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Nat Biotechnol. 2017 Aug;35(8):773-780. doi: 10.1038/nbt.3876. Epub 2017 Jul 3.

Long time-lapse nanoscopy with spontaneously blinking membrane probes.

Author information

1
Department of Cell Biology, Yale University School of Medicine, New Haven, Connecticut, USA.
2
Department of Chemistry, Yale University, New Haven, Connecticut, USA.
3
Graduate School of Medicine, The University of Tokyo, Bunkyo-ku, Japan.
4
PRESTO, Japan Science and Technology Agency, Kawaguchi, Saitama, Japan.
5
Graduate School of Pharmaceutical Sciences, The University of Tokyo, Bunkyo-ku, Japan.
6
CREST, Japan Agency for Medical Research and Development (AMED), Chiyoda-ku, Tokyo, Japan.
7
Department of Biomedical Engineering, Yale University, New Haven, Connecticut, USA.
8
Department of Molecular, Cellular, and Developmental Biology, Yale University, New Haven, Connecticut, USA.

Abstract

Imaging cellular structures and organelles in living cells by long time-lapse super-resolution microscopy is challenging, as it requires dense labeling, bright and highly photostable dyes, and non-toxic conditions. We introduce a set of high-density, environment-sensitive (HIDE) membrane probes, based on the membrane-permeable silicon-rhodamine dye HMSiR, that assemble in situ and enable long time-lapse, live-cell nanoscopy of discrete cellular structures and organelles with high spatiotemporal resolution. HIDE-enabled nanoscopy movies span tens of minutes, whereas movies obtained with labeled proteins span tens of seconds. Our data reveal 2D dynamics of the mitochondria, plasma membrane and filopodia, and the 2D and 3D dynamics of the endoplasmic reticulum, in living cells. HIDE probes also facilitate acquisition of live-cell, two-color, super-resolution images, expanding the utility of nanoscopy to visualize dynamic processes and structures in living cells.

PMID:
28671662
PMCID:
PMC5609855
DOI:
10.1038/nbt.3876
[Indexed for MEDLINE]
Free PMC Article
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