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Ultrafast superresolution fluorescence imaging with spinning disk confocal microscope optics
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 su...
Autores principales: | , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
The American Society for Cell Biology
2015
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4436784/ https://www.ncbi.nlm.nih.gov/pubmed/25717185 http://dx.doi.org/10.1091/mbc.E14-08-1287 |
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author | Hayashi, Shinichi Okada, Yasushi |
author_facet | Hayashi, Shinichi Okada, Yasushi |
author_sort | Hayashi, Shinichi |
collection | PubMed |
description | 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 microtubules, 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. |
format | Online Article Text |
id | pubmed-4436784 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2015 |
publisher | The American Society for Cell Biology |
record_format | MEDLINE/PubMed |
spelling | pubmed-44367842015-07-16 Ultrafast superresolution fluorescence imaging with spinning disk confocal microscope optics Hayashi, Shinichi Okada, Yasushi Mol Biol Cell Articles 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 microtubules, 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. The American Society for Cell Biology 2015-05-01 /pmc/articles/PMC4436784/ /pubmed/25717185 http://dx.doi.org/10.1091/mbc.E14-08-1287 Text en © 2015 Hayashi and Okada. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0). “ASCB®,” “The American Society for Cell Biology®,” and “Molecular Biology of the Cell®” are registered trademarks of The American Society for Cell Biology. |
spellingShingle | Articles Hayashi, Shinichi Okada, Yasushi Ultrafast superresolution fluorescence imaging with spinning disk confocal microscope optics |
title | Ultrafast superresolution fluorescence imaging with spinning disk confocal microscope optics |
title_full | Ultrafast superresolution fluorescence imaging with spinning disk confocal microscope optics |
title_fullStr | Ultrafast superresolution fluorescence imaging with spinning disk confocal microscope optics |
title_full_unstemmed | Ultrafast superresolution fluorescence imaging with spinning disk confocal microscope optics |
title_short | Ultrafast superresolution fluorescence imaging with spinning disk confocal microscope optics |
title_sort | ultrafast superresolution fluorescence imaging with spinning disk confocal microscope optics |
topic | Articles |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4436784/ https://www.ncbi.nlm.nih.gov/pubmed/25717185 http://dx.doi.org/10.1091/mbc.E14-08-1287 |
work_keys_str_mv | AT hayashishinichi ultrafastsuperresolutionfluorescenceimagingwithspinningdiskconfocalmicroscopeoptics AT okadayasushi ultrafastsuperresolutionfluorescenceimagingwithspinningdiskconfocalmicroscopeoptics |