage [85, 86]. Figure 3.2 (page 31) shows a model of the developmental cycle
of biofilms which our data supports.
Repeatability and sample size information In total, 24 different biofilm colo-
nies, selected randomly by visual inspection, were imaged. Only colonies that
had distinct boundaries, visible by widefield microscopy, were imaged at ran-
dom time points that ranged between 36 and 120 h post inoculation. The di-
ameters ranged from 25 µm to 95 µm and their thicknesses from 16 µm to 55 µm.
Either single two–dimentional cross–sections taken near the half-height of the
colony, or z–stacks at different depths were taken. Data shown in Figure 4.8
and Figure 4.11 consist of the complete pool of imaged biofilm colonies (N =
24), where 19 of them were grown under flow velocity of 0.042 cm s−1 and the
remaining 5 under 0.14 cm s−1 . Data shown in Figure 4.10 and Figure 4.12 rep-
resent the mean and standard deviations from 10 technical replicate measure-
ments at each point of the colony. Data shown in Figure 4.6 consist of 30 mea-
surements on alginate beads: ten repeated measurements taken at three differ-
ent points within each bead of different composition.
Limitations This study was restricted to measurements of colonies that had
distinct boundaries, visible by wide field microscopy. The ROIs used to calculate
the average Brillouin shifts were defined manually by visual inspection. The flat,
undifferentiated portions of the biofilms, or the biofilm substratum were not
investigated. The elastic background scatter near the coverslip was too strong
to suppress, hence only colonies that were at least 20 µm thick were imaged.
For the same reason, measurements very close to the substrate of thick biofilms
were not performed. Further, the exact age of each individual colony cannot
62
be precisely determined in our study, as the attachment of different colonies
might have occurred at different time points after the initial cell inoculation. In
terms of characterising the colony size, it remains unclear whether some of the
larger colonies should be treated as single colonies, or as aggregates of smaller
colonies, nonetheless, we believe that our classification is consistent.
4.5 Conclusion
We have demonstrated that Brillouin microscopy is suitable to probe the inter-
nal micromechanical properties of bacterial biofilms. The stiffness of P. aerungi-
nosa biofilm colonies growing in a flow cell was measured in vivo, without dis-
rupting the flow conditions or damaging the samples. To our knowledge, this is
the first study that has revealed internal patterns of stiffness in P. aerunginosa
bacterial biofilms at a µm scale. We have shown that the stiffness patterns dif-
fer between colonies, and areas of reduced stiffness can exist within large, over
45 µm in diameter, colonies. Such observations would not be possible by bulk
rheology methods – which are extensive used in studying the mechanical prop-
erties of biofilms – due to their averaging effect, nor by AFM, as it only probes
the external surfaces [113].
Thus, Brillouin imaging has significant potential in the field of microbiology
and in particular in mechanical studies. Given that it does not require labelling,
it can be applied to almost any biofilm, either formed in the laboratory or natu-
rally occurring. This means that biofilms can be repeatedly measured to obtain
dynamic information on the change in stiffness over time, or as a consequence
of exposure to different treatments or stressors, e.g. dispersal agents. For ex-
ample, it has been found that PA biofilms are able to efficiently recover from
mechanical damage, and that their recovery abilities are not influenced by most
of the common treatment agents [101]. Other studies have found an oscillatory
63
growth and metabolic behaviour [77, 192]. In all cases, it is not clear how this
mechanical remodelling takes place from a biophysical point of view.
We believe that information stemming from Brillouin imaging can offer new
insights towards the qualitative and quantitative understanding of the mechan-
ics at the multicellular bacterial level that drives the biofilm mode of life. Bril-
louin microscopy can complement studies by rheology, fluid flow or molecular
transport. Such a combinatorial approach could be beneficial to the develop-
ment of drugs and drug delivery methods for the removal of biofilms, as it is
currently unclear which biofilm components are the most promising targets for
treatment [101].
Further studies should be done in colonies that undergo dispersion, since
it is at that point where drastic impacts of changes in the mechanical proper-
ties are expected. A number of interesting questions could be answered, for ex-
ample, does dispersion happen only beyond certain stiffness levels, and which
are the sites where dispersion begins. Both natural and laboratory–induced (by
nitric oxide, for example [82]) dispersions could be studied. Lastly, in the fu-
ture, Brillouin microscopy could work in tandem with other techniques, such as
FCS or anisotropy microscopy, that can provide information on local diffusivity,
porosity and viscosity, towards a better understanding of the role of mechanical
properties in signalling and transport of nutrients within biofilm communities
[193], or, with Raman spectroscopy for giving a reasonable chemical and biolog-
ical interpretation of the observed stiffness profiles [195, 194].
64
Chapter 5
SPIM – TRAST for Oxygen
Concentration Measurements
This chapter deals with the application of Transient State (TRAST) monitoring
on Single Plane Illumination Microscopy (SPIM) for measuring oxygen concen-
tration in living biofilms. The theory, concepts and protocols are presented, fol-
lowed by a discussion of findings of this study. Results of this chapter have been
partially published in ref. [196].
5.1 Introduction
5.1.1 Single Plane Illumination Microscopy (SPIM)
SPIM is a versatile tool for imaging complex, three dimensional biological sam-
ples. The unique characteristic of this modality is that it illuminates the sample
by a thin, diffraction–limited laser light sheet that propagates perpendicularly
to the detection axis. This is achieved by the use of two objectives, one for creat-
ing the light–sheet (termed illumination objective) and a second (termed detec-
tion objective) for the imaging of the illuminated plane onto the camera (Figure
65
CHAPTER 5. SPIM – TRAST FOR O2 MEASUREMENTS
5.1b). The light–sheet is usually created by a combination of a cylindrical lens
and an objective lens, or a fast–scanning laser. The thickness of the light sheet
is determined by the NA of the illumination objective. Typically, light sheets of
width approximately 1 µm are used, which can be created by illumination objec-
tives of low NA (0.2 – 0.5), and imaged with objectives of NA ≈ 1. Thinner light
sheets can either be created with higher NA objectives, which, however, have
smaller working distances possibly limiting their applicability, or by structured
illumination techniques, such as Bessel beams or lattices [197, 198].
The achievable resolution of SPIM is comparable to standard epi–fluorescence
microscopes, and three–dimensional imaging is possible by reconstructing the
projections of multiple views taken while rotating and translating the specimen
between observations. A rather good signal quality can be achieved with SPIM,
since no out–of–focus fluorescence is excited or detected [199]. Additionally, it
is well suited for deep imaging within organisms, and, since tissues are exposed
to only a thin plane of light, photobleaching and phototoxicity are minimal, thus
allowing for long term observations [24].
Initially, SPIM was mostly used in developmental biology due to its ability
to image whole, large samples, such as zebrafish embryos or flies over a large
number of days. Today, however, SPIM is routinely used in various other spec-
troscopic applications beyond imaging, such as for single molecule detection
studies [200], FCS and FCCS [201, 202, 203, 204], lifetime imaging [205] and
FRET [206, 207]. A comprehensive review of the method and a number of its
applications can be found at ref. [131].
Here, an in–house built SPIM system [202] is used and further developed for
TRAST imaging to be implemented. The choice of SPIM as an imaging modality
was driven by its capability to provide the desired optical slicing for thick sam-
ples such as bacterial biofilms, as well as the decreased phototoxicity that makes
SPIM suitable for long term imaging of living samples.
66
w T EMCCD
(1)
Iexc (2)
Iem
(3) w
(1) (2) (3)
(a) Pulse width
Det. obj.
(b)
488 nm
Software
function generator Beam expansion Cyl. lens Ill. obj.
Figure 5.1: (a) Schematic of the concept behind TRAST monitoring by modulated illu-
mination: increasing the duty cycle (a = w/T ) of a pulse train while main-
taining a constant total illumination time leads to a decrease of emitted flu-
orescence (Iem ), since molecules have less time to relax to the ground state
before the onset of the following pulse. (b) Diagram of a TRAST–SPIM setup,
not to scale. After [196]. © IOP Publishing. Reproduced with permission. All
rights reserved.
5.1.2 Transient States (TRAST) spectroscopy
TRAST imaging by modulated illumination is a recently developed technique
that allows kinetic information of transient states to be extracted, by measuring
the time–averaged fluorescence response of a sample illuminated with different
pulsed excitation patterns.
Prior to the development of TRAST imaging, triplet–states had been mea-
sured mainly by transient absorption spectroscopy, phosphorescence microscopy
or FCS, all of which have their own limitations. Transient absorption spectroscopy
uses a pump pulse to excite the sample, followed by a delayed probe pulse. By
recording the change in absorption spectrum as a function of time and wave-
length, information regarding the number of decay paths per wavelength as well
as the relaxation times of the decay processes can be revealed. However, this
technique is limited to mainly cuvette experiments, samples of high concentra-
tions (> µM), and requires relative complicated experimental setups including
fast pulsing lasers [208]. Phosphorescence microscopy is largely restricted to
67
carefully prepared and deoxygenized samples, since phosphorescence is typi-
cally much weaker than fluorescence, and is very susceptible to oxygen quench-
ing [209]. Lastly, FCS is able to extract triple state information from the fluctua-
tions generated by transitions to and from the dark triplet states of the fluores-
cent molecules, which appear superimposed on those due to diffusion. How-
ever, FCS is limited to measurements in samples of very low concentrations
(typically in the pM – nM range), requires bright dyes, and, since measurements
are time–resolved, fast and sensitive electronics are needed [210]. In contrast,
TRAST uses the time–averaged fluorescence recorded over long periods (ms - s),
circumventing both the need for high time–resolution or sensitive detectors, as
well as limitations in the dye concentrations.
In general, the excitation rate of a fluorophore is given by the product of its
excitation cross–section (σexc ) and the excitation intensity (I exc ) that is k 01 =
σexc I exc . Therefore, by manipulating the excitation intensity one can control
the population of molecules driven to the excited states. Under modulated ex-
citation a triplet–state population is built–up during the pulses, which then de-
cays in–between them. An equilibrium of the populations of energy states is
formed, which depends on the pulse train characteristics. More specifically, as
the duty cycle (i.e. the ratio of pulse width over period, that is α = w/T ) in-
creases, there is an increase in the triplet state population since fluorophores
do not have sufficient time to return to the ground state before the onset of the
next pulse. As fewer molecules populate the ground state at the onset of the
next pulse, less molecules are available to be excited to the singlet states and
fluoresce, leading to weaker recorded signals [48, 211], as illustrated in Figure
5.1a.
This expected decrease in fluorescence as a function of duty cycle has been
analytically derived for rectangular pulses [211]. It is therefore possible to use
68
this model for pixel–wise fitting on a series of fluorescence images taken with
increasing duty cycles in order to extract the triplet state dynamics, and more
specifically the intersystem crossing time and the triplet relaxation time. To
date, TRAST imaging has been implemented in a number of microscopic se-
tups. It was first validated on confocal microscopy [48] then on a total internal
reflection microscope [212] and most recently on a SPIM microscope [213].
Due to their long life times – which are in the order of µs - ms (Table 2.2)
– fluorophores in transient states are very likely to interact with their immedi-
ate environment [210]. Thus, triplet kinetics can change considerably due to
changes in accessibility of quencher molecules or micro–viscosities, rendering
TRAST imaging suitable for monitoring environmental changes [48, 49]. As a
proof of concept, it was shown that the changes in triplet–state parameters of
the fluorophore rhodamine 6G due to different concentrations of potassium io-
dide (KI) in aqueous enviroments can be measured with TRAST monitoring [48].
Later, Rhodamine 110 samples were measured in aqueous solutions at different
concentrations of dissolved oxygen [212], and, in a more recent study, the triplet
kinetic rates of a number of different bulk dyes incuding Fluorescein, Eosin Y
and Atto 495 were measured in fully aerated and deoxygenated conditions [213].
To date, not many applications of TRAST imaging of biomedical interest ex-
ist; the first one came in 2010, when Geissbuehler et al. measured the oxygen
consumption during the contraction of a single smooth muscle cell on a wide–
field optical setup [211]. Two years later, TRAST imaging was used to assess
the internalization and recycling of proteins in single living cells by identifying
the relative quantities of a transmembrane receptor based on the differences in
triplet lifetimes of two fluorescent species (autofluorescence versus label) [214].
Lastly, in 2014, TRAST monitoring was used to study patterns of altered oxygen
consumption in cancer versus healthy cells [215].
69
5.2 Material and Methods
5.2.1 Simplified energy system
In this thesis, a simplified, three–state energy system which can adequately de-
scribe most fluorophores is used. The Jablonski diagram of such a model is
shown in Figure 5.2. The ground state is considered to be the singlet S 0 . S 1
and T0 are the excited singlet and triplet states, respectively. The more higher–
energy excited states have been omitted, as their contribution is negligible [216].
The system of equations that describes such a three–level energy system is:
kisc=τ isc-1
S1
T0
-1 -1
k01=τ 01
k10=τ 10
k T =τ T-1
S0
Figure 5.2: Jablonski diagram for a simplified, three–state model such as the one con-
sidered in the present study.
 
