Dokument: Method development in Fluorescence Spectroscopy
| Titel: | Method development in Fluorescence Spectroscopy | |||||||
| URL für Lesezeichen: | https://docserv.uni-duesseldorf.de/servlets/DocumentServlet?id=64553 | |||||||
| URN (NBN): | urn:nbn:de:hbz:061-20250206-093613-7 | |||||||
| Kollektion: | Dissertationen | |||||||
| Sprache: | Deutsch | |||||||
| Dokumententyp: | Wissenschaftliche Abschlussarbeiten » Dissertation | |||||||
| Medientyp: | Text | |||||||
| Autor: | Voort, Nicolaas [Autor] | |||||||
| Dateien: |
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| Beitragende: | Prof. Dr. Seidel, Claus [Gutachter] Prof. Dr. Monzel, Cornelia [Gutachter] | |||||||
| Stichwörter: | Fluorescence Spectroscopy Super-resolution FRET | |||||||
| Dewey Dezimal-Klassifikation: | 500 Naturwissenschaften und Mathematik » 540 Chemie | |||||||
| Beschreibung: | Although orders of magnitude more modest, this thesis attempts to follow the example of the invention of the microscope by Anthony van Leeuwenhoek [49] leading to the discovery of the microbiome which ultimately led to improvements in the human conditions by means of public sanitation. It does so by developing new methods that enables the user to glean information previously inaccessible. Specifically, 1) FRET-nanoscopy accesses the Ångström level using fluorescent imaging, and thus can study biomolecules under physiological conditions at the highest resolution on a single-molecule basis. This method was applied to obtain the length of the activated hGBP protein at 28 nm and perform precise measurements of the 3D position of fluorescent dyes on origamis to enable isotropic 3D resolution. 2) Cell Lifetime FRET Image Spectroscopy (CELFIS) is used to measure the abundance of molecular species to a 1% level in live cells under a large range of input conditions, such as concentration and cell phenotype. This method is of general use to unravel the complex input-response machinery, which is pivotal information for understanding cells. The method is applied to elucidate the signaling response of CD95 and to obtain the dimerization constant of CTLA4. 3) Contributions were made to existing methods, including robust photobleaching step analysis in live cells, extraction of quantitative data on molecular species from gated STED data and optimized acquisition procedures for live-cell FCS. Methods 1) and 2) are elaborated upon below.
FRET-nanoscopy The ultimate goal of nanoscopy is to deliver molecular resolution compatible with live systems at a single-molecule level. Fluorophore-fluorophore interactions via FRET is a well-established method to achieve structural resolution below the diffraction limit, but poses a challenge for single-nanometer nanoscopy as it leads to ambiguities of the fluorophore position if not properly treated. FRET-nanoscopy takes a step towards the ultimate goal of nanoscopy by providing Ångström resolution based on localizing single emitters in aberrationfree STED data (colocalization STED (cSTED)) and by providing an experimental method and theoretical framework to calculate FRET distances under STED conditions, harnessing the potential of FRET. Synergistically, the information from FRET and cSTED is combined to make the transition from 2D to 3D at a molecular scale using Optical Pythagoras (OP). The method works as follows. FRET-nanoscopy requires a donor and acceptor FRET pair that can be depleted by the same STED laser. Alexa 568, Alexa 594 or Atto 594 are used as donors whereas Atto 643 or Atto 647N are used as acceptors. By localizing all emitters on a two-channel STED image, the fluorophore xy-position is resolved with high precision (<4 nm) at any length scale, achieving seamless resolution from 4 nm and upwards. Using a self-built particle averaging routine, broken or misfolded samples can be separated from intact samples with high fidelity yielding high purity data. When multiple molecular species are present in the ensemble, such as upside-up and upside-down origami platforms, these can be isolated and clustered separately. For each molecular species, the average particle is calculated to increase our precision to 0.4 nm. We use the breadboard property of DNA-origamis to confirm that our measurements match the predicted position within this precision. As the depletion laser changes the fluorescence lifetime decay of the dyes, we develop new analysis procedures to obtain accurate intensity- and lifetime-based FRET indicators for accurate distances <0.5 nm under STED conditions. Uniquely, FRET measures distances independent of molecular orientation with respect to the imaging plane (hypotenuse), whereas localization measures the projected xy-distance (adjacent side), using Pythagoras’ theorem they synergistically combine to determine the angle of the protein (OP). Lastly, we apply our approach to the protein hGBP1 in vitro, which undergoes a conformational change to an extended state upon activation, inaccessible to either STED or FRET alone. Using cSTED, we measure the most likely donor and acceptor distance to be 28 nm. CELFIS The field of molecular biology is rapidly evolving from studying binary yes/no relationships to probing complex signal transduction mechanisms. To study a system where the outcome depends quantitively on the concentration of input signaling molecules, a method is required that can measure interactions with high sensitivity, cover large concentration ranges and gather sufficient statistics to study the natural variability in live cells. CELFIS fulfills these conditions by analyzing the change in donor lifetime decay due to FRET from live cell data, ϵ(t), to detect changes in FRET fractions as low as 0.2 %. The FRET fraction is transformed into the fraction of oligomers by 1) using Accessible Volume Simulations (AV) to calculate the portion of the time where donor and acceptor are close enough and 2) accounting for the abundance of donor-acceptor species in addition to donor-donor or non-matured species. CELFIS gathers high statistics using our highly automated acquisition and data processing pipeline, measuring >3000 cells in one experiment while obtaining phenotype information such as cell fate and protein expression level. We apply CELFIS to detect oligomerization of CD95 in response to addition of CD95L to reveal that 12 % oligomer formation in median is sufficient to trigger apoptosis. Furthermore, we study dimerization of the CTLA4 membrane receptor protein and find that the dimerization fraction depends on concentration, enabling us to obtain the dimerization constant Kdimer. An upper limit in the concentration accessible by FRET is posed by unspecific interaction due to proximity FRET. By using three independent monomer controls, we quantify and correct for proximity FRET, allowing us to measure quantitative oligomer fractions up to concentrations ∼10 000 receptors/µm2. CELFIS is easily transferable to measure molecular interaction <10 nm in any cellular organelle. | |||||||
| Lizenz: |  Dieses Werk ist lizenziert unter einer Creative Commons Namensnennung 4.0 International Lizenz | |||||||
| Fachbereich / Einrichtung: | Mathematisch- Naturwissenschaftliche Fakultät » WE Chemie » Physikalische Chemie und Elektrochemie | |||||||
| Dokument erstellt am: | 06.02.2025 | |||||||
| Dateien geändert am: | 06.02.2025 | |||||||
| Promotionsantrag am: | 04.02.2023 | |||||||
| Datum der Promotion: | 12.10.2023 |
