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Nat Methods. 2016 Apr;13(4):359-65. doi: 10.1038/nmeth.3797. Epub 2016 Mar 7.

High-density three-dimensional localization microscopy across large volumes.

Author information

1
Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, Virginia, USA.
2
Coleman Technologies, Newton Square, Pennsylvania, USA.
3
Penn Image Computing and Science Laboratory (PICSL), University of Pennsylvania, Philadelphia, Pennsylvania, USA.
4
Department of Radiology, University of Pennsylvania, Philadelphia, Pennsylvania, USA.

Abstract

Extending three-dimensional (3D) single-molecule localization microscopy away from the coverslip and into thicker specimens will greatly broaden its biological utility. However, because of the limitations of both conventional imaging modalities and conventional labeling techniques, it is a challenge to localize molecules in three dimensions with high precision in such samples while simultaneously achieving the labeling densities required for high resolution of densely crowded structures. Here we combined lattice light-sheet microscopy with newly developed, freely diffusing, cell-permeable chemical probes with targeted affinity for DNA, intracellular membranes or the plasma membrane. We used this combination to perform high-localization precision, ultrahigh-labeling density, multicolor localization microscopy in samples up to 20 μm thick, including dividing cells and the neuromast organ of a zebrafish embryo. We also demonstrate super-resolution correlative imaging with protein-specific photoactivable fluorophores, providing a mutually compatible, single-platform alternative to correlative light-electron microscopy over large volumes.

PMID:
26950745
PMCID:
PMC4889433
DOI:
10.1038/nmeth.3797
[Indexed for MEDLINE]
Free PMC Article
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