P1 PT P0
 
 + − τ10 τT τ01 
P
 0  
 

d   
  P P P

 P 1  =  − τ101 − τi sc1 + τ010 (5.1)
 

dt    
   
PT
 
P1 PT
τ − τT
 
i sc
where P 0 , P 1 and P T are the populations of S 0 , S 1 and S T respectively, τ10 is the
lifetime for the singlet state relaxation, τi sc is the intersystem crossing lifetime
from the excited singlet state to the triplet state, τ01 is the excitation lifetime,
and τT is the triplet state lifetime. The solution of the above system is a second
order differential equation involving the four transition rates [48, 211]:
d 2PT τ10 τT d P T τ10 q T τT

µ ¶ µ ¶
1
τ10 τT + τ10 + τT + + 1+ + P T = q T τT (5.2)
dt 2 τ01 dt τ01 τ01 τ01
70
where q T = τ10 /τi sc is the triplet quantum yield.
5.2.2 TRAST monitoring theory
Equation (5.2) can be solved for the case of illumination with rectangular pulses
[211], and an expression for the time–averaged fluorescence emission intensity
of a fluorophore that is illuminated by an isodose of light delivered by a train of
pulses can be described by:
³ ³ ´´
eq
1 − exp − Tτ−w
 
τT P T exp(k w) − 1
T 2
I em = γ 1 − ³ ´ × , (5.3)
T −w
1 − exp − τT + k 2 w w
where w denotes the pulse width and T the period of the pulse train. Addition-
ally, k 2 describes the population rate of the triplet state after onset of excitation,
eq
P T is the relative triplet state population at equilibrium for constant excitation,
and γ is a scaling factor that depends on the concentration of fluorophores and
the detection efficiency, defined as:
τ01 + τ10 + q T τT
µ ¶
k2 = − (5.4)
τT (τ01 + τ10 )
eq q T τT
PT = (5.5)
τ01 + τ10 + q T τT
qf
γ = ξΓ (5.6)
τ01 + τ10 + q T τT
where
τi sc
qf = (5.7)
τi sc + τ10
71
is the quantum efficiency of the fluorophores, Γ is the concentration of fluo-
rophores and ξ is a conversion factor between the emitted intensity and the dig-
ital readout of the camera.
The time–averaged fluorescence intensity in a TRAST experiment depends
on all the transition rates, as seen on equation (5.3). One of the assumptions
made for the three–state model used is that the excitation time, τ01 , is limited
by the illumination intensity and the absorption cross–section of the molecule.
The excitation rate for a particular dye and experimental settings, is therefore
considered constant and can be calculated by:
hc
τ01 = (5.8)
λP l aser σ(λ)
where h is the Planck constant, c the speed of light, λ the excitation wavelength,
P l aser the excitation power and σ(λ) the wavelength–dependent molecular ab-
sorption cross–section. Using equation (5.8) and for laser powers that match
our experimental setup, τ01 for Eosin Y is calculated to be in the range of a few
ns, in agreement to the previously measured value of 1.55 ns [217].
On the other hand, the triplet relaxation rate and intersystem crossing are
more difficult to define, and, as discussed earlier, depend largely on environ-
mental factors such as the availability of quenchers and the viscosity of the sol-
vent. The triplet relaxation time for Eosin Y has been found to change from ap-
proximately 2 to 30 µs between a completely aerated and deaerated solution,
respectively [212, 213]. Likewise, a range for intersystem crossing times can
be found in literature – from approximately 2 ns to 5 ns [212, 218, 219, 213].
It should be noted that both the measured triplet relaxation and intersystem
crossing times additionally depend on the experimental method.
72
1 1
τΤ (μs) τisc (ns)
2 0.8 2
0.8 4
Intensity (AU)
3
Intensity (AU)
8
10 4
0.6 14 0.6
20 5
0.4 0.4
0.2 0.2
(a) (b)
0 0
0 10 20 30 40 50 0 10 20 30 40 50
Pulse width (μs) Pulse width (μs)
Figure 5.3: (a) The effect of triplet relaxation time on TRAST curves. Parameters used
in modelling: T = 50 µs, τ01 = 2.2 ns, τi sc = 2.5 ns, τ10 = 3 ns [213]. (b) The
effect of intersystem crossing time on TRAST curves. Parameters used in
modelling: T = 50 µs, τ01 = 2.2 ns, τT = 3 µs, τ10 = 3 ns [213].
The effect of triplet relaxation and intersystem crossing time on the appear-
ance of TRAST curves is shown in Figure 5.3, where out of the two parameters,
it can clearly be seen that triplet relaxation time plays a much more crucial role
in the intensity of fluorescence under isodose pulsed illumination. The range of
values modelled for the two parameters is experimentally–relevant, and covers
the cases of deaerated to fully aerated aqueous samples. For each plot, all the
other parameters apart from the investigated one were considered to remain
unchanged.
5.2.3 Derivation of TRAST contrast
As can be seen in Figure 5.3a, a change in triplet relaxation time affects mostly
images taken with higher duty cycles, due to the inability of molecules to recover
between light pulses. On the other hand, when very short pulses are used, there
is sufficient time for molecules to relax and hence there is no decrease in average
fluorescence for different triplet relaxation times.
Therefore, it is expected that the greatest differences in fluorescence inten-
sity will be found between images taken with the shortest and longest pulse
73
widths. The ratio of fluorescence images acquired with different laser beam
dwell times on a scanning confocal microscope has been successfully used be-
fore to distinguish samples of different triplet populations [49]. Here, in order to
simplify and speed up the collection process, we propose and examine an acqui-
sition protocol that is based on the maximum relative decrease in fluorescence
intensity, that is the relative decrease between images taken with the shortest
(t p =1 µs) and longest (t p =50 µs) pulses, which correspond to duty cycles of 1%
and 100%, respectively. We can therefore define the TRAST contrast as:
I shor t − ICW
CT = (5.9)
I shor t
where I shor t and ICW are the intensities recorded from a train of pulses with the
aforementioned characteristics. The values of ICW can be calculated analyti-
cally by setting w = T in equation (5.3), which is reduced to:
A
I em |w=T ≡ ICW = H (5.10)
B +C τT
where H is a constant factor depending on the concentration of the fluorophores
and acquisition efficiency, and,
A = τi sc
B = τi sc (τ01 τi sc + τ10 τi sc + τ01 τ10 + τ210 ) (5.11)
C = τ210 + τ10 τi sc .
On the other hand, I shor t needs to be numerically solved.
Figure 5.4 shows the solution of equation (5.9) for a range of different triplet
relaxation times. It can be seen that TRAST contrast increases from 64.7% to
94.7% for an increase in triplet relaxation time from 2 µs to 20 µs.
74
100
Numerical solution
95
90
85
CT (%)
80
75
70
65
60
2 4 6 8 10 12 14 16 18 20
τT (μs)
Figure 5.4: TRAST contrast, defined as C T = (I shor t − ICW )/I shor t , where I shor t is the
sample fluorescence when illuminated using a low duty cycle (1%), and ICW
when using continuous illumination (total illumination time constant), for
a range of triplet relaxation times.
5.2.4 TRAST contrast as a measure of [O2 ]
The effect of molecular oxygen on the triplet dynamics has been a matter of
interest for many years, since the longer lifetimes associated with triplet states
lead to higher quenching probabilities and therefore offer a good candidate for
studies of the molecular microenvironment. Using FCS, a five–fold decrease in
triplet relaxation lifetime when measuring Rh6G in an oxygen atmosphere ver-
sus a de–aerated one has been observed [210]. Similar behaviour for Eosin Y has
been reported [215] and it has been shown that the significant decrease in triplet
state relaxation rate is mainly due to quenching by oxygen [212].
Here, we are examining the relationship between molecular oxygen concen-
trations and TRAST contrast, in order to be able to quantify local oxygen con-
centrations by applying equation (5.9) that only requires the acquisition of two
fluorescence images. It is possible to re–write equation 2.18 as:
τT 0
τT = (5.12)
1 + k q τT 0 [O 2 ]
75
95 95
TT0 kq
12 μs 0.6 10-9 M-1 s-1
90 16 μs 90 0.8 10-9 M-1 s-1
20 μs 10-9 M-1 s-1
CT (%)
CT (%)
30 μs
85 85
80 80
(a) (b)
75 75
0 50 100 150 200 250 0 50 100 150 200 250
O2 (μM) O2 (μM)
Figure 5.5: TRAST contrast plotted against oxygen concentrations and the effect of (a)
triplet relaxation time in absence of quencher and (b) bimolecular quench-
ing constant.
where τT 0 is the triplet lifetime in the absence of the quencher.
By combining equations (5.10) and (5.12), we can find the relationship be-
tween TRAST contrast and absolute values of oxygen concentration. When char-
acteristic values for Eosin Y are considered (τ01 = 2.2 ns, τi sc = 2.5 ns, τ10 = 3
ns [213], k q = 109 M−1 s−1 [220] and τT 0 = 16.5 µs1 ), a linear fit (R2 = 0.99) can
suffiently be used to describe the relationship for values of oxygen concentra-
tion ranging from deaerated to air–saturated solutions (0 – 250 µM [221]). The
resulting curve depends on the bimolecular quenching constant, k q , and the
triplet relaxation time in absence of oxygen, τT 0 , both of which were here treated
as constants [221]. More specifically, k q , changes the slope of the curve while
τT 0 sets the y–intercept, as can be seen in Figure 5.5.
5.2.5 Experimental setup
All experiments were performed using an in–house-built SPIM setup. A 488 nm
laser (OBIS LX, Coherent Inc., Santa Clara, CA, USA) is used and its beam is ex-
panded about 5 times by two sets of achromatic lenses. It then passes through
an achromatic cylindrical lens ( f = 75 mm, Edmund optics, Singapore). The

