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Steps towards simultaneous atomic force and fluorescence spectroscopy of single DNA [Elektronische Ressource] / Alexander Gaiduk

De
209 pages
Steps towards simultaneous atomic-force and fluorescence spectroscopy of single DNA Thesis Department of Mathematics and Natural Sciences Heinrich-Heine-University Düsseldorf Alexander Gaiduk from Minsk June 2006 Institute of Physical Chemistry, Heirich-Heine University Düsseldorf Printed with the permission of Department of Mathematics and Natural Sciences Heinrich-Heine-University Düsseldorf 1. Referee: Prof. Dr. C.A.M. Seidel 2. Referee: Prof. Dr. K. Kleinermanns 3. Referee: Prof. Dr. Th. Basche Date of the oral examination: 29 November 2006 Посвящается моим родителям, Анатолию Ивановичу и Валентине В а сил ьевн е Гай д ук Acknowledgments First of all I would like to thank my supervisor Prof. Dr. C.A.M. Seidel for giving me the opportunity to work on interesting interdisciplinary projects, to study and improve my knowledge in various fields of physics and chemistry. I am grateful for your enthusiasm, for fruitful discussions and ideas, for your help in understanding the results as well as for your faith in me and your kind support in everyday life outside lab. I am truly grateful to… Dr. Suren Felekyan for his help in various aspects of single molecules fluorescence detection and analysis. I would like to thank you for always showing interest in my work and for your critical remarks.
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Steps towards simultaneous atomic-force and
fluorescence spectroscopy of single DNA
Thesis

Department of Mathematics and Natural Sciences
Heinrich-Heine-University Düsseldorf


Alexander Gaiduk
from Minsk
June 2006
Institute of Physical Chemistry,
Heirich-Heine University Düsseldorf















Printed with the permission of
Department of Mathematics and Natural Sciences
Heinrich-Heine-University Düsseldorf


1. Referee: Prof. Dr. C.A.M. Seidel
2. Referee: Prof. Dr. K. Kleinermanns
3. Referee: Prof. Dr. Th. Basche

Date of the oral examination: 29 November 2006










Посвящается моим родителям, Анатолию Ивановичу и
Валентине В а сил ьевн е Гай д ук
Acknowledgments
First of all I would like to thank my supervisor Prof. Dr. C.A.M. Seidel for giving me the
opportunity to work on interesting interdisciplinary projects, to study and improve my
knowledge in various fields of physics and chemistry. I am grateful for your enthusiasm, for
fruitful discussions and ideas, for your help in understanding the results as well as for your
faith in me and your kind support in everyday life outside lab.

I am truly grateful to…
Dr. Suren Felekyan for his help in various aspects of single molecules fluorescence detection
and analysis. I would like to thank you for always showing interest in my work and for your
critical remarks. Thank you for your patience, frankly conversations and a plenty of useful
advice. I will always remember “training evenings”, “grilling on Rhein”, history of Armenia
and many other things.

Dr. Matthew Antonik, for introducing and guiding me in exciting worlds of AFM, LabView
tricks and biology as well as for being a nice roommate. I will really miss your extravagant
jokes and our talks about science, history, languages, games, culture, and politics.
Dr. Ralf Kühnemuth for teaching fluorescence methods, as well as for his useful comments
and valuable suggestions. It was a pleasure for me to work together with a tactful person and
an experienced scientist like you. I hope we will have another chance to canoe with your nice
family and to drink coffee in the middle of the lake again.
My progress would not have been possible without previous hard work of Dr. Matthew
Antonik and Dr. Ralf Kühnemuth, who had started the complicated task of the equipment
combination. I want to thank both of you for sharing your knowledge and experience. Thank
you a lot for your help and support.

Volodymyr Kudryavtsev for his programming work and explanations. I have been enjoying
discussions on the art of photography and visual perception. I will not forget our “just five
kilometres” bicycle trips and “even sunburn”.

Carl Sandhagen, Martin Schramm and Marcel Merkwitz for their help with useful electronic
devices and computers.

Alessandro Valeri for his help with the fluorescence spectrometer and bulk fluorescence
spectra measurements, for being a nice company on whatever places and situations, for being
positive and humorous.

Denis Dörr and Dr. Stanislav Kalinin for the exchange of ideas as well as for pleasant chats
and joint activities.

Dr. Marcelle König, Dr. Enno Schweinberger, Dr. Anna Wozniak and Dr. Filipp Oesterhelt
for useful tips as well as for the organization of most of the parties and social activities at the
department.

