List of Figures ................................................. v
List of Abbreviations ......................................... vii
Abstract ....................................................... ix
1 Introduction ................................................. 1
1.1 The Problem of Resolution ............................... 1
1.2 Solutions for the Lateral Dimensions .................... 4
1.3 Solutions for the Third Dimension ....................... 5
1.4 Optical Near-fields to Overcome the Resolution Limit .... 7
1.5 Resolution, Accuracy, and Precision ..................... 8
1.6 Microtubules as Optical Probes .......................... 9
2 Calibrating the Optical Near-fields in Total Internal
Reflection Fluores cence Microscopy ......................... 11
2.1 Total Internal Reflection Fluorescence Microscopy ...... 12
2.2 Quantitative Measurement of the Optical Near-fields -
Experiments ............................................ 15
2.3 Calibrating the Optical Near-fields - Results .......... 22
2.4 Correcting for Aberration Effects - Theory ............. 27
2.5 Discussion ............................................. 33
3 Determining Molecular Positions and Geometries in
the Third Dimension by Fluorescence Lifetime Modulations
near Metallic Surfaces ...................................... 37
3.1 Fluorescence Lifetime Modulations in Proximity
to Surfaces ............................................ 38
3.2 Measuring Fluorescence Lifetime of Microtubules
Elevated in Different Configurations - Experiments ..... 40
3.3 Converting Fluorescence Lifetimes to Vertical
Positions Theory ....................................... 48
3.4 Determining Molecular Geometries - Results ............. 51
3.5 Discussion ............................................. 54
4 Summary and Outlook ......................................... 59
A Protocols for Calibrating the Optical Near-fields in Total
Internal Reflection Fluorescence Microscopy ................. 63
A.l Microtubule Preparation ................................ 63
A.2 Microtubule Immobilization ............................. 63
В Protocols for Determining Molecular Positions and
Geometries in the Third Dimension by Fluorescence Lifetime
Modulations near Metallic Surfaces .......................... 65
B.l Microtubule Preparation ................................ 65
B.2 Sample Preparation ..................................... 66
B.3 Control Experiment I ................................... 68
Bibliography ................................................... 69
Index .......................................................... 75
Acknowledgements ............................................... 77
Erklarung ...................................................... 79
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