1
Value from own measurements in deaerated bulk solution
76
resulting beam over–illuminates the back focal aperture of the low NA illumina-
tion objective (SLMPlan 20X / 0.25, Olympus, Singapore) to obtain a light sheet
≈ 1.2 µm thick. The illumination objective has a working distance of WD = 21
mm. This provides the necessary space to bring the light sheet into the focal
plane of the detection objective (LUMPLFLN 60x/1.0 W, WD = 2.0 mm, Olympus,
Singapore). The sample mounting unit consists of a custom built sample cham-
ber (Physics mechanical workshop, NUS, Singapore) and motorized linear x–,
y– and z–stages together with a rotation stage (XYZ–linear stages: 3×8MT184–
13DC and rotation stage: 8MR174–1–20, Standa Ltd., Lithuania). The detec-
tion objective was mounted on a piezo flexure objective scanner (P–721 PIFOC,
Physik Instruments, Singapore) to control the objective in nm precision. A flu-
orescence emission filter (BrightLine HC525/50, Semrock Inc., New York) was
mounted behind the detection objective, before a standard tube lens (part no.
LU074700, f = 180 mm, Olympus, Singapore) used to image the sample onto the
camera. An Andor iXon X3 860 EMCCD (Andor Technology, Belfast, UK) cam-
era was used in the experiments. The camera pixel size at the object plane is
400 nm, and n × n pixel binning is possible during or post acquisition.
Modulation of the laser output is digitally controlled using a custom–build
program on LabView (National Instruments, Austin, TX) that can create finite–
length pulse trains of desired characteristics. The pulse sequence is fed from the
computer to both the laser and the camera via a terminal box (NI CB–68LPR, Na-
tional Instruments, Austin, TX) connected to a data acquisition board (NI PCI–
6221, National Instruments, Austin, TX). The camera was triggered by the onset
of the first pulse and the exposure time was equal to the full length of the pulse
train. Pulses of widths as short as 250 ns were validated using a Si switchable
gain detector (PDA36A–EC, Thorlabs) connected to an oscilloscope.
77
5.2.6 Sample preparation
Bulk solutions
Eosin Y (Sigma–Aldrich, Singapore) was chosen for our experiments due to its
high triplet–yield that facilitates the build–up of triplet state population at rela-
tively low incident powers [215, 218]. Key characteristics of Eosin Y are shown in
Table 5.1. Stock samples of mM concentration were prepared in ethanol (Sigma
Chemicals, St. Louis, U.S.A.). The samples were further diluted in double-distilled
water (ddH2 O) to final concentration of 2 µM. In the full TRAST monitoring
experiments, bulk solution was placed in heat–sealed sample bags made from
thin transparent foil with a refractive index matching that of water (Katco Ltd.,
United Kingdom). Each of the bags contained approximately 50 µL of the solu-
tion. The samples were air–saturated by bubbling atmosphere air in for approx-
imately 30 min using a common aquarium pump. When necessary, complete
deaeration of the sample was performed by addition of Na2 SO3 at final concen-
tration of 2 M [222].
Property Value
Chemical formula C20 H6 Br4 Na2 O5
Molar mass 647.89052
Absorption maximum (λa ) 524 nm
Fluorescence maximum (λ f ) 543.5 nm
Quantum yield 67%
Molar extinction coefficient at 524 nm 112,000 cm−1 /M
Molar extinction coefficient at 488 nm 30,000 cm−1 /M
CAS Number 17372–87–1
Table 5.1: Characteristic properties of Eosin Y [57, 223].
Partial oxygen removal, which is necessary for the TRAST contrast vs oxy-
gen concentration calibration curve, was achieved by titrating L–Ascorbic Acid,
78
catalysed by Ascorbate Oxidase [211, 224], according to:
L-Ascorbic Acid + O2 −→ 2 H2 O + 2 Dehydroascorbate (5.13)
L–Ascorbic Acid was purchased from Sigma–Aldrich and dissolved 65 µM. Ascor-
bate Oxidase from Cucurbita sp. (lyophilised powder purchased from Sigma–
Aldrich) was reconstituted in 20 mM sodium phosphate (NaH2 PO4 ) buffer. The
enzyme has an activity of 1,000 to 3,000 units per mg protein. One unit will ox-
idize 1.0 µM of L–Ascorbic Acid to dehydroascorbate per min at pH 5.6 at 25o C .
The samples were placed in larger bags that can hold approximately 800 µL. Ab-
solute values of oxygen concentration were measured using an pre–calibrated
optical fibre oxygen sensor (FireSting2, Pyro–Science) while at the same time,
fluorescence intensity were measured using different pulse widths using the
SPIM setup. The bags were left open at the top to allow for probe insertion (Fig-
ure B.1, Appendix B).
Three units of Ascorbate Oxidase were added prior to placement of the sam-
ple in the microscope and aliquots of L–Ascorbic Acid were injected and gently
mixed inside the bag by pipetting, before the onset of acquisition. Measure-
ments were taken during the course of atmospheric oxygen uptake, while the
absolute values were monitored by the probe. Measurements were repeated by
replenishing the solution.
Hydrogel beads
Calcium alginate hydrogels were prepared using the methods described in [174].
Alginate acid (Acros Organics, U.K.) solutions (3% wt/vol) in water were pre-
pared and autoclaved. Filter–sterilised CaCl2 (100 mM) solution was poured in
a 6 well plate (Nunclon Flat, Thermoscientific) in columns approximately 2 mm
tall. Droplets of 10 – 20 µL of alginate acid were dropped into the well using
a pipette. The cross linking was almost instantaneous. The drops were left to
79
gelate in the CaCl2 for approximately 20 min before they were washed twice
with sterile water. The beads were then kept in sterile water, and imaging ex-
periments were carried out the same day. For imaging, the beads were trans-
ferred to heat–sealed bags made from thin transparent foil with a refractive in-
dex matching that of water (Katco Ltd., United Kingdom) that included the bulk
dye solution.
Biofilms
A flow cell setup system similar to the one described at ref. [177] was used.
A square Fluorinated Ethylene Propylene (FEP) tube (inner diameter: 2.1 mm,
outer diameter: 2.3 mm) was used as the flow cell (Adtech Polymer Engineer-
ing Ltd, UK). P. aeruginosa (PAO1) were grown overnight in LB medium (Difco)
40 g/L in a shaking incubator at 37o C, 200 rpm. The culture was diluted in min-
imal medium ([15 mM (NH4 )2 SO4 , 34 mM Na2 HPO4 , 22 mM KH2 PO4 , 58 mM
NaCl, 1 mM MgCl2 , 0.1 mM CaCl2 and 0.001 mM FeCl3 ], supplemented with 2
g/L of Glucose plus 2 g/L of Casamino acids) to an OD600 ≈ 2.0 and 300 µL were
injected into the tubing, near the entry port of the flow cell. The inoculum re-
mained in the flow cell to settle for 1 h without flow, at room temperature. The
flow of the minimal medium was then resumed at a rate of ≈ 6 ml/h. A 6–Roller
peristaltic pump (Langer Instruments, U.S.A.) was used to supply the flow.
The biofilms were allowed to grow on the surfaces of the flow cell. Prior to
imaging, flow was stopped and one of the four faces of the flow cell was carefully
cut out using a surgical scalpel and placed in a thin foil, heat–sealing bag which
contained approximately 50 µL of the dye (Eosin Y, 2 µM). The substratum ma-
terial (FEP tube) was chosen due to its optical and mechanical properties; that
is, its refractive index is close to that of water, and it is a rigid material to sus-
tain flow during the growing phase, yet light enough to be placed in the FEP bag
80
(a) Detection
Illumination objective z
(b) y
objective
x
Figure 5.6: (a) Illustration of mounting concept for bacterial biofilms in SPIM. One of
the surfaces of the square tube which has attached biofilm on, is cut and
removed using a scalpel and put in a bag made of thin FEP matching the
refractive index of water and filled with dye solution. Drawing is not to scale.
(b) Schematic showing the optical sectioning of a colony.
during imaging.
All connecting FEP tubings (30 mm long, 2.1 mm ID and 2.3 mm OD) were
autoclaved and flushed with 10% (v/v) bleach, then thoroughly rinsed in sterile
milli–Q and then primed with the minimal medium before the start of every
experiment.
5.2.7 Acquisition and analysis
In a pulse train, the duty cycle (α) is given by α = w/T , where w is the pulse
duration and T the period (Figure 5.1a). The total illumination time is given by
Ti l l = N ·w, where N is the number of pulses within the train. In the TRAST mon-
itoring experiments of this study, the illumination time was kept constant (Ti l l =
100 ms). This was achieved by changing both the duty cycle and the number of
pulses accordingly, while the period of the pulse train was kept unchanged. The
pulse width duration, w, varied from 1 to 50 µs, the number of pulses, N , from
200,000 to 2,000 while the period, T , was kept constant at 50 µs. The total acqui-
sition time at each point of the curve can therefore be calculated by T ac q = N ·T .
The laser power before objective was 27.3 mW.
81
7000
Fluorescence intensity (AU)

6800
6600
6400
6200
6000
5800
5600
Baseline <1 minute 2 minutes 3 minutes 4 minutes

Interval between images
Figure 5.7: Eosin Y (2 µM) in double–distilled water (ddH2 O) samples repeatedly im-
aged after allowing different time intervals between images. Values shown
are the mean and standard deviations of the recorded intensity across 50 ×
50 pixel images. Three independent measurements were taken for each con-
dition. The baseline measurement is shown as a reference of the expected
values in absence of bleaching.
Up to fifteen images were taken per experiment at increasing pulse widths
to compile each TRAST curve (see Table A.1, Appendix A). The exposure time
of the camera was set equal to T ac q for each acquisition, so that intensity was
integrated during the full pulse train. A pause of approximately 2 min was in-
troduced between image acquisitions to ensure that dye in the focal volume has
been replenished, thus avoiding artefacts from bleaching. This was determined
by a calibration experiment where we repeatedly imaged samples at different
time intervals and we found that the intensity is fully recovered when at least
two minutes are allowed between images (Figure 5.7). The acquired images were
loaded and analysed by a custom–made Matlab script [225] that can perform a
pixel–wise fitting on the solution of the three–state model under the condition
of isodose illumination.
To calculate TRAST contrast, images using only the lowest duty cycle (α =
1%) and continuous illumination (α = 100%) were collected (see Appendix B.1).
Ti l l was kept constant, as above. The relative intensity decrease was then calcu-
lated in a pixel–wise manner, according to equation (5.9).
82
5.3 Results
5.3.1 Measurements in bulk solutions
To validate the setup, we performed TRAST measurements in homogeneous
bulk solutions of Eosin Y (2 µM) in double–distilled water (ddH2 O), taken in both
air–saturated and deaerated conditions. Complete deaeration was performed
by addition of Na2 SO3 . Figure 5.8a shows typical TRAST curves for measure-
ments in the aforementioned conditions. The error bars on the plot correspond
to the standard deviation of the intensity of each image, 20 × 20 pixels in size.
We found a significant increase in triplet relaxation time from 2.16 ± 0.02 µs to
16.52 ± 0.06 µs when oxygen was removed (Figure 5.8b,c). Our results are com-
pared to previously published results in Table 5.2. For the fitting, the initial value
of τ01 was calculated from (5.8), and the initial values for τi sc , τ10 , and τT were
given based on literature review.
Sample Method [Ref.] τT (µs) τi sc (ns)

