Abstract ........................................................ v
1 Introduction ................................................. 1
1.1 The Challenge of Life Science Research .................. 1
1.2 Outline ................................................. 4
I Theoretical Basis ............................................ 7
2 Basic principles of fluorescence spectroscopy ................ 9
2.1 One-photon excitation ................................... 9
2.2 Multi-photon processes ................................. 10
2.3 Fluorescence Lifetime .................................. 11
2.4 Some dyes .............................................. 13
2.4.1 Autofluorescent Proteins ........................ 13
2.4.2 Organic Chromophores ............................ 14
2.4.3 Quantum Dots .................................... 14
3 Fluorescence Correlation Spectroscopy ....................... 15
3.1 Historic overview and principles ....................... 15
3.2 1-Photon excitation .................................... 16
3.2.1 The auto-correlation function ................... 16
3.2.2 Derivation of correlation functions ............. 19
3.2.2.1 Determining the concentration
correlation function - general
approach ............................... 23
3.2.2.2 Translational diffusion ................ 25
3.2.2.3 Auto-correlation function for
multiple species ....................... 27
3.2.2.4 Active transport ....................... 28
3.2.2.5 Unimolecular reactions ................. 29
3.2.2.6 Other processes ........................ 32
3.2.3 Cross-correlation ............................... 34
3.2.4 Coincidence ..................................... 36
3.2.5 Triple-Correlation .............................. 37
3.2.6 Triple-Coincidence .............................. 38
3.3 Caveats ................................................ 39
3.3.1 Influence of solvent viscosity η and
refractive index n .............................. 39
3.3.2 Influence of different brightnesses ............. 42
3.3.2.1 Crosstalk but no change in brightness
upon binding ........................... 42
3.3.2.2 Crosstalk and change in brightness
upon binding ........................... 45
3.3.2.3 Uncorrelated background ................ 47
4 Fluorescence Resonance Energy Transfer ...................... 49
4.1 spFRET ................................................. 49
4.2 triFRET ................................................ 54
4.2.1 Linear energy transfer cascade (B -> Y -> R) .... 59
4.2.2 Energy transfer to two acceptors, no triFRET
(R <- В -> Y) ................................... 60
4.2.3 Linear energy transfer cascade (B -> Y -> R)
and direct FRET (B -> R) ........................ 61
5 Time-correlated single-photon counting ...................... 63
5.1 Principles of TCSPC .................................... 63
5.2 Caveats of TCSPC ....................................... 64
5.2.1 Timing .......................................... 64
5.2.2 Pulse pileup .................................... 65
5.2.3 Effects of laser repetition frequency ........... 65
5.3 Example of a TCSPC measurement ......................... 66
5.4 Analysis of Fluorescence Decays ........................ 67
5.5 Single molecule measurements ........................... 68
5.5.1 Continuous flow mode ............................ 68
5.5.2 FIFO (Time-Tag) Mode ............................ 68
5.5.3 Burst analysis .................................. 70
6 Optical Trap ................................................ 73
6.1 Short historic overview ................................ 73
6.2 Principles ............................................. 73
6.3 Biological implications ................................ 77
II Experimental Realisation ................................... 79
7 Experimental Setup .......................................... 81
7.1 Experimental Setup ..................................... 81
7.2 Laser sources .......................................... 84
7.2.1 Continuous-wave one-photon excitation ........... 84
7.2.2 Two-photon excitation ........................... 85
7.2.3 Pulsed one-photon excitation .................... 85
7.3 Mirrors and filters .................................... 87
7.4 Detectors .............................................. 89
8 Data acquisition electronics and software ................... 91
8.1 Hardware correlator .................................... 91
8.2 TCSPC card ............................................. 92
8.3 Data analysis software ................................. 93
8.3.1 Commercial programmes ........................... 93
8.3.2 Custom-written software ......................... 93
9 Sample Preparation .......................................... 97
9.1 Sample cells ........................................... 97
9.1.1 Types of sample cells ........................... 97
9.1.1.1 Cover slides ........................... 97
