Molekulare Orientierung als Kontrastmechanismus in der Fluoreszenzmikroskopie und konfokale Multidetektor-Scanning-Mikroskopie

Abstract

This doctoral thesis addresses two novel approaches in fluorescence microscopy. The first part introduces a new method that can be used to enhance the effect of photoselection. This method, termed ExPAN (excitation polarization angle narrowing), exploits stimulated emission to narrow the angular distribution of excited fluorophores. ExPAN could be of significant interest for applications like fluorescence anisotropy measurements, which are commonly applied in life science, e.g. to study receptor-ligand interactions or investigate protein dynamics. In the context of this work, ExPAN was applied in conjunction with a new fluorescence microscopy approach that utilizes molecular orientation as a contrast mechanism. Using excitation light with continuously rotating polarization, it is possible to obtain information about the orientation of dye molecules. When excited in this manner, molecules with a preferred orientation exhibit a periodically modulated fluorescence signal as a result of photoselection. This work showed that the additional modulation information can be utilized to distinguish molecules with different orientations even when their signals overlap spatially. The approach was explored by experiments with single molecules on a surface using a modified widefield microscope and additionally by simulation studies. The results indicated that it is possible to distinguish signals from dye molecules with a distance down to 80 nm. It was demonstrated that ExPAN enhances the effect of photoselection and can be used to distinguish molecules even when their orientations are very similar. Furthermore, a modulated fluorescence signal was also observed in fixed biological samples and with surface labeled microparticles in aqueous solution. In the second part of this thesis a method is presented for improving the resolution of confocal laser scanning microscopes. This method, termed multi-detector scanning (MDS), is based on the concept of image scanning microscopy (ISM). Using ISM it is theoretically possible to double the resolution of a fluorescence microscope. Since ISM requires an array detector, most of the previously developed implementations use CCD or CMOS cameras for detection. Instead of a camera, several single-photon detectors that are combined to an array detector by a fiber optic bundle, are used in this work.This configuration allowed it for the first time to use the method in conjunction with Fluorescence-lifetime imaging microscopy (FLIM). FLIM has proven to be an important microscopy technique in life science and has been applied in a wide variety of studies including protein-protein interaction and cell metabolism research. Therefore, the improvement in spatial resolution of FLIM through ISM is of potential interest for numerous biological applications. Within the scope of this work, a multi-detector scanning microscope was constructed and characterized. Resolution improvements of 168 nm with MDS and 146 nm with MDS and deconvolution were achieved in fixed biological samples. Furthermore, the measurement of fluorescence lifetimes was demonstrated with the experimental setup. Thus, the application of MDS has the potential to increase the spatial resolution of FLIM and could serve as a tool for measuring biological systems in more detail.