Eosin Y, Present study 2.16 ± 0.02 1.86 ± 0.02
air–saturated solution SPIM–TRAST [213] 2.08 ± 0.04 1.34 ± 0.06
Confocal–TRAST [213] 2.38 ± 0.11 1.32 ± 0.14
Picosecond double– – 2.38 ± 0.28
pulse excitation [218]
Flash photolysis [216] – 1.19 ± 0.11
TRAST [215] 1.89 ± 0.04 1.18 ± 0.03
Eosin Y, Present study 16.52 ± 0.06 3.03 ± 0.01
deaerated solution TRAST [215] 29.41 ± 3.40 2.96 ± 0.25
Table 5.2: Triplet relaxation and intersystem crossing times for Eosin Y in air–
saturated and deaerated solutions, together with values from litera-
ture review.
Next, we measured the TRAST contrast in solutions of varying oxygen con-
tent, prepared by titration of Ascorbic Acid, in order to acquire a calibration
83
1 20
Aerated (b)
0.9 Aerated
Deaerated 15
0.8
0.7 10
Intensity (AU)
0.6 5
0.5
0
0.4 2.05 2.15 2.25
0.3 25
0.2 Deaerated (c)
20
(a)
0.1
5 10 15 20 25 30 35 40 45 50 15
0.02 10
Residual
0.01
0 5
-0.01
0
-0.02
5 10 15 20 25 30 35 40 45 50 16.3 16.5 16.7
Pulse width (μs) Triplet time (μs)
Figure 5.8: (a) TRAST curves from measurements in homogeneous, bulk solutions of
Eosin Y (2 µM) in double–distilled water, in air–saturated and de–aerated
conditions. (b, c) Extracted triplet times from pixel–wise fitting for the air–
saturated and deaerated solution, respectively. After [196]. © IOP Publish-
ing. Reproduced with permission. All rights reserved.
90
80
Relative decrease in intensity (%)
70
60
50
40
30
Oxygen concentration [μM]
Figure 5.9: TRAST contrast in samples of varying oxygen content. Two independent
measurements of Eosin Y (2 µM) in double–distilled water (open and solid
diamonds) and linear fits. After [196]. © IOP Publishing. Reproduced with
permission. All rights reserved.
84
curve between absolute values of oxygen concentration and TRAST contrast
readings, as shown in Figure 5.9. The error bars on the x–axis were derived from
the fluctuation of the oxygen content as measured by the probe over the time
of the measurement (including the pause between acquiring the low duty cycle
image and the continuous illumination image). The error bars on the y–axis de-
note the standard deviation between pixels (128 × 128 pixel images). Two inde-
pendent measurements on freshly prepared samples are shown (open and solid
diamonds) together with their linear fits (dashed lines). The slope of the linear
fit was (−0.96 ± 0.07) 10−3 . We investigated the effect of viscosity by measuring
TRAST contrast in solutions of increasing sucrose concentrations [226]. No sig-
nificant change was found for samples of viscosities of 1.30 and 1.80 cP, where
the slope was (−1.06±0.15) 10−3 and (−0.77±0.45) 10−3 , respectively, calculated
from two independent measurements in fresh samples for each case.
5.3.2 Measurements in heterogeneous samples
Since differences in the material properties of the sample other than its oxy-
gen content might affect the fluorescence intensity, next, we performed control
measurements in non–homogenous samples that, however, do not produce or
consume oxygen locally. Thus, calcium alginate hydrogel beads, which are com-
monly used as model synthetic matrices in biofilm studies as their composition
resembles that of biofilms [174, 180, 181], were prepared and studied.
We selected a ROI that includes the interface of the bead with the aqueous
solution (Figure 5.10a). A full TRAST imaging experiment showed that the triplet
relaxation lifetimes inside the hydrogel bead were not different from those in the
surrounding solution (Figure 5.10b), leading to a single–population distribution
(Figure 5.10c). The extracted values of triplet relaxation time and intersystem
crossing (τT = 2.09 ± 0.08 µs and τi sc = 1.92 ± 0.11 ns, respectively) resemble
those found in fully aerated bulk solutions. This is an expected result, since there
85
2.4 100
(a) (b) (c) Triplet time (μs)
2.3
80
2.2
60
2.1
2.0 40
1.9 20
1.8
0
μs 1.8 2.0 2.2 2.4
65 2.4 1
(d) (e) (f) 0.99
2.3
60
2.2 0.98
2.1 0.97
55
2 0.96
50 0.95
1.9
1.8 0.94
45
% ns 0.93
Figure 5.10: (a) Transmission image of a calcium alginate (3% w/w) hydrogel bead in
Eosin Y (2 µM) solution. (b) Triplet relaxation time map and (c) frequency
histogram. (d) TRAST contrast image. (e) Frequecy histograms of the ex-
tracted intersystem crossing times. (f) R2 of the fit. The pixel size on panels
(b-f) is 1.6 × 1.6 µm (after 4 × 4 pixel binning). White pixels represent areas
where the fitting has failed. Scale bar: 8 µm. After [196]. © IOP Publishing.
Reproduced with permission. All rights reserved.
was no production or consumption of oxygen in the sample, and oxygen diffuses
freely within the hydrogel. A TRAST contrast image is shown in Figure 5.10d. The
average contrast inside and outside the bead is 57±2% and 60±2%, respectively.
A map of the intersystem crossing times is shown in Figure 5.10e and lastly, R 2 ,
as a measure for goodness of fit, is shown in Figure 5.10f.
5.3.3 Measurements in bacterial biofilms
We used TRAST imaging to extract the transient kinetics, and TRAST contrast
to quantify the oxygen distribution inside a P. aeruginosa biofilm that had been
growing under continuous flow of minimal medium for ≈ 80 h after cell inocu-
lation. Prior to imaging, the flow was stopped and the samples were transferred
to the FEP bag for imaging. Images were taken ≈ 3 h after flow was stopped.
The height of the microcolonies was in the range of 30 – 60 µm and all images
86
were taken at mid–point depth for each microcolony (geometry shown in Figure
5.6b).
Figure 5.11(I) a single biofilm colony imaged in an air–saturated solution.
A wide range of triplet relaxation times were found, evident from the pixel–to–
pixel variations, as extracted from local fitting with equation (5.3). However, the
average triplet relaxation time inside the colony was significantly longer com-
pared to that outside (5.53 ± 1.17 µs and 3.41 ± 0.23 µs, respectively).
After complete oxygen removal by the addition of Na2 SO3 , there was no sta-
tistical difference in the triplet relaxation times inside and outside the colony
(9.09 ± 0.27 µs and 9.15 ± 0.17 µs, respectively), as shown in Figure 5.11(II). In-
stead, a narrow distribution of triplet times was observed (Figure 5.11c) whose
values were significantly longer than those measured prior to deaeration. Sim-
ilarly, the values of intersystem crossing times where limited to 4.75 – 5.68 ns
after deaeration, versus 2.10 – 7.45 ns before.
Using equation (5.9), TRAST contrast was calculated for this colony, and is
shown in figure 5.12a. It was found that inside the biofilm the average value was
significantly larger compared to outside (68 ± 7% and 56 ± 3%, respectively). On
the other hand, TRAST contrast was almost uniform (77±1% and 79±1%, inside
and outside of a colony, respectively) when measured after complete oxygen re-
moval (results not shown).
Lastly, using the calibration curve derived earlier (Figure 5.9), and after set-
ting the value of τT 0 as extracted from the deaerated biofilm measurement, a
map of absolute oxygen concentrations was constructed and shown in Figure
5.12b. It can be seen that oxygen gradients have been established, and oxygen
depletion extended outside the area where bacteria lie (dashed lines). The nec-
essary assumptions for converting TRAST contrast to absolute values of oxygen
87
I
11
5
y
3
x (a) (b) (c) μs
70 1
(d) 7
60 Triplet time (μs) 0.99
6.5
6 0.98
50
5.5 0.97
40 5
0.96
4.5
30 4 0.95
20 3.5 0.94
3
0.93
10 2.5
(e) 2 (f) 0.92
0 ns
2 4 6 8 10
II
10. 5
10
9.5
8.5
(a) (b) (c) μs
100 5.7 0.997
(d) Triplet time (μs) 5.6
0.996
80 5.5
5.4 0.995
60 5.3
0.994
5.2
5.1 0.993
40
5
0.992
4.9
20 0.991
4.8
(e) (f)
0 ns
8 8.5 9 9.5 10 10.5
Figure 5.11: A P. aeruginosa biofilm colony imaged in solution of Eosin Y (2 µM) in mini-
mal medium. Panel I: Air–saturated conditions. Panel II: Deaerated condi-
tions. (a) SPIM images. (b) Fluorescence images taken with 100 ms contin-
uous illumination. (c) Triplet relaxation time maps and (d) frequency his-
tograms, showing the pixel–to–pixel variation, extracted from full TRAST
experiments. (e) Intersystem crossing time maps. (f) R2 of fit. Images taken
≈ 80 h post–inoculation. The pixel size was 0.8×0.8 µm (after 2×2 pixel bin-
ning) and white pixels represent areas where the fitting has failed. Dashed
lines mark the boundaries of the colonies to aid visualisation. Scale bar:
4 µm. After [196]. © IOP Publishing. Reproduced with permission. All
rights reserved.
88
90 300
2 00
50
15
80 250
0
10
10
25 200
0
70 0
20 150
60 50 0
15
10 100
0
50 5 0 10
100
10
10
0 50
50
40
(a) (b)
10
50
10
0 0
10
0
% μM
0
Figure 5.12: (a) TRAST contrast image and (b) contours of oxygen concentration in-
side the colony of Figure 5.11(I). Dashed lines mark the boundaries of the
colony to aid visualisation. Scale bar: 4 µm. After [196]. © IOP Publishing.
Reproduced with permission. All rights reserved.
concentrations in living systems are discussed in the next section.
5.4 Discussions
Reducing the irradiance requirements The first published protocols for TRAST
imaging required long, up to 3 min, acquisition times and high laser excitation
powers, up to 500 kW/cm2 [49], which can lead to photodamage of the sam-
ple. To date, a number of steps have been taken to decrease these requirements
so that TRAST imaging can find more applications in biomedical studies. In a
study published in 2010, the exposure time was reduced to 1.2 ms per image,
however the irradiance remained rather high, at 60 kW/cm2 [211]. More recent
studies [213, 215] utilised Eosin Y that has a higher triplet yield, and managed
to decrease the laser irradiance requirement to 0.44 kW/cm2 and ≈ 10 kW/cm2 ,
however, their protocols still required over 20 and up to 57 images per TRAST
curve, respectively.
In this thesis, to further reduce illumination time, we have proposed and val-
idated a different acquisition protocol and explored its applicability for quan-
tifying oxygen concentrations. Specifically, we calculate and use TRAST con-
89
