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Mol Biol Cell. 2015 May 1;26(9):1743-51. doi: 10.1091/mbc.E14-08-1287. Epub 2015 Feb 25.

Ultrafast superresolution fluorescence imaging with spinning disk confocal microscope optics.

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Products Development Department 6, R&D Division, Olympus Corporation, 2951 Ishikawa-cho, Hachioji, Tokyo 192-8507, Japan.
Laboratory for Cell Polarity Regulation, Quantitative Biology Center, RIKEN, Furuedai 6-2-3, Suita, Osaka 565-0874, Japan


Most current superresolution (SR) microscope techniques surpass the diffraction limit at the expense of temporal resolution, compromising their applications to live-cell imaging. Here we describe a new SR fluorescence microscope based on confocal microscope optics, which we name the spinning disk superresolution microscope (SDSRM). 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. 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. Therefore a single SR image requires only a single averaged image through the rotating disk. On the basis of this theory, we modified a commercial spinning disk confocal microscope. The improved resolution around 120 nm was confirmed with biological samples. The rapid dynamics of micro-tubules, mitochondria, lysosomes, and endosomes were observed with temporal resolutions of 30-100 frames/s. 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.

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