STED and related concepts for far-field optical nanoscopy

Far-field fluorescence microscopy is the most frequently applied microscopy technique in life sciences. Its strength is the unique combination of highly attractive features such as molecular specificity, simple sample preparation, possibility of 3D imaging and operation under ambient, live cell compatible, conditions. The main shortcoming in comparison to methods, such as electron microscopy, is its resolution which is limited by diffraction to Deltar ~ lambda /(2NA) where lambda denotes the wavelength and NA refers to the numerical aperture. This leads typically to a resolution of approx 200nm laterally and 500nm axially. In 1994 STED (stimulated emission depletion) was invented providing the possibility to combine the advantages of far-field fluorescence microscopy with virtually unlimited resolution.