trast, that is the relative decrease in fluorescence intensity between two images:
one taken with a low duty cycle (α = 1%) and one with continuous illumination
(α = 100%), as per equation (5.9). This allows the total illumination to be low-
ered by a factor of up to 25 in comparison to previous studies. Measurements
presented in our study were performed at an irradiance of ≈ 7 kW/cm2 and the
total illumination time for each image was 200 ms.
It can be argued that the robustness of the fit could improved by increasing
the number of acquired images. However, acquiring additional images leads
to larger light doses and longer acquisition times, which can be deleterious to
samples. This trade–off has indeed been discussed in a previous study, where
authors note that they had to compromise on image quality by reducing laser
power in order be able to acquire additional images [211]. It should be also
noted that by using TRAST contrast as a measure, one does not have to use equa-
tion (5.3) for fitting; instead, the simpler equation (5.9) can be used, leading to
reduction of post–processing computational time as well.
Quantifying local oxygen concentration with TRAST contrast Our simula-
tions indicate that, when transition rates characteristic of Eosin Y are consid-
ered, a linear relationship holds between TRAST contrast and absolute oxygen
concentration (Figure 5.5).
We first tested and validated this relationship in calibration experiments with
bulk dyes (Figure 5.8). Subsequently, we examined its applicability in heteroge-
neous samples by imaging alginate hydrogel beads whose composition is simi-
lar to that of biofilms [174, 180, 181]. We found that local differences in the ma-
terial properties of the sample did not affect the measurements (Figure 5.10), an
expected result since TRAST contrast is a concentration–independent method
[49].
90
For the correct application of the calibration curve on complex samples,
both its slope and intercept must be known. To estimate oxygen concentrations
in biofilms we have here used the slope extracted from bulk dye measurements
and the intercept from deaerated biofilm measurements.
Oxygen concentration distribution inside biofilm colonies Single colonies
of P. aeruginosa biofilms were studied. First, full TRAST experiments showed
that the triplet relaxation times inside the biofilm are significantly longer than
those measured in the surrounding medium. Second, we used TRAST contrast
to quantify the oxygen concentration. It should be noted here, that in complex
living systems, a number of environmental factors and dynamic processes can
potentially affect the triplet–singlet formations, therefore, one needs to be care-
ful with the interpretation of results. In order to assess any possible cross–talk
artefacts from other processes, we deliberately depleted oxygen in the sample
and measured the triplet relaxation times. We no longer found any gradients
or local differences (Figure 5.11(II)c–d), and the biofilm became indiscernible
in the triplet relaxation time map, implying that, in our case, oxygen concentra-
tion is the major factor affecting triplet relaxation time and TRAST contrast. This
is a similar assumption to those made in previous published studies [215, 211].
The absolute values of τT 0 in the deaerated biofilms were different than those
found in deaerated bulk dye solutions. This discrepancy could be attributed to
by–products or other mechanisms associated with biofilm processes.
Figure 5.12 shows that in the biofilm imaged here, anoxic areas existed within
the colony and that oxygen depletion extended beyond the area of bacterial
cells. This finding is in agreement with a previous study that measured oxygen
concentration at the base of biofilms. The authors found that P. putida colonies
have anoxic centers and a “relatively homogeneous oxygen distribution of 5%
to 7% air saturation” when they measured below colonies, under low flow to
91
stagnant conditions [128]. An older study using microprobes has also reported
gradients of oxygen depletion towards the deeper parts of biofilms [117]. These
anoxic regions could be attributed to the respiration of oxygen–consuming bac-
teria that results in rapid reduction in the oxygen concentration from the surface
to the interior of the micro colonies. Further, it has previously been shown that
genes associated with anaerobic respiration were induced in the interior of mi-
cro colonies [82].
Interestingly, we also found areas inside the biofilm with higher oxygen con-
centrations compared to those close to the boundary. This could be attributed
to transport channels that are known to exist in colonies [44, 228], as well as
to the porosity of colonies [229]. By convection through these channels, in-
ner parts of biofilms can receive nutrients and metabolites. Furthermore, it is
known that some colonies have hollow parts in their interior, due to dispersal
of the bacteria [85]. In this way, the bacteria on the outer surface of the micro-
colony would consume and reduce the local oxygen content, while oxygen could
leak into the interior of the colonies through the channels described above.
Overall, the cross sections presented here show a highly heterogeneous oxy-
gen distribution with steep gradients both inside and outside the visible bound-
aries of the colony (Figure 5.12). Oxygen concentration reaches air–saturation
levels at approximately 15 – 20 µm away from the visible colony edge. It should
be noted that unlike any of the existing studies, our measurements were done at
approximately half the depth of a 60 µm thick mushroom–like colony.
Avoiding photo–bleaching artefacts TRAST imaging is based on relative dif-
ferences in intensities. For the correct interpretation of TRAST measurements,
it is crucial that there are no photobleaching artefacts that further decrease flu-
orescence. If no precausions or corrections against photobleaching are taken,
92
the perceived fluorescence decrease would be overestimated, leading to an un-
derestimation of oxygen concentration. In previous studies which investigated
phenomena happening in fast (tens of seconds) timescales, bleach correction
was retrospectively performed. Such an approach can decrease the total exper-
imental time to a few seconds, however, it introduces the need of “extensive im-
age processing” [211]. In our measurements we allow for dye replenishment in
the region of interest instead (Figure 5.7). In all results presented here, a pause
of 2 min was introduced between images, which is the limiting factor for our to-
tal experimental time. Despite being slower than protocols that include correc-
tions for image bleaching, ours is suitable to study phenomena that happen on
longer time scales, such as the formation of anaerobic areas in growing biofilms.
A post–acquisition bleach correction could readily be employed, if necessary.
Therefore, the theoretical temporal resolution of the TRAST contrast method is
limited only by the acquisition time (T ac q ) needed for two images: one with a
pulse train of low duty cycle (in our experiments 200,000 pulses of 1% duty cy-
cle with 50 µs period, i.e. Ti l l = 100 ms and T ac q = 10 s) and one with constant
illumination with the equivalent dose (Ti l l = T ac q = 100 ms).
Limitations The main limitation in the presented methodology is that the mea-
surements on biofilms were done in a closed system, that is the thin foil sealed
bag. In such arrangements it is expected that nutrients will be depleted over
time, influencing the activity and oxygen consumption rate. Additionally, since
there is no flow of media during imaging, the system is dominated by diffusion.
This could contribute to our observation of gradients extending into the bulk
liquid. It is possible that the oxygen profile of biofilms imaged under flow would
be quite different, and this is something worth investigating in future studies.
Lastly, in order to convert the TRAST contrast to absolute values of oxygen
concentration, the slope of the calibration curve needs to be known in a pixel–
93
wise manner, while in our analysis we have used a constant value that was calcu-
lated from calibration measurements in bulk aqueous solutions. Nonetheless, it
has been reported that the local viscosity in L. lactis biofilms is “not modified in
comparison with that of water” [227]. Another study has measured the viscosity
of the EPS matrix of S. maltophilia to be 1.50 cP, using FCS [230]. We found no
significant change in the slope of the linear fit when we measured bulk solutions
up to 1.80 cP, prepared by addition of sucrose. No data about local dynamic vis-
cosities in P. aeruginosa biofilms exist to our knowledge, however, based on the
above, it is reasonable to assume a viscosity of our biofilm samples close to that
of water.
5.5 Conclusion
In this chapter we have implemented TRAST imaging on a SPIM system, driven
by its capability to provide optical slicing for thick samples with minimal pho-
totoxicity. For the first time, we have shown high–resolution maps of triplet re-
laxation times inside bacterial biofilms. Furthermore, we have defined TRAST
contrast and presented a new acquisition protocol that allows quantification
of molecular oxygen concentrations based on a calibration curve that was vali-
dated for different micro–environmental conditions. In contrast to most other
existing techniques for measuring oxygen, the technique presented here is not
limited to measuring close to the surface or the substrate of biofilms. Using
TRAST contrast, we were able to image the oxygen concentration profile inside
a P. aeruginosa biofilm, in a non–destructive way. Steep concentration gradients
and zones of complete deaeration inside the colony were found, that would im-
possible to identify at such high resolution with other methods.
We believe that the approach taken here can be used to further our under-
standing of the role of oxygen during biofilm development and life cycle, and
