Title: Building a cryostate for Cryo fluorescence microscopy
Other Titles: Aufbau eines Kryostats für Fluoreszenzmikroskopie bei tiefen Temperaturen
Language: English
Authors: Sparer, Lukas 
Qualification level: Diploma
Advisor: Schütz, Gerhard 
Assisting Advisor: Lopez Martinez, Montserrat 
Issue Date: 2021
Sparer, L. (2021). Building a cryostate for Cryo fluorescence microscopy [Diploma Thesis, Technische Universität Wien]. reposiTUm. https://doi.org/10.34726/hss.2021.78906
Number of Pages: 55
Qualification level: Diploma
Fluorescence microscopy has been one of the most important tools for understanding how biological systems are organized and how they function. Over the last two decades, the resolution of fluorescence microscopy has been enhanced by the development of super resolution methods that extend the limit of optical microscopy beyond the diffraction limit, providing unprecedented levels of information on the organization of molecular networks in cells. These methods include Structured Illumination Microscopy (SIM), Stimulated Emission Depletion Microscopy (STED),and techniques based on the localization of individual fluorescent molecules: Photoactivated Localization Microscopy (PALM) and Stochastic Optical Reconstruction Microscopy (STORM), referred to collectively as Single Molecule Localization Microscopy (SMLM) techniques. Super-resolution methods usually involve the use of chemical fixation, which has been shown to introduce artefacts that can be visible at the resolutions achieved. An alternative to chemical fixation is cryofixation, which is a superior structural preservation method, but requires to preserve the specimen under cryogenic temperatures during the measurement, which requires a big adaption of the experimental set-up. Favorably, cryogenic temperatures slow down the photobleaching of fluorophores, which has the potential to improve the resolution. In this work, after a short introduction about fluorescence super resolution microscopy, the main topic will be how we built a setup for super resolution microscopy at cryogenic temperatures. After that, preliminary measurements with fluorescent dyes AF-647 (Alexa Fluor) and Atto-647N will be shown and discussed how their photophysical properties are affected by low temperatures.
Keywords: Single molecule microscopy; fluorescence microscopy; cryogenic temperature
URI: https://doi.org/10.34726/hss.2021.78906
DOI: 10.34726/hss.2021.78906
Library ID: AC16184838
Organisation: E134 - Institut für Angewandte Physik 
Publication Type: Thesis
Appears in Collections:Thesis

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