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Nat Methods. 2020 Feb;17(2):225-231. doi: 10.1038/s41592-019-0676-4. Epub 2020 Jan 6.

Nanoscale subcellular architecture revealed by multicolor three-dimensional salvaged fluorescence imaging.

Author information

1
Department of Cell Biology, Yale School of Medicine, New Haven, CT, USA.
2
Department of Neuroscience, Yale School of Medicine, New Haven, CT, USA.
3
Howard Hughes Medical Institute, Yale School of Medicine, New Haven, CT, USA.
4
Section of Hematology, Department of Internal Medicine, Yale School of Medicine, New Haven, CT, USA.
5
Cell Biology and Biophysics Unit, European Molecular Biology Laboratory, Heidelberg, Germany.
6
Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, VA, USA.
7
Kavli Institute for Neuroscience, Yale School of Medicine, New Haven, CT, USA.
8
Nanobiology Institute, Yale University, West Haven, CT, USA.
9
Auckland Bioengineering Institute, University of Auckland, Auckland, New Zealand.
10
Department of Cell Biology, Yale School of Medicine, New Haven, CT, USA. joerg.bewersdorf@yale.edu.
11
Kavli Institute for Neuroscience, Yale School of Medicine, New Haven, CT, USA. joerg.bewersdorf@yale.edu.
12
Nanobiology Institute, Yale University, West Haven, CT, USA. joerg.bewersdorf@yale.edu.
13
Department of Biomedical Engineering, Yale University, New Haven, CT, USA. joerg.bewersdorf@yale.edu.

Abstract

Combining the molecular specificity of fluorescent probes with three-dimensional imaging at nanoscale resolution is critical for investigating the spatial organization and interactions of cellular organelles and protein complexes. We present a 4Pi single-molecule switching super-resolution microscope that enables ratiometric multicolor imaging of mammalian cells at 5-10-nm localization precision in three dimensions using 'salvaged fluorescence'. Imaging two or three fluorophores simultaneously, we show fluorescence images that resolve the highly convoluted Golgi apparatus and the close contacts between the endoplasmic reticulum and the plasma membrane, structures that have traditionally been the imaging realm of electron microscopy. The salvaged fluorescence approach is equally applicable in most single-objective microscopes.

PMID:
31907447
DOI:
10.1038/s41592-019-0676-4

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