94
that this method has potential to be used as a diagnostic tool in the future, such
as for example to identify aerobic or anaerobic zones in multispecies biofilms
[231] or to study oxygen consumption rates of genetically modified strains of
bacteria.
In future studies, TRAST imaging could work in tandem with other tech-
niques that can provide information about local diffusion and dynamics – such
as for example FCS or anisotropy measurements that can also be established on
camera systems [201, 232]. This could allow accounting for possible artefacts
due to local heterogeneities within the imaged region. Similarly, TRAST imaging
could be combined with sensors that can monitor other factors that could affect
the results, such as for example pH, or other quenchers like thiol derivatives.
95
Chapter 6
Afterword
6.1 Overall conclusions
This thesis aimed to introduce novel spectroscopy techniques in the study of
bacterial biofilms. It is now known that active biofilms are dynamic systems
whose development is characterised and driven by physical and chemical gra-
dients. Hence, methods that can provide spatially and temporally resolved in-
formation are of great value, even more so when they can be applied in vivo.
Two questions in particular were raised and addressed: “How is the stiff-
ness of colonies organised internally?”, and, “How is oxygen distributed inside
biofilms?”. Focus was put on studying these two parameters as they are both
associated with biofilm dispersal which is of high interest in strategising meth-
ods for inhibiting and removing biofilms. To answer these questions, two novel
spectroscopic techniques, namely Brillouin Imaging and Transient State (TRAST)
imaging were employed and further developed.
Regarding stiffness, no correlation with colony size was found when com-
paring the averages of Brillouin shifts of two–dimensional cross–sections of 24
randomly selected colonies growing under continuous flow of minimal media.
Given the high degree of complexity of the samples and the large number of vari-
96
CHAPTER 6. AFTERWORD
ables, a multivariate analysis could be more helpful to reveal quantitative data.
However, the sample size and the spatial–averaging of the Brillouin shift pro-
hibit such an approach. To this end, we focused our study on single colonies,
where we observed two distinct spatial patterns: in smaller colonies, stiffness
increased towards their interior, indicating a more compact structure of the cen-
tre of the colony, whereas, larger (over 45 µm) colonies were found to have less
stiff interiors. We have also identified differences in the stiffness profile of a sin-
gle colony when imaged at different time points. Regarding oxygen distribu-
tion, we have found that oxygen in the deeper parts of the colony is depleted,
which was also known from older studies using microsensors. However, only
with the combination of TRAST with SPIM and after a calibration procedure, as
was done here, was it possible to quantify the steep oxygen gradients that exist
within the colony and extend outside its boundaries. The above highlights that
spectroscopy combined with optical microscopy can provide valuable informa-
tion to microbiologists that would not be available otherwise.
We believe that our results can be beneficial to the study of the complex dy-
namics that drive biofilms and their dispersal. For example, it has been found
that c–di–GMP, a dynamic intracellular signaling molecule, is associated with
the onset of dispersal. More specifically the concentration of c–di–GMP is de-
creased when P. aeruginosa biofilms are exposed to nitric oxide (NO)–releasing
compounds that cause dispersal [233]. Further, a very recent study, using a ra-
tiometric, image–based quantification method, showed that c–di–GMP distri-
bution within colonies is heterogeneous. In particular, high amounts of c–di–
GMP were found to localise towards the outer boundaries of mature colonies,
whereas in smaller colonies a more uniform distribution was found to exist [234].
The localisation of the decrease of c–di–GMP – a cue for dispersal onset – in the
centres of mature colonies, coincides with both our findings from Brillouin mi-
97
croscopy that indicate softer cores in larger colonies, as well as with our TRAST
imaging results that found anoxic zones within colonies.
6.2 Limitations and outlook
For the techniques presented here to find wider applications in microbiology,
progress has to be made in two domains: First, a rigorous theoretical under-
standing of the phenomena underlying the measured quantities has to be de-
veloped. As discussed, in the case of Brillouin microscopy the exact connection
between the measured Brillouin shift and the mechanical modulli in heteroge-
neous samples is, to date, not known. Likewise, in TRAST imaging, the contri-
bution of other factors in the relative changes of triplet relaxation times needs to
be quantified and accounted for. Second, further engineering refinement of the
techniques is needed to allow for more robust and repeatable measurements to
be made. In Brillouin imaging, the current bottleneck is temporal resolution due
to the weak Brillouin signal. This can be overcome in the future by employment
of additional background suppression techniques, such as molecular absorp-
tion cells [157, 158] or notch filters [161]. On the other hand, in SPIM – TRAST
imaging the current bottleneck was the development of a flow cell, suitable for
SPIM, that allows for measurements under constant flow.
Despite these limitations, we envision an exciting future for these techniques
in microbiology, as they can readily be applied to any biofilm, either grown in
nature or developed in the laboratory. A possible combination of both presented
methods under the same platform could give answers relating to the degree of
spatiotemporal correlation between the onset of dispersal and the depletion of
oxygen or other metabolites, especially if markers that can identify transitions
between different stages of the biofilm lifecycle are used [82]. Further, Brillouin
microscopy could used to evaluate different levels of rigidity deriving from spe-
98
cific genes and their mutants [235], or to assess how stiffness is affected at the
microscopical level by different production rates of extracellular polysaccha-
rides, such as Psl or alginate [236]. Other studies could investigate the differ-
ences in mechanical properties induced by biofilms growing on different sub-
strates [237], or, to study the internal changes in biofilm stiffness resulting from
exposure to different dispersal agents, which, to date, have only been done in
bulk [101]. Similarly, TRAST imaging can be used to study oxygen distribution
and consumption inside multispecies biofilms [231] or to monitor changes in
the physiology of bacteria occurring as a response to oxygen depletion [44].
Therefore, the methods that this thesis has dealt with bring unique value
with their high spatial resolution and their non–destructive nature, and, are
compatible with existing microbiological protocols. Hence, their adaptation in
defined future studies can help to shed some light on emerging questions re-
garding the biofilm mode of life.
99
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Appendix A
Typical TRAST illumination scheme
Here we include instructions and a lookup table that can be used to recreate the
illuminating conditions for a TRAST monitoring experiment.
Steps:
1. Decide the total illumination time (Ti l l ). For Eosin Y bulk solutions in the
range of 1 – 2 µM illuminated with a 488 nm laser at ≈ 5 mW before objective,
100 ms is a good starting point.
2. Decide the period of the pulse train (T). This should be longer than the time
scale of the kinetics you want to measure. For our experiments, 50 µs is a suit-
able value.
3. Create a lookup table, similar to the one shown in Table A.1. Depending on
the number of images you wish to acquire, create a distribution of pulse widths
(w) that is denser near the time scale of the kinetics you want to measure and
extends up to the period of the pulse train. The duty cycle (α) for each image
can be calculated as α = T /w and the number of pulses for maintaining con-
A-1
APPENDIX A. TYPICAL TRAST ILLUMINATION SCHEME
stant total illumination as N = Ti l l /w.
4. For each image to be acquired, the acquisition time of your camera has to
cover the integrate the whole train of pulses, therefore it needs to be set to T ac q =
N ·T.
Image no. Pulse width Duty cycle Number of pulses Pulse train length
w (µs) α (%) N Tac q (s)
1 0.5 1 200,000 10.000
2 1 2 100,000 5.000
3 2 3 66,667 3.333
4 2 4 50,000 2.500
5 3 6 33,333 1.667
6 4 8 25,000 1.250
7 5 10 20,000 1.000
8 8 16 12,500 0.625
9 10 20 10,000 0.500
10 13 25 8,000 0.400
11 16 32 6,250 0.313
12 20 40 5,000 0.250
13 30 60 3,333 0.167
14 35 70 2,857 0.143
15 40 80 2,500 0.125
16 50 100 2,000 0.100
Table A.1: Lookup table for TRAST image acquisition settings.
A-2
Appendix B
Protocol for TRAST Contrast vs
Oxygen calibration
In this appendix, instructions, considerations and photographs for recreating
the parallel measurements with a fibre sensor and SPIM are presented. Results
from these measurements are used to construct the calibration curve for quan-
tifying oxygen concentrations (Figure 5.9, page 84).
Steps:
1. Prepare suitable FEP bags. The bags need to be able to hold approximately
800 µL and should be left open at the top to allow insertion of the probe (Figure
B.1a).
2. Calibrate your optical fibre sensor according to manufacturer specifications.
In this thesis a FireSting2 (Pyro–Science) sensor was used and was calibrated by
the two–point method: first, the air–saturation point was set by measurement in
water after bubbling atmosphere air for approximately 30 min using a common
aquarium pump. The zero–point was set by measurement in a water sample
with Na2 SO3 at final concentration of 2 M.

