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Stimulated emission depletion (STED) resolves fluorescent features that are closer than the far-field optical diffraction limit by applying a spatially modulated light field keeping all but one of these features dark consecutively. For estimating the efficiency of transient fluorophore darkening, we developed analytical equations considering the spatio-temporal intensity profile of the STED beam. These equations provide a quick analysis and optimization of the resolution and contrast to be gained under various conditions, such as continuous wave or pulsed STED beams having different pulse durations. Particular emphasis is placed on fluorescence fluctuation methods such as correlation spectroscopy (FCS) using STED.

Original publication




Journal article


Opt Express

Publication Date





26417 - 26429


Algorithms, Computer-Aided Design, Equipment Design, Equipment Failure Analysis, Image Interpretation, Computer-Assisted, Microscopy, Fluorescence, Reproducibility of Results, Sensitivity and Specificity