Fluorescence depletion mechanisms in super-resolving STED microscopy

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We prove that stimulated emission is by far the dominant quenching mechanism for providing super-resolution in fluorescence microscopy with a red-shifted depletion beam. Our evidences are based on simultaneously measuring fluorescence quenching and photon gain in the quenching beam. Measurements were performed for several fluorescent dyes including fluorescent proteins over a wide spectral range of their emission spectra. We found that, for each fluorophore, the wavelength dependence of both signals closely follows that of the stimulated emission cross-section.