B-1
APPENDIX B. PROTOCOL FOR TRAST CONTRAST VS OXYGEN CALIBRATION
Figure B.1: (a) FEP bags that can hold approximately 800 µL. The tape is added for
structural support. A standard–sized 2–µL Eppendorf tube is shown for
comparison. (b) A trial run on bench top using the probe sensor. (c-d) Par-
allel measurements with an optical sensor and SPIM.
3. Perform a trial run on the bench using only the fibre sensor to make sure that
the titration experiment works as expected and oxygen concentrations are cap-
tured. Hold the bag with a reverse tweezer and place the sensor tip in the middle
of the bag. Pipette Ascorbic Acid and mix gently while measuring (Figure B.1b).
B Make sure the tip of the probe does not come in contact with the walls
of the bag at any time.
4. Transfer the set–up to the SPIM. Place the bag in the centre of the chamber
and hold it steadily using a reverse tweezer. Ensure that the bag is clean and
non–wrinkled to avoid distorting the light sheet. Lower the sensor tip near the
middle of the bag (Figure B.1c–d).
B-2
APPENDIX B. PROTOCOL FOR TRAST CONTRAST VS OXYGEN CALIBRATION
B Make sure that the probe tip does not lie in the laser beam path and
does not come in contact with the bag walls. The bag should not touch
objective or the walls of the chamber.
5. Measure oxygen concentration continuously with the point sensor while per-
forming the titration. At each point, capture two images, one taken while ex-
citing the sample with a low duty cycle pulse train (eg: 200,000 pulses, pulse
width = 0.5 µs, period = 50 µs, illumination time = 100 ms) and one with con-
tinuous wave illumination while keeping the total illumination time equal as
before. Calculate the relative decrease in fluorescence intensity between these
two images (using eq. 5.9, page 74) and correlate it with the absolute value of
oxygen concentration that was measured by the probe sensor at that time point.
B-3

