RESOLFT nanoscopy with water-soluble synthetic fluorophores

by Philipp Johannes Alt
Doctoral thesis
Date of Examination:2017-12-15
Date of issue:2018-01-17
Advisor:Prof. Dr. Stefan Hell
Referee:Prof. Dr. Stefan Hell
Referee:Prof. Dr. Jürgen Troe
Persistent Address: http://dx.doi.org/10.53846/goediss-6679

 

 

Files in this item

Name:Philipp Johannes Alt - RESOLFT nanoscopy wit...pdf
Size:9.26Mb
Format:PDF
Description:Dissertation - Philipp Johannes Alt - RESOLFT nanoscopy with water-soluble synthetic fluorophores

The following license files are associated with this item:

Abstract

English

Fluorescence microscopy is an important and widely used tool in the life sciences due to its unique ability to observe cellular processes in living specimens with target-specific image contrast. The development of high resolution methods in the far-field has increased its importance further by enabling the visualization of structures featuring sizes below the diffraction limit of light. In particular, RESOLFT (reversible saturable optical linear fluorescence transitions) nanoscopy using low light intensities has become a method of choice for live-cell high-resolution fluorescence imaging. RESOLFT nanoscopy requires labels with specialized properties which, to date, have only been observed in reversibly photoswitchable fluorescent proteins (RSFPs). Attempts to extend the palette of RESOLFT labels using synthetic organic fluorophores have been limited to proof of concept studies, mostly owing to their insolubility in water. However, organic fluorophores bear the potential for higher brightness, broader emission and excitation wavelength range as well as higher photostability than RSFPs. In this work the first demonstration of RESOLFT nanoscopy with photoswitchable diarylethene-based fluorophores in aqueous environments is presented. The photophysical behavior of these novel fluorophores was characterized and compared to RSFPs commonly used for RESOLFT. Furthermore, the fluorophores were successfully applied to label biological structures in fixed mammalian cells. Imaging based on the RESOLFT concept with these fluorophores yielded two- to threefold improved spatial resolution compared with confocal imaging. The fluorophores presented in this work thus lay the foundation for the further development of synthetic fluorophores for RESOLFT nanoscopy in living specimens.
Keywords: high-resolution microscopy; nanoscopy; RESOLFT; water solubility; organic photoswitchable fluorophores
 
Statistik