9.1.1.2 Sandwich Cells ......................... 97
9.1.1.3 Large volume sample chambers ........... 98
9.1.2 Coating of coverslides .......................... 99
9.1.2.1 Quartz coverslides ..................... 99
9.1.2.2 Coating with BSA (bovine serum
albumin) ............................... 99
9.1.2.3 Silanised coverslides .................. 99
9.1.2.4 Coating with Poly-L-Lysine ............ 100
9.1.2.5 Coating with PEG (polyethylene
glycol) ............................... 100
9.2 Chromophores .......................................... 100
9.2.1 Autofluorescent proteins ....................... 101
9.2.2 Synthetic chromophores ......................... 102
III Experimental Results ...................................... 105
10 Scanning cross-correlation ................................. 107
10.1 Introduction .......................................... 107
10.2 Immobilising single molecules ......................... 109
10.3 Experimental Setup .................................... 110
10.4 Theory ................................................ 111
10.5 Results and Discussion ................................ 112
10.6 Conclusions and Outlook ............................... 117
11 Structural changes in UvrB monitored by spFRET ............. 119
11.1 Nucleotide excision repair (NER) ...................... 119
11.2 The crystal structure of a sequence homologue ......... 121
11.3 The padlock model ..................................... 122
11.4 Sample preparation .................................... 123
11.5 Experimental details .................................. 124
11.6 Bulk measurements ..................................... 126
11.7 Single-molecule measurements .......................... 133
11.7.1 Binding of UvrB317 to dsDNA .................... 133
11.7.2 Binding of UvrB276 to dsDNA .................... 139
11.7.3 Binding of ssDNA ............................... 142
11.8 Discussion ............................................ 142
11.8.1 Binding of different cofactors ................. 144
11.8.2 Selective binding of DNA ....................... 144
11.8.3 ATPγS prevents helicase activity ............... 145
11.8.4 Binding of ssDNA ............................... 145
11.8.5 Overview ....................................... 145
12 Three-colour Measurements .................................. 149
12.1 DNA ................................................... 149
12.1.1 The biological system .......................... 149
12.1.2 Experimental details ........................... 151
12.1.3 Bulk measurements .............................. 152
12.1.3.1 Fluorescence spectra .................. 152
12.1.3.2 Fluorescence lifetime ................. 155
12.1.4 Correlation techniques ......................... 158
12.1.5 Singlc-molcculc triFRET ........................ 163
12.1.6 Discussion ..................................... 166
12.2 EcoRI ................................................. 167
12.2.1 The biological system .......................... 167
12.2.2 Sample preparation ............................. 168
12.2.3 Experimental details ........................... 169
12.2.4 Single-molecule measurements ................... 170
12.2.4.1 The Educts ............................ 171
12.2.4.2 The products .......................... 171
12.2.4.3 Kinetics of Alexa546-EcoRI cutting
Alexa488-DNA-Cy5 ...................... 172
12.2.5 Discussion ..................................... 173
12.3 UvrABC ................................................ 174
12.3.1 The biological system .......................... 174
12.3.1.1 GFP - UvrB - Alexa 546 and DNA -
Cy5 ................................... 174
12.3.1.2 GFP - UvrC, UvrB - Alexa 546 and
DNA - Cy5 ............................. 175
12.3.2 Experimental details ........................... 176
12.3.3 Singlc-molcculc measurements ................... 177
12.3.3.1 GFP - UvrB - Alexa 546 and DNA -
Cy5 ................................... 177
12.3.3.2 GFP - UvrC, UvrB - Alexa 546 and
DNA - Cy5 ............................. 180
12.3.4 Discussion ..................................... 181
13 Biomimetic containers for "single" molecules ............... 183
13.1 Vesicle preparation ................................... 184
13.2 Experimental setup .................................... 185
13.2.1 One laser line for both trapping and
fluorescence excitation ........................ 185
13.2.2 Long term stability of the optical trap and
the vesicles ................................... 186
13.2.3 Separate laser lines for trapping and
excitation ..................................... 186
13.3 FCS in small confined compartments .................... 187
13.4 TCSPC in single vesicles .............................. 188
13.4.1 High chromophore concentrations ................ 188
13.4.2 "Single" molecules ............................. 189
13.4.3 Influence of different environments ............ 190
