Stimulated emission depletion microscopy narrows the region from which detected fluorescence is allowed to originate. After excitation, a second beam drives stimulated emission in the surrounding region; a central minimum of that depletion beam leaves a smaller region able to emit the collected fluorescence.
Scanning this region builds an image with detail beyond an ordinary excitation-only diffraction limit. The depletion light must be rejected from the detection channel, and the central minimum must be well aligned with the excitation focus. Increasing depletion intensity can narrow the effective emitting region, but bleaching, background and specimen changes limit the useful improvement.[ref]
Breaking the diffraction resolution limit by stimulated emission: stimulated-emission-depletion fluorescence microscopy by Stefan W. Hell and others. 1994 proposal, abstract and Figure 2: excitation followed by spatially selective depletion
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