Preview

PDF, English - main document Download (22MB) | Terms of use

Abstract

Advances in nanoscopy techniques have enabled the visualization of the cellular interior and its dynamics in nanoscale detail. Their success relies on availability of suitable fluorescent probes with well-distinguishable on and off states, complementing the specific demands and requirements of each application. Thus, the design and optimization of fluorescent dyes with corresponding bioconjugation and labeling strategies is essential for future discoveries aided by nanoscopy. In this thesis, photoswitchable, photoactivatable and cleavable dyes, in combination with different labeling strategies —from affinity probes to small molecules— were characterized for their applications in nanoscopy techniques. As photoswitchable probes, bioconjugates of diarylethenes with different photophysical and photochemical properties were applied in SMLM, RESOLFT and MINFLUX nanoscopy. Nanobody bioconjugates of a slow-switching red- shifted thienyl-substituted diarylethene, decorated with multiple carboxylic acid groups, were utilized in SMLM to demonstrate the impact of linkage error in the apparent width of vimentin filaments. MINFLUX nanoscopy was conducted with the diarylethene as the photoswitchable marker. Faster-switching phenyl-substituted diarylethenes decorated with non-charged polar solubilizers were investigated and triphenylphosphonium-bearing derivatives were applied in live-cell confocal imaging of mitochondria. By combining the antibody conjugates of a phenyl and a thienyl-substituted diarylethene, two-color fixed-cell confocal and SMLM imaging was enabled, despite partial spectral overlap, yet divergent photoactivation properties. In RESOLFT nanoscopy a series of trimethylammonium-substituted diarylethenes with different linker lengths were applied. In supramolecular complex assembly with cucurbit[7]uril, the probes exhibited improved photofatigue resistance and enhanced fluorescence brightness. As photoactivatable probes, HaloTag-reactive derivatives of a rhodamine,carborhodamines and a siliconrhodamine with emission spectra ranging from green to far red were applied in live-cell labeling. Images of NUP96 and vimentin proteins were acquired via MINFLUX nanoscopy and analyzed to investigate their performance, revealing superior apparent labeling efficiency and low single-digit nanometer resolution. For click labeling, a series of live-cell compatible photoactivatable xanthones bearing tetrazine moieties were investigated in SMLM and MINFLUX nanoscopy. Linkage-error-free labeling via genetic code expansion was exemplified in comparison with different labeling strategies targeting vimentin filaments. In a non-light mediated approach, a chemically cleavable dithiol linker was incorporated in a fluorophore–nanobody–fluorescent protein assembly exhibiting FRET, and was examined by confocal microscopy, FLIM and STED techniques. Chemical cleavage was utilized for multiplexing with one fluorophore for imaging different cellular structures. These results, involving a wide range of fluorescent probes with divergent (photo)physical and (photo)chemical properties, present an overview of their applicability in nanoscopy — including strengths and weaknesses in a range of currently most utilized techniques, as well as insights into upcoming challenges, possible improvements and solutions.