13.5 Discussion ............................................ 191
13.6 Potential applications - A feasibility study .......... 191
13.7 Conclusions ........................................... 195
14 Summary and Outlook ........................................ 197
A Glossary of acronyms ....................................... 201
В Glossary of physical terms ................................. 203
B.l Latin ................................................. 203
B.2 Greek ................................................. 205
Bibliography .................................................. 207
List of publications .......................................... 223
Acknowledgements .............................................. 225
Erklärung (Declaration) ....................................... 227
List of Figures
1.1 Protein synthesis and structural elements .................. 2
1.2 Energy landscape for protein folding ....................... 3
1.3 Myoglobin as an example for structural dynamics of
proteins ................................................... 4
2.1 Jablonski diagram for one-photon excitation ................ 9
2.2 Fluorescence spectrum and intensity depending on
the excitation wavelength ................................. 10
2.3 Jablonski diagram and Feynman diagram for two-photon
excitation ................................................ 11
2.4 Comparison of one-and two-photon excitation and emission
spectra ................................................... 12
2.5 Approximate sizes of various molecules .................... 13
3.1 Molecular mechanisms causing fluorescence fluctuations .... 15
3.2 Comparison between one- and two-photon absorption
processes ................................................. 16
3.3 Calculation of the auto-correlation function .............. 17
3.4 Dependence of the auto-correlation curve on
the concentration and the size of the molecules ........... 18
3.5 Simulated auto-correlation curve .......................... 33
3.6 Principle of dual-colour cross-correlation ................ 35
3.7 Influence of viscosity and refractive index on
the auto-correlation curve ................................ 40
3.8 Influence of viscosity and refractive index on
brightness and apparent concentration ..................... 41
3.9 Changes in the correlation amplitudes in the presence
of spectral crosstalk ..................................... 44
3.10 Changes in the correlation amplitudes in the presence
of spectral crosstalk and quenching ....................... 46
4.1 FRET: Schematic diagram and spectra of two fluorophores ... 50
4.2 The orientation factor к2 ................................. 52
4.3 Potential arrangement of the chromophores in a simple
triple FRET cascade ....................................... 54
5.1 Information of a single fluorescence photon ............... 63
5.2 Principle of TCSPC and possible applications .............. 64
5.3 Pile-up effects in fluorescence decay curves .............. 65
5.4 Normalised fluorescence decay for TMR ..................... 66
5.5 Convoluting the instrument and the impulse response
function yields the measured data ......................... 67
5.6 Different continuous flow measurements .................... 69
5.7 Burst analysis: Identification of single-molecule
passages through the focus ................................ 70
5.8 Photon arrival times vs. intensity for identifying
bursts .................................................... 71
6.1 Optical trapping of a dielectric sphere ................... 74
6.2 Ray optical explanation of an optical trap ................ 74
6.3 Scattering force, gradient force and total force exerted
on a sphere ............................................... 76
6.4 Direction and magnitude of gradient force, scattering
force and their vector sum ................................ 77
7.1 Schematic FCS Setup ....................................... 81
7.2 Microscope and detection unit for one- and two-channel
detection ................................................. 82
7.3 Detection units for one-, two- and three-channel
detection ................................................. 83
7.4 Available laser wavelengths for fluorescence excitation ... 84
7.5 Cross-section of the Ti:Sa laser beam ..................... 85
7.6 Auto-correlation curves and intensity traces of Alexa488
for cw and pulsed one-photon excitation ................... 86
7.7 Brightness of Alexa 488 depending on the excitation mode
and incident power ........................................ 87
7.8 Stokes Raman spectra of the OH band in water .............. 87
7.9 Wavelength of the Raman band of water depending on
the incident wavelength ................................... 88
8.1 Screenshots of the two-channel programme .................. 94
8.2 Programme handling ........................................ 95
