ABSTRACT
In Chapter 2 we discussed localization microscopy approaches that switch the majority of fluorophores in the field of view to a dark state using optical or chemical switching mechanisms. A small fraction of fluorophores enters the bright state stochastically, enabling the reconstruction of a high-resolution image from many diffraction limited images. Stimulated emission depletion nanoscopy (STED) is also based on controlling the on/off state of fluorophores, but in a deterministic rather than stochastic way. This approach to circumvent the diffraction limit was proposed in 1994 [1] and first realized in 1999 [2, 3]; it uses stimulated emission to reversibly silence fluorophores in predefined regions in the sample to facilitate separation at sub-diffraction length scales.
