Advanced Fluorescence Microscopy pp 213-230
Application of STED Microscopy to Cell Biology Questions
The increasing interest in “seeing” the molecular environment in biological systems has led to the recent quest for breaking the diffraction barrier in far-field fluorescence microscopy. The first nanoscopy method successfully applied to conventional biological probes was stimulated emission depletion microscopy (STED). It is based on a physical principle that instantly delivers diffraction-unlimited images, with no need for further computational processing: the excitation laser beam is overlaid with a doughnut-shaped depleting beam that switches off previously excited fluorophores, thereby resulting in what is effectively a smaller imaging volume. In this chapter we give an overview of several applications of STED microscopy to biological questions. We explain technical aspects of sample preparation and image acquisition that will help in obtaining good diffraction-unlimited pictures. We also present embedding techniques adapted for ultrathin sectioning, which allow optimal 3D resolutions in virtually all biological preparations.