8.3 Screenshots of the three-channel programme ................ 96
9.1 Different types of sample cells ........................... 98
10.1 Two different scanning patterns .......................... 109
10.2 Experimental setup for two-colour scanning fluorescence
cross-correlation ........................................ 110
10.3 GFP and DsRed immobilised in a polyacrylamide matrix ..... 113
10.4 Stepwise bleaching of a single immobilized DsRed or
GFP molecule ............................................. 114
10.5 FCS curves of the freely diffusing molecules in
solution ................................................. 114
10.6 FCS curves acquired during a line scan of immobilized
molecules ................................................ 115
10.7 Comparison of the cross-correlation amplitudes of
fusion proteins and the unlinked chromophores ............ 115
10.8 Scanning FCCS curves of Cy2-Cy5-labelled cholera toxin
on a cell membrane ....................................... 116
11.1 Damage recognition in nucleotide excision repair ......... 120
11.2 Schematic structure of the UvrB-DNA complex .............. 122
11.3 Crystal structure of UvrВ and label positions ............ 124
11.4 Absorption and emission spectra of GFP and Rhodamine
Red X .................................................... 125
11.5 Overlap integral ......................................... 126
11.6 Normalised fluorescence emission spectra of
GFP-UvrB276 ............................................... 127
11.7 Normalised fluorescence emission spectra of double-
labelled UvrB317 .......................................... 128
11.8 Larger view of the peaks in figure 11.7 .................. 128
11.9 Normalised fluorescence emission spectra of double-
labelled UvrB276 .......................................... 129
11.10 Larger view of the peaks in figure 11.9 ................. 129
11.11 Auto-correlation curves for UvrB317GFP only with
different binding partners .............................. 130
11.12 Fluorescence decay curves for both mutants for
different buffer conditions ............................. 131
11.13 Fluorescence decay curves and deconvolutions for both
mutants ................................................. 132
11.14 Distribution of FRET efficiencies for Alexa 488 and
for UvrB317GFP only ........................................ 133
11.15 FRET efficiencies for UvrB317 in the presence of Mg2+,
ATPγS and UvrA2 ......................................... 134
11.16 FRET efficiencies for UvrB317 in the presence of ATP,
UvrA2 and DNA ........................................... 135
11.17 Inter-chromophore distances for UvrB317 in
the presence of Mg2+, ATPγS, ATP and UvrA2 ............... 136
11.18 Inter-chromophore distances for UvrB317 in
the presence of ATP, UvrA2 and both damaged and
intact DNA .............................................. 137
11.19 Inter-chromophore distances for UvrB317 in
the presence of different cofactors ..................... 140
11.20 Inter-chromophore distances for UvrB276 in
the presence of different cofactors ..................... 141
11.21 Inter-chromophore distances for UvrB317 and UvrB276
in the presence of ssDNA ................................ 143
12.1 Positions of the three dyes attached to the 28-mer
dsDNA .................................................... 150
12.2 FRET efficiencies for the different chromophore
combinations ............................................. 150
12.3 Normalised emission spectra of the three chromophores
and transmission spectra of the filters .................. 152
12.4 Normalised spectra of the chromophore triplets and
the single dyes attached to the dsDNA .................... 153
12.5 Efficiency of triFRET for RhG-TAMRA-Cy5 .................. 154
12.6 Annealing kinetics of the TAMRA-labelled ss-14-mer and
the corresponding RhG-Cy5-labeled oligonucleotide ........ 155
12.7 Fluorescence decay curves for different chromophore-
combinations on the DNA .................................. 156
12.8 Determining the molecular brightness via FCS ............. 158
12.9 Cross-correlation amplitudes for the three pairwise
cross-correlations ....................................... 160
12.10 Coincidence and triple-coincidence ...................... 161
12.11 Annealing kinetics of the TAMRA-labelled ss-14-mer
and the corresponding RhG-Cy5-labeled oligonucleotide
monitored by FCS ........................................ 162
12.12 2D-histograms for RhG acting as primary donor ........... 164
12.13 2D-histograms for TAMRA acting as primary donor ......... 165
12.14 2D-histograms of different oligomer mixtures all
giving the same equimolar chro-mophore concentrations ... 166
12.15 The system under investigation: EcoRI-Alexa 546 and
21-mer Alexa 488-dsDNA-Cy5 .............................. 167
12.16 Reaction scheme for monitoring the binding and
cleavage of DNA by EcoRI ................................ 169
