Strong signal increase in STED fluorescence microscopy by imaging regions of subdiffraction extent

• DOI: 10.1073/pnas.1621495114
• Publication Date: 2017-02-14T02:00:27Z
• Authors: Fabian Göttfert; Tino Pleiner; Jörn Heine; Volker Westphal; Dirk Görlich; Steffen J. Sahl; Stefan W. Hell
• Keywords: 0303 health sciences; Photobleaching; Lasers, Dye; Fluorescence; Xenopus laevis; 03 medical and health sciences; Microscopy, Fluorescence; Image Processing, Computer-Assisted; Animals; Organic Chemicals; Algorithms; Cells, Cultured; Fluorescent Dyes

Significance In stimulated emission depletion (STED) fluorescence microscopy, the diffraction resolution barrier is overcome by applying a doughnut-shaped light beam that transiently switches fluorescence off, confining molecular emission to subdiffraction-sized regions around the doughnut center. Unfortunately, the doughnut intensities required for high resolution exacerbate photobleaching. Our remedy, called MINFIELD, exploits the fact that fluorescence off-switching by STED does not require the high intensities of the doughnut crest. By recording subdiffraction-sized areas or volumes in the sample, molecular exposure to high intensities is avoided and photobleaching is reduced by more than 100-fold. Fluorescence analysis of cellular structures becomes possible in unprecedented detail. Providing larger signal and faster recording times, MINFIELD-STED microscopy should open a unique range of superresolution imaging applications in the life sciences.