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NOVEL SPECTROSCOPY

TECHNIQUES FOR FUNCTIONAL

A THESIS SUBMITTED FOR THE

NUS GRADUATE SCHOOL FOR INTEGRATIVE

have been used in the thesis.

a Creative Commons Attribution Non-Commercial No Derivatives licence. Re-

must make clear to others the licence terms of this work.

Results of this thesis have been partially published in:

- A. Karampatzakis, J. Sankaran, K. Kandaswamy, S. A. Rice, Y. Cohen, T. Wohland “Mea-

surement of oxygen concentrations in bacterial biofilms using transient state monitor-

- A. Karampatzakis, C. Z. Song, L. P. Allsopp, A. Filloux, S. A. Rice, Y. Cohen, T. Wohland,

P. Török “Probing the internal micromechanical properties of Pseudomonas aeruginosa

biofilms by Brillouin imaging” npj Biofilms and Microbiomes, 3, 1–7, (2017).

along the way.

First and foremost, I would like to express my sincere thanks to my super-

my work despite the difficulties along the way.

Many thanks to my thesis advisor committee members Nanguang Chen (NUS)

most of the biological work for this thesis was done.

Sanchez, Anand Singh, Kumaravel Kandaswamy, Sarala Neomi Tantirimudalige,

Pei–Ju Wung, Giuseppe Antonacci and Guillaume Lepert.

NGS Scholarship, and for being extremely helpful and accommodating.

List of Figures xviii

List of Abbreviations and Symbols xix

1.1 Historical perspective . . . . . . . . . . . . . . . . . . . . . . . . . . 1

2 Photophysics and Photochemistry 8

2.1 Light scattering . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8

2.1.2 Brillouin scattering . . . . . . . . . . . . . . . . . . . . . . . . 10

2.1.3 Brillouin modulus and mechanical properties . . . . . . . . 12

2.2 Luminescence phenomena . . . . . . . . . . . . . . . . . . . . . . . 15

2.2.1 Energy states . . . . . . . . . . . . . . . . . . . . . . . . . . . 15

2.2.2 Singlet vs Triplet states . . . . . . . . . . . . . . . . . . . . . 18

2.2.4 Quenching by Oxygen . . . . . . . . . . . . . . . . . . . . . . 21

3.1 The biofilm mode of life . . . . . . . . . . . . . . . . . . . . . . . . . 25

3.2 EPS and mechanical properties of biofilms . . . . . . . . . . . . . . 29

3.3 The role of oxygen in biofilms . . . . . . . . . . . . . . . . . . . . . . 32

4 Brillouin Microscopy for Mechanical Measurements 35

4.1.1 Confocal microscopy . . . . . . . . . . . . . . . . . . . . . . . 35

4.1.2 Brillouin spectroscopy . . . . . . . . . . . . . . . . . . . . . . 37

4.1.3 The conventional use of term ’stiffness’ . . . . . . . . . . . . 40

4.2 Material and Methods . . . . . . . . . . . . . . . . . . . . . . . . . . 42

4.2.1 Experimental setup . . . . . . . . . . . . . . . . . . . . . . . . 42

4.2.2 Sample preparation . . . . . . . . . . . . . . . . . . . . . . . 45

4.2.3 Acquisition and analysis . . . . . . . . . . . . . . . . . . . . . 47

4.3.1 Measurements in hydrogel beads . . . . . . . . . . . . . . . 51

4.3.2 Characterisation of the stiffness of P. aeruginosa biofilms . 51

4.3.3 Brillouin shift measured at increasing depths . . . . . . . . 53

4.3.4 Temporal changes of stiffness . . . . . . . . . . . . . . . . . . 55

4.3.5 Effect of flow rate . . . . . . . . . . . . . . . . . . . . . . . . . 58

5 SPIM – TRAST for O2 Measurements 65

5.1.1 Single Plane Illumination Microscopy (SPIM) . . . . . . . . 65

5.1.2 Transient States (TRAST) spectroscopy . . . . . . . . . . . . 67

5.2 Material and Methods . . . . . . . . . . . . . . . . . . . . . . . . . . 70

5.2.1 Simplified energy system . . . . . . . . . . . . . . . . . . . . 70

5.2.2 TRAST monitoring theory . . . . . . . . . . . . . . . . . . . . 71

5.2.3 Derivation of TRAST contrast . . . . . . . . . . . . . . . . . . 73

5.2.4 TRAST contrast as a measure of [O2 ] . . . . . . . . . . . . . . 75

5.2.5 Experimental setup . . . . . . . . . . . . . . . . . . . . . . . . 76

5.2.6 Sample preparation . . . . . . . . . . . . . . . . . . . . . . . 78

5.2.7 Acquisition and analysis . . . . . . . . . . . . . . . . . . . . . 81

5.3.1 Measurements in bulk solutions . . . . . . . . . . . . . . . . 83

5.3.2 Measurements in heterogeneous samples . . . . . . . . . . 85

5.3.3 Measurements in bacterial biofilms . . . . . . . . . . . . . . 86

6.1 Overall conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . 96