A type of optical microscopy called stimulated emission depletion (STED) microscopy has been developed by Stefan Hell and co-workers in Germany. In STED microscopy, the first STED beam is focused on the area of interest which may contain fluorescing nanoparticles or single molecules. To get fluorescence resolution higher than the diffraction limit, a second STED laser pulse that is donut-shaped and red-shifted is directed at the focus of the first pulse after a short delay. The second STED beam de-excites and depletes the fluorescence emission from the first pulse via stimulated emission except in the region of the donut hole where the STED pulse has zero or low intensity. Fluorescence emission will then occur only at the center of the focal spot of the exciting light pulse, resulting in an effective resolution for fluorescence emission that can be much smaller than the diffraction spot size of the exciting pulse.

STED setup

STED apparatus showing reduced spot size for fluorescence emission

See also: Fluorescence, Stimulated emission.

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