I would like to thank many times to Bärbel Hoffman and Veronika Mendorf for your
optimism and taking care of all the paper work.

Ralf Müller for being a good fellow and for his kind help during the move.

Opas Tojira for useful comments on my presentation in Berlin.

Harekrushna Sahoo for a real Indian food.

My uncle, Dr. Peter Gaiduk for his advices and support.

Prof.Dr. Sergey Gaponenko for initiating my interest in single molecules research.

I would like to express my gratitude once again to Ralf Kühnemuth, Suren Felekyan, Stefan
Marawske, Matthew Antonik, Elena and Bernd Potthoff – people, who have carefully read my
thesis. Dear Elena, thank your for your lessons.

Also I would like to thank everyone at the department of Physical Chemistry in Düsseldorf
for their warm opened relationship and readiness to help. Special thanks to the dancing
community of HHU Düsseldorf for the great time on a dance floor.

I thank my parents Anatoli Gaiduk and Valentina Gaiduk, my sister Olga and my friends, for
their support and encouragement.

Finally, I would like to express my love and thank to my wife Ala Valetava, a wonderful
woman. I thank you for your patience, understanding, support and making my life much more
beautiful and full of tenderness.

Summary 1
Summary
The development of single molecule fluorescence detection allows studying the
properties of molecules without ensemble averaging. The detailed information about the
fluorescence lifetime, the fluorescence quantum yield, the quenching mechanisms and the
motion parameters of individual fluorophores and fluorophore mixtures can be delivered by
Multiparameter Fluorescence Detection (MFD). The structure, dynamics and functionality of
complex biological molecules can also be probed with MFD.
An additional dimension can be added to the MFD for studies of complex biological
molecules by applying an external force. A precise mechanical manipulation of the sample
providing the information about the force would enhance the control of the experiments and
the analytical power of the analysis.
Goals and objectives
This thesis is devoted to the combination of fluorescence microscopy and spectroscopy
with atomic force microscopy (AFM) and spectroscopy techniques. The aim of the
combination is simultaneous force and fluorescence studies of single biological molecules (in
particular DNA).
This complex problem requires consecutive realization of several steps:
• preparation of transparent surfaces for a stable attachment of biomolecules,
• investigation of the atomic force cantilevers optical properties,
• development of fluorescence analysis techniques capable of resolving subnanometer
distance changes between fluorophores,
• establishment of the atomic force spectroscopy method in the lab,
• development of simultaneous atomic-force and fluorescence spectroscopy experiment.
Surface preparation
Since there are two microscopy techniques with different spatial resolutions to be
combined, there are two possible approaches to identify a DNA molecule on the surface. The
first one implies finding a molecule with atomic force microscope, while the second one uses
optical microscopy for the localization of DNA. Each approach has different requirements to
the transparency, roughness and cleanness of glass substrates as well as glass modification
procedures.
Glass cleaning and silanization provide surfaces for stable DNA molecule binding. The
fluorescence of the surface after cleaning and silanization is very low and the optical signal
detected after laser illumination consist mainly of Raman scattered light. Thus optical 2 Summary
microscopy allows finding a single DNA molecules labelled with the dye on the surface. A
relatively rough glass surface (up to 3.4 nm RMS) does not promote the identification of
single DNA by means of AFM.
Fluorescence from the cantilevers
Knowledge of the optical properties of AFM tips is relevant for the combination of
optical and force spectroscopy. Detailed studies show whether the amount of scattered and
luminescence light from an AFM tip would overwhelm the signal of a single fluorophore. The
problem of the cantilever tips fluorescence is studied by means of the newly developed
Multiparameter Fluorescence Imaging (MFI) technique. This technique is used for 3-
dimentional optical imaging and characterization of the AFM tip. Cantilevers of two types of
material (Si and Si N ) were tested. 3 4
The Si tips have the lowest signal intensity and they are preferred for the combination of
MFD and force spectroscopy. However, commercial Si cantilevers do not have mechanical
properties (low stiffness) required for the sensitive force measurements. The Si N tips have 3 4
higher relative brightness which drops quickly with the distance from the tip. Since the sharp
edges are typically the brightest scattering sources on a tip, unsharpened and blunt tips should
be used when possible for combined applications. Alternatively, tips could be modified or
replaced with materials which are more suitable for optical experiments, effectively
substituting the native tip properties.
A time gating or a molecule separation from the tip by a linker can be employed to
eliminate the additional signal from the tip.
In addition, modelling of the background in a multi-component fit of the data with fixed
