Dokument: Steps towards simultaneous atomic-force and fluorescence spectroscopy of single DNA
| Titel: | Steps towards simultaneous atomic-force and fluorescence spectroscopy of single DNA | |||||||
| URL für Lesezeichen: | https://docserv.uni-duesseldorf.de/servlets/DocumentServlet?id=3612 | |||||||
| URN (NBN): | urn:nbn:de:hbz:061-20070215-082747-4 | |||||||
| Kollektion: | Dissertationen | |||||||
| Sprache: | Englisch | |||||||
| Dokumententyp: | Wissenschaftliche Abschlussarbeiten » Dissertation | |||||||
| Medientyp: | Text | |||||||
| Autor: | Gaiduk, Alexander [Autor] | |||||||
| Dateien: |
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| Beitragender: | Prof. Dr. Seidel, Claus A. M. [Gutachter] | |||||||
| Stichwörter: | single molecule, fluorescence imaging, optical spectroscopy, AFM, force spectroscopy, simultaneous AFM and fluorescence spectroscopy, photon counting, probability distribution analysis, FRET, DNA | |||||||
| Dewey Dezimal-Klassifikation: | 500 Naturwissenschaften und Mathematik » 570 Biowissenschaften; Biologie | |||||||
| Beschreibung: | The development of single molecule fluorescence detection allows studying the properties of molecules without ensemble averaging. The detailed information about the fluorescence 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 is 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. 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 in the sample preparation and in the experimental techniques handling, as well as in the analysis of the fluorescence and force data. The glass treatment steps (cleaning and silanization) are developed that keep the fluorescence of the surface very low. On the other hand glass silanization provides surfaces for stable DNA molecule binding. Thus optical microscopy allows finding single DNA molecules labelled with the dye on the surface. Knowledge of the optical properties of AFM tips is relevant for the combination of optical and force spectroscopy. A new Multiparameter Fluorescence Imaging (MFI) technique is developed, which allows detailed studying of conditions where the amount of scattered and luminescence light from an AFM tip would not overwhelm the signal of a single fluorophore. 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. A newly developed probability distribution analysis (PDA) is capable of predicting the shot noise limited shapes of histograms generated from single photon counting data. The PDA takes into account the effects of background and stochastic processes and can successfully extract the originating distance value behind shot noise limited FRET signal distributions. The precision in the distance measurements is better than 1 % of the Förster radius, so that the distribution by 5Å due to mobility of the dyes on flexible linkers is easily revealed in FRET analysis. 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 indicate a hysteresis between the retraction and approach force spectroscopy curves. The dsDNA B-S transition force is increased 8.9% and 15% upon SYBR Green binding at an average concentration of 0.28-0.55 dyes/bp and 1-2 dyes/bp (comparing to the literature value of the B-S transition force of 65 pN). Consecutive pulling on a single DNA and the simultaneous optical signal registration (more than 10 pulls in one experiment) were achieved. As observed in the simultaneous experiments, the structural changes of a DNA molecule correlate with the intensity and the lifetime change in the fluorescence of the DNA binding dye. | |||||||
| Lizenz: |  Urheberrechtsschutz | |||||||
| Fachbereich / Einrichtung: | Mathematisch- Naturwissenschaftliche Fakultät » WE Biologie | |||||||
| Dokument erstellt am: | 18.01.2007 | |||||||
| Dateien geändert am: | 12.02.2007 | |||||||
| Promotionsantrag am: | 29.11.2006 | |||||||
| Datum der Promotion: | 29.11.2006 |
