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Nat Methods. 2016 Aug;13(8):679-84. doi: 10.1038/nmeth.3899. Epub 2016 Jul 4.

Nanoscale imaging of RNA with expansion microscopy.

Author information

1
Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA.
2
Media Lab, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA.
3
McGovern Institute, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA.
4
Department of Bioengineering, University of Pennsylvania, Philadelphia, Pennsylvania, USA.
5
Division of Health Sciences and Technology, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA.
6
Wyss Institute for Biologically Inspired Engineering, Boston, Massachusetts, USA.
7
Department of Systems Biology, Harvard Medical School, Boston, Massachusetts, USA.
8
Department of Genetics, Harvard Medical School, Boston, Massachusetts, USA.
9
Department of Brain and Cognitive Sciences, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA.

Abstract

The ability to image RNA identity and location with nanoscale precision in intact tissues is of great interest for defining cell types and states in normal and pathological biological settings. Here, we present a strategy for expansion microscopy of RNA. We developed a small-molecule linker that enables RNA to be covalently attached to a swellable polyelectrolyte gel synthesized throughout a biological specimen. Then, postexpansion, fluorescent in situ hybridization (FISH) imaging of RNA can be performed with high yield and specificity as well as single-molecule precision in both cultured cells and intact brain tissue. Expansion FISH (ExFISH) separates RNAs and supports amplification of single-molecule signals (i.e., via hybridization chain reaction) as well as multiplexed RNA FISH readout. ExFISH thus enables super-resolution imaging of RNA structure and location with diffraction-limited microscopes in thick specimens, such as intact brain tissue and other tissues of importance to biology and medicine.

PMID:
27376770
PMCID:
PMC4965288
DOI:
10.1038/nmeth.3899
[Indexed for MEDLINE]
Free PMC Article

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