Abstract
Resolution describes the possibility to separate structural features in the imaging process. In far-field optical microscopes the resolution is limited by wavelength and numerical aperture. Stimulated Emission Depletion Microscopy (STED) is a method to resolve structures below the limits of optical resolution and is therefore attributed to super-resolution. STED uses a differential method of two different diffraction patterns, where one pattern excites and the second pattern de-excites fluorochromes. The residual excited area is controllable by intensity down to a theoretically unlimited resolution.
Among the major steps in the development of STED microscopy is the use of continuous wave lasers (CW-STED). A recent investigation on the time course of the fluorescence emission probability in CW-STED has revealed the benefit of using temporal gating of fluorescence detection (gSTED) to further improve the resolution of CW-STED and to reduce the STED laser intensity in the sample for a given resolution.
There are many practical benefits of STED microscopy. It works directly on standard fluorophores and fluorescent proteins such as Alexa 488, Oregon Green 488 and FITC, eYFP, Citrin, and eGFP. As a point scanning implementation, STED provides optical section capabilities like those in confocal microscopy to allow super-resolution imaging within tissue. The method produces details smaller than 50 nm in a direct optical implementation that produces super-resolution raw data, to which contrast enhancing processes such as deconvolution can be applied.