![]() STED can be accelerated, but faster image acquisition requires orders of magnitude stronger illumination than conventional imaging, which risks photodamage. Thus the image acquisition time needs to be 10 s of imaging for a single frame. However, most subcellular structures are 3 μm/s ( Allen et al., 1982). Because our method requires only small optical modifications, it will enable an easy upgrade from an existing spinning disk confocal to a SR microscope for live-cell imaging.įluorescence microscopy is now an essential tool for the cell biologist. The rapid dynamics of microtubules, mitochondria, lysosomes, and endosomes were observed with temporal resolutions of 30–100 frames/s. The improved resolution around 120 nm was confirmed with biological samples. On the basis of this theory, we modified a commercial spinning disk confocal microscope. Therefore a single SR image requires only a single averaged image through the rotating disk. However, the SDSRM is 10 times faster than a conventional SIM because SR signals are recovered by optical demodulation through the stripe pattern of the disk. Theoretically, the SDSRM is equivalent to a structured illumination microscope (SIM) and achieves a spatial resolution of 120 nm, double that of the diffraction limit of wide-field fluorescence microscopy. Here we describe a new SR fluorescence microscope based on confocal microscope optics, which we name the spinning disk superresolution microscope (SDSRM). Most current superresolution (SR) microscope techniques surpass the diffraction limit at the expense of temporal resolution, compromising their applications to live-cell imaging. ![]()
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