STED Microscopy of FRET Pairs
von Maria Loidolt-Krüger
Datum der mündl. Prüfung:2018-03-19
Betreuer:Prof. Dr. Stefan Hell
Gutachter:Prof. Dr. Stefan Hell
Gutachter:Prof. Dr. Sarah Köster
EnglischFörster resonance energy transfer (FRET) is a popular tool in life sciences, for example to detect protein-protein interactions and ligand binding, or to construct fluorescent biosensors for metabolites or ions. Obtaining such functional information provided by FRET from diffraction-unlimited images would be advantageous, because, for one thing, the spatial averaging of fluorescence signals could be reduced with a smaller detection volume, and, for another thing, FRET signals from neighboring subdiffraction areas could be distinguished. Thus, the goal of this thesis was to investigate the feasibility of measuring FRET using stimulated emission depletion (STED) microscopy. Numerical simulations of a single FRET pair under continuous-wave and pulsed STED illumination were performed to study the interplay of FRET and STED photophysics, including the influence of STED intensity and pulse delay. Organic fluorophores were screened to identify a STED-compatible FRET pair, and the dyes Atto532 and Star635P were chosen for further experiments. Fluorescence lifetime imaging (FLIM) STED microscopy of single FRET pairs bound to dsDNA at defined distances was performed with various STED laser powers and STED pulse delays. Thus measured fluorescence decay curves were compared with simulated ones to confirm the validity of the simplified photophysical model that was established to describe the measurement process. Based on this model, the measurement strategy was devised and the measurement settings were chosen. The spectral detection channels of donor, FRET and acceptor signals differ in spatial resolution; this excludes the use of intensity-based FRET quantification methods. Only the analysis of acceptor fluorescence decay curves can yield diffraction-unlimited FRET information. Large spectral shifts of the fluorescence emission of both fluorophores were observed in single-molecule measurements. STED imaging showed that emission shifts to shorter wavelengths can decrease the STED efficiency and thus the image resolution. The spectral shifts also change the spectral overlap integral of donor emission and acceptor absorption and thereby the FRET efficiency; the magnitude of this effect was estimated. In summary, a photophysical model for the interference of STED and FRET was established. Experimental requirements for STED imaging of FRET pairs were identified and a suitable data analysis strategy was proposed. Additionally, photoconversion of organic fluorophores was characterized and its effect on STED and FRET investigated.
Keywords: Microscopy; STED; FRET