We report immunofluorescence imaging with a spatial resolution well beyond the diffraction limit. An axial resolution of approximately 50 nm, corresponding to 1/16 of the irradiation wavelength of 793 nm, is achieved by stimulated emission depletion through opposing lenses. We
We overcame the resolution limit of scanning far-field fluorescence microscopy by disabling the fluorescence from the outer part of the focal spot. Whereas a near-UV pulse generates a diffraction-limited distribution of excited molecules, a spatially offset pulse quenches the excited
Lens-based optical microscopy failed to discern fluorescent features closer than 200 nm for decades, but the recent breaking of the diffraction resolution barrier by sequentially switching the fluorescence capability of adjacent features on and off is making nanoscale imaging routine. Reported
Beitraege zur Theorie des Mikroskops und der mikroskopischen Wahrnehmung.
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