Cargando…

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...

Descripción completa

Detalles Bibliográficos
Autores principales: Hayashi, Shinichi, Okada, Yasushi
Formato: Online Artículo Texto
Lenguaje:English
Publicado: The American Society for Cell Biology 2015
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
_version_ 1782372129938866176
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