|Title:||Standardised Monomeric Probes for Brightness-based Microscopy Methodologies||Language:||English||Authors:||Mellai, Mauro||Qualification level:||Diploma||Advisor:||Schütz, Gerhard||Assisting Advisor:||Brameshuber, Mario||Issue Date:||2021||Citation:||
Mellai, M. (2021). Standardised Monomeric Probes for Brightness-based Microscopy Methodologies [Diploma Thesis, Technische Universität Wien]. reposiTUm. https://doi.org/10.34726/hss.2021.78800
|Number of Pages:||53||Qualification level:||Diploma||Abstract:||
Brightness analysis experiments via fluorescence microscopy are routinely conducted in today’s biological and biophysical research worldwide. However, a standardised brightness reference to estimate the oligomerisation state of a protein is missing, regardless to the general awareness and its importance. This research project tried to pave the way for creating one. Two fundamental aspects were investigated: (i) if and how specific chemical binding methods linking a fluorescent dye to a protein can influence a monomer’s brightness level; (ii) to determine optimal imaging conditions for recording a reliable monomer brightness distribution. The latter could be used as the starting point for a future development of a more complete reference system for higher oligomeric state of proteins. The experiments were conducted on supported lipid bilayer with three different monomeric proteins and were recorded with a fluorescence microscope using the same settings to assure comparability between the measurements taken at two different excitation laser powers. Next, image processing stages yielded the brightness of each protein for the two imaging conditions studied and estimated the relative diffusion coefficient for the proteins. A careful choice of the laser power should be the primary concern in any experiment involving brightness analysis, as it is a key factor in gathering useful data. In fact, it was observed that single-molecule signals bleach more in the centre of the region of interest than at the periphery, which strongly influences the recorded brightness distribution. In addition, a misleading or careless selection of too high imaging power could strongly alter the surface density estimation leading to incorrect scientific interpretations and conclusions. In fact, high laser power bleaches fluorescent single-molecule signals quicker plummeting statistics after the early frames very fast, poor statistics in image collection makes conclusions prone to errors. With the gained knowledge, it has become possible to define reproducible single-molecule references for brightness-based fluorescence microscopy experiments.
|Keywords:||Single molecule microscopy; brightness analysis||URI:||https://doi.org/10.34726/hss.2021.78800
|DOI:||10.34726/hss.2021.78800||Library ID:||AC16228440||Organisation:||E134 - Institut für Angewandte Physik||Publication Type:||Thesis
|Appears in Collections:||Thesis|
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