Dokument: Development and applications of new fluorescence techniques to characterize single biomolecules in solution and living cells
| Titel: | Development and applications of new fluorescence techniques to characterize single biomolecules in solution and living cells | |||||||
| URL für Lesezeichen: | https://docserv.uni-duesseldorf.de/servlets/DocumentServlet?id=5552 | |||||||
| URN (NBN): | urn:nbn:de:hbz:061-20070903-120633-4 | |||||||
| Kollektion: | Dissertationen | |||||||
| Sprache: | Englisch | |||||||
| Dokumententyp: | Wissenschaftliche Abschlussarbeiten » Dissertation | |||||||
| Medientyp: | Text | |||||||
| Autor: | Dr. Kudryavtsev, Volodymyr [Autor] | |||||||
| Dateien: |
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| Beitragende: | Prof. Dr. Seidel, Claus A. M. [Gutachter] Prof. Dr. Oesterhelt, Filipp [Gutachter] | |||||||
| Stichwörter: | single molecules, Multiparameter fluorescence detection, iving cells, fluorescence correlation spectroscopy, time-correlated single photon counting | |||||||
| Dewey Dezimal-Klassifikation: | 500 Naturwissenschaften und Mathematik » 540 Chemie | |||||||
| Beschreibung: | This thesis presents the development and applications of novel experimental and analytical techniques for the quantitative investigation of single biological molecules in solution and living cells. In contrast to ensemble experiments, single molecule measurements eliminate averaging over a population of molecules and deliver direct structural and dynamic information. Multiparameter fluorescence detection (MFD) is now established as a technique in single-molecule fluorescence spectroscopy and allows for the simultaneous measurement of the full fluorescence information: the spectral properties, fluorescence brightness and quantum yield, fluorescence lifetime and fluorescence anisotropy.
A general strategy to identify and quantify single molecules in dilute solution employing MFD was developed. Twelve identical oligonucleotides were measured at the single molecule level. Including free dye molecules and incompletely labeled oligonucleotides, 16 different compounds were identified and separated from the mixture, based on the multidimensional information contained in the fluorescence of the analyzed molecules. MFD was shown to be a powerful tool for studies of dynamic and conformational properties of single biomolecules. gamma-subunit rotation of F0F1-ATP synthase was studied during ATP synthesis and hydrolysis. F0F1-ATP synthases from Escherichia coli were labelled with fluorescence reporters and incorporated into liposomes. It was found that the -subunit rotates stepwise during proton transport–powered ATP synthesis, showing three distinct distances to the b subunits in repeating sequences. The average durations of these steps correspond to catalytic turnover times upon ATP synthesis as well as ATP hydrolysis. The direction of rotation during ATP synthesis is opposite to that of ATP hydrolysis. A new general strategy based on MFD was introduced to register and quantitatively analyse fluorescence images. MFD delivers the complete accessible spectroscopic information in each pixel of an image and thus increases the information content per image, which often is crucial for a correct interpretation of functional information. By analyzing 2D histograms of the fluorescence parameters, specific pixels can be selected for subsequent sub-ensemble analysis to improve statistical accuracy of the estimated parameters. MFD can be combined with fluorescence correlation spectroscopy (FCS), which reveals molecular concentrations as well as the molecule’s mobility parameters. An improved time-correlated single photon counting (TCSPC) technique for simultaneous fluorescence lifetime and correlation experiments was developed. It allows calculation of the full correlation curve from the same photons emitted from single or very few, molecules.A fast multi-tau software correlator was developed to correlate the data. The algorithm uses the asynchronous single photon intensity information of the TCSPC data without requiring the construction of a multichannel scaler (MCS) trace, as needed for conventional intensity correlation algorithms. The method offers two main advantages for experiments at low concentrations over conventional single molecule fluorescence detection: 1)Short (nanosecond time-scale) and long (ms time scale) correlation times are now simultaneously accessible in a TCSPC experiment. The presented technique bridges the gap between anisotropy decay analysis and currently available commercial correlators. 2)The number of independent fluorophores on a single molecule can be determined by measuring coincident photons simultaneously with all other fluorescence parameters. | |||||||
| Lizenz: |  Urheberrechtsschutz | |||||||
| Fachbereich / Einrichtung: | Mathematisch- Naturwissenschaftliche Fakultät » WE Chemie » Physikalische Chemie und Elektrochemie | |||||||
| Dokument erstellt am: | 03.09.2007 | |||||||
| Dateien geändert am: | 03.09.2007 | |||||||
| Promotionsantrag am: | 01.04.2003 | |||||||
| Datum der Promotion: | 31.01.2007 |
