Super‐Resolution Fluorescence Microscopy

This chapter covers the basic principles of super-resolution fluorescence microscopy but recognises that essential operational details can only be learned within an experienced imaging facility of a research group where super-resolution microscopes are the central tool. The first practical implementation of super-resolution imaging in far-field microscopy with lenses was by STED, or stimulated emission depletion, in 1994. RESOLFT is an acronym that stands for reversible saturable optical fluorescence transitions. STED microscopy differs from RESOLFT in that it uses nonlinear excitation saturation to switch fluorophores into the dark, ground, state by emission depletion. While STED microscopy was being developed by Stefan Hell's research group, Heintzmann and Gustafsson were independently developing structured illumination microscopy (SIM). Normally SIM is limited only by the readout time of the camera to about 100 ms per image. Most super-resolution methods exhibit a common drawback: with respect to the total number of emitted photons, they are less efficient than standard widefield microscopy.