STED microscopy in the visible range

Abstract

Light microscopy is a key scientific instrument in the life sciences. However, the resolution of far-field light microscopy is limited by diffraction. Exploiting a saturated depletion of the molecular excited state by stimulated emission, stimulated emission depletion (STED) breaks the resolution barrier in the important subfield of fluorescence microscopy. To this end, STED microscopy utilizes a doughnut-shaped beam featuring a central zero which is capable of quenching the fluorescence solely in the focal periphery. While STED microscopy was initially restricted to near infrared-emitting fluorophores, in this thesis STED microscopy is shown to be viable with visible (green-yellow-red) fluorophores. In particular, STED is established with the green and yellow fluorescent proteins (GFP, YFP) which are endogenous cellular markers of outstanding biological importance. The spectral conditions for STED with these fluorophores are given. The expansion of STED to the visible range has enabled the first application to biophysics and to addressing unsolved problems of cell biology. For example, STED microscopy revealed that the synaptic vesicle protein synaptotagmin remains an integral patch following fusion with the plasma membrane. Moreover, membrane microdomains were resolved with unprecedented (65 nm) spatial resolution. Furthermore, a novel doughnut-shape of the STED beam utilizing a helical phase ramp and a central singularity was established for STED. Finally, due to the smaller wavelength for stimulated emission, the viability of STED with visible dyes pushes the resolution down to smaller attainable values.