Stimulated Emission Depletion Microscopy

As one of the mainstream methods of super-resolution microscopy, stimulated emission depletion (STED) microscopy can effectively break through the diffraction limit of traditional far-field optical microscopy and increase the resolution of the microscope to the level of nanometre. In the course of about 20 years, STED microscopy has continued to get evolved and has been widely used in many fields, such as life science and physical research, which effectively broadens the scope of our understanding of the microworld. We believe this review will provide a guide for broad readers in related technology. Key Concepts STED microscopy can achieve three-dimensional super-resolution imaging with a common resolution of 10–70 nm. Parallelisation illumination can achieve high-speed imaging using multiple excitation foci to illuminate the specimens. Conventional STED imaging in deep tissue remains challenging mostly due to the fact that light gets scattered in tissues. Under ultrahigh depletion radiation, background signals are generated by secondary excitation of the depletion illumination which has so far prohibited STED microscopy from reaching its theoretically molecular resolution. In practice, the attainment of a higher resolution is notably retarded by the existence of photobleaching effect, a kind of photochemical reaction that permanently inhibits fluorophore molecules to fluoresce.