pre-determined background constants can be used for taking into account the optical signal
from a cantilever in the data analysis.
Fluorescence detection and high precision distance measurements
The combination of fluorescence and force spectroscopy techniques can provide
complementary data in studies of the structure and dynamics of complex biomolecules.
Förster (fluorescence) resonance energy transfer (FRET) is used to measure distances in
macromolecules. For FRET experiments a single molecule or molecular complex is labelled
with two different dyes and the efficiency of energy transfer from one dye to the other is
monitored. The main difficulty in extracting molecular information from fluorescence
intensity distributions is the inability to unambiguously distinguish molecular fluctuations
from either stochastic variations or background counts. A newly developed probability
distribution analysis (PDA) is capable of predicting the shot noise limited shapes of Summary 3
histograms generated from single photon counting data. The PDA takes into account the
effects of background and stochastic processes for the high precision quantitative analysis.
The PDA can successfully extract the originating value behind shot noise limited FRET
signal distributions and determine the underlying fluorescence signal ratio with a precision of
better than 2%. This precision translates into a precision in the distance measurements better
than 1 % of the Förster radius. A broadening of the distribution by 5Å due to mobility of the
dyes on flexible linkers is easily revealed. However, detailed studies of the influence of
background counts on PDA results are still required.
The PDA is also applied to study the spectral shifts of fluorescent molecules, which
makes the method attractive for pH monitoring in a living cell or for probing a
microenvironment of fluorescent molecules.
Force spectroscopy establishment
The custom-build setup for the force spectroscopy is based on a commercial AFM
system and an additional acquisition board. Pulling experiments on single DNA molecules
reveal structural transitions in the molecule upon the applied force.
The influence of the dye (SYBR Green I, groove binder) is studied. The results were
found to be consistent with previous optical tweezers reports on dsDNA force spectroscopy
using this dye, which indicate a hysteresis between the retraction and approach force curves.
The dsDNA B-S transition force is increased up to 8.9% upon SYBR Green binding at an
average concentration of 0.28–0.55 dyes/bp and 15% upon SYBR Green binding at an
average concentration of 1-2 dyes/bp (comparing to the literature value of the B-S transition
force of 65 pN).
Combined fluorescence and force spectroscopy
The piezo hysteresis in the sample plane is quantified and the way to eliminate it
without a closed loop control is proposed. The position uncertainty of molecules binding on
the AFM tip is discussed.
Several experimental approaches to realize the simultaneous force spectroscopy and
multiparameter fluorescence detection have been demonstrated. Successful experiments are
performed depositing DNA molecules on the tip or on the surface. Consecutive pulling on a
single DNA and the simultaneous optical signal registration (more than 10 pulls) were
achieved. As observed in the simultaneous experiments, the structural changes of a DNA
molecule correlate with the intensity and the lifetime change of the fluorescence of the DNA
binding dye.
4 Summary
Perspectives
Confocal scanning adds spatial resolution to the analysis available in MFD.
Simultaneous 3D-mapping of all fluorescence parameters with MFI technique can be
implemented for studies in cells and membranes. Additionally, image correlation analysis is
capable of analysing temporal and spatial fluctuations in the raster scan images and extends
the fluorescence correlation analysis to time scales of seconds or minutes.
Long Si or carbon cantilever tips have the best optical properties for combined
experiments in order to minimize the influence of the tip’s fluorescence signal. Sharp AFM
probes and their proper chemical treatment will limit the attachment area of molecules on the
tip.
The method of the fluorescence-directed force spectroscopy can be implemented to
study different biological molecules and cells. Simultaneous MFD and force spectroscopy
proposes a way to study the structure of macromolecules and fast dynamic processes. A
molecule can be driven into a certain conformation and its behaviour can be monitored based
on the fluorescence signal. Differently, unstable intermediate states can be probed by force
spectroscopy and detailed temporal information about the fluorescence can be obtained in a
single run experiment. The data analysis of the combined experiment then includes the
correlation on the time scale of force events and changes in fluorescence parameters. Thus, an
additional dimension (force values) is added to the standard two-dimentional MFD
histograms.
The combination of force spectroscopy and FRET probability distribution analysis
propose high spatial resolution in both mechanical and fluorescence measurements of the
combined experiment.

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