| Zugriffsnummer | 20263 |
| Dokumenttyp | Dissertation |
| Peer Review | unbekannt |
| Sprache | Englisch |
| Titel | Confocal microscopy and quantitative single molecule techniques for metrology in molecular medicine |
| Autor(in); Institution |
Rüttinger, Steffen; 8.3, Biomedizinische Optik, PTB-Berlin
|
| Quelle/Jahr | (2006), 1 - 141 |
| Dissertationsvermerk | Dissertation, Technische Universität Berlin, 2006 |
| Persistent Identifier | |
| Verlag | Berlin: Techn. Univ. |
| Freie Schlagworte | FRET ; distance measurement ; FCS ; confocal microscopy ; single molecule detection |
| Zusammenfassung | In the last decade, scientists from medicine, biology, chemistry, and physics have achieved great progress in exploring the structure of complex molecules, in particular of biomolecules. Beside structure, however, it is more and more evident that understanding of the dynamics of biomolecules is important to comprehend their function. Without analysis of the physical mechanisms of biomolecular dynamics it seems to be impossible to achieve progress in the understanding of the functioning of biological macromolecules. Within the frame of this work two experimental techniques have been investigated for their purposes to study biomolecular dynamics quantitatively on the level of single molecules: 1. Fluorescence Correlation Spectroscopy (FCS) was investigated as a tool to study fluctuations in the local concentration of molecules and their dynamic properties like diffusion. 2. F¨orster Resonace Energy Transfer (FRET) was investigated as a technique to determine distances between two molecules in close proximity (1 nm to 10 nm) under native conditions as well as dynamic changes in the conformation of biomolecules. It is worth to note that the application of both methods on single molecules is important since ensemble measurements would barely help to gain the desired insight in the molecular dynamics and function due to the huge heterogeneity inherent to biological systems. The scope of this work was to evaluate the accuracy and the limits of quantitative FCS and FRET on the single molecule level. The experimental requirements will be given and hidden pitfalls are discussed. The needs for careful calibration and its realisation are shown. In particular, methods to obtain the detection volume of a confocal microscope and the experimental conditions influencing its shape are discussed, as knowledge of the so-called confocal volume is a prerequisite for quantitative FCS analysis. Secondly, a novel approach for FRET distance measurements is presented which accounts for incomplete labeled samples and experimental conditions, thereby increasing the accuracy of FRET distance measurements. |