12.17 Normalised emission spectra of the three chromophores
and transmission spectra of the filters ................. 170
12.18 Two-dimensional histograms for Alexa 488 - DNA - Cy5
and EcoRI - Alexa546 .................................... 171
12.19 Two-dimensional histograms for a mixture of Alexa
488 - DNA - Cy5 and EcoRI -Alexa546 with EDTA and
MgCl2 ................................................... 172
12.20 Kinetics of the endonucleolytic cleavage reaction ....... 173
12.21 Crystal structure of UvrB and label positions for
triFRET ................................................. 175
12.22 NER cascade and lapel positions for triFRET using
UvrABC and DNA .......................................... 176
12.23 Two-dimensional histograms for GFP - UvrB - Alexa 546
and DNA - Cy5 ........................................... 178
12.24 Two-dimensional histograms for GFP - UvrB - Alexa 546
and unlabelled DNA ...................................... 179
12.25 Two-dimensional histograms for GFP - UvrB and DNA -
Cy5 ..................................................... 179
12.26 Two-dimensional histograms for GFP - UvrC, UvrB -
Alexa 546 and DNA - Cy5 ................................. 180
12.27 Two-dimensional histograms with reduced ATP ............. 181
13.1 DsRed immobilised in PAA ................................. 183
13.2 Different kinds of vesicles .............................. 184
13.3 Principle of trapping a vesicle with the Ti:Sa laser ..... 185
13.4 Survival probability of vesicles ......................... 186
13.5 FCS curves of Fluorescein contained within a vesicle ..... 187
13.6 Intensity and fluorescence lifetime decay curves for
fluorophores within a vesicle ............................ 189
13.7 Bleaching of a DsRed-oligomer contained within
a vesicle ................................................ 189
13.8 Fluorescence decay of GFP-DsRed in different
environments ............................................. 190
13.9 One-channel setup for characterising E. coli ............. 192
13.10 Burst distribution for diffusing E. coli and bacteria
in a directed flow ...................................... 193
13.11 Sorting E. coli by fluorescence lifetime ................ 194
List of Tables
3.2 Coefficients for the rotational correlation function ...... 33
3.4 Brightness and concentration of green and red
chromophores .............................................. 42
7.1 Properties of different laser diode heads ................. 86
8.1 Binary data format of the SPC-600 TCSPC card for 32-bit
resolution ................................................ 92
8.2 Binary data format of the SPC-600 TCSPC card for 48-bit
resolution ................................................ 92
9.1 Characteristics of two commonly used fluorescent
proteins ................................................. 101
9.2 Characteristics of various organic chromophores .......... 102
9.3 Characteristics of various rhodamine dyes ................ 103
11.1 Fluorescence lifetimes of GFP-UvrB in different buffer
conditions (linear fit) .................................. 127
11.2 Fluorescence lifetimes of GFP-UvrB in different buffer
conditions (deconvolution) ............................... 130
11.3 FRET efficiency and average distance between
the chromophores ......................................... 132
11.4 Mean inter-chromophore distances for UvrB317 ............. 139
11.5 Mean inter-chromophore distances for UvrB276 ............. 139
11.6 Mean inter-chromophore distances for UvrB276 in
the presence of ATP7S .................................... 141
11.7 Inter-chromophore distances for UvrB317 and UvrB276
in the presence of ssDNA ................................. 142
11.8 Structural changes predicted and observed
experimentally upon binding of Mg2+, ATP and UvrA2 ....... 146
11.9 Structural changes predicted and observed
experimentally upon DNA binding .......................... 147
12.1 Approximate distances between the different
chromophores ............................................. 151
12.2 Comparison between calculated and measured FRET
efficiencies ............................................. 154
12.3 Fluorescence lifetimes of the different chromophore
combinations ............................................. 157
12.4 Comparison between calculated and measured FRET
efficiencies ............................................. 157
12.6 Relative brightness values for the three chromophore
pairs .................................................... 159
12.7 FRET efficiencies determined from the single-molecule
measurements ............................................. 165
12.8 Approximate distances between the different
chromophores ............................................. 169
12.9 Forstcr radii for triFRET in UvrB ........................ 177
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