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Nat Methods. 2015 Mar;12(3):244-50, 3 p following 250. doi: 10.1038/nmeth.3256. Epub 2015 Jan 19.

A general method to improve fluorophores for live-cell and single-molecule microscopy.

Author information

1
Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, Virginia, USA.
2
1] Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, Virginia, USA. [2] Department of Biochemistry, University of Leicester, Leicester, UK.
3
1] Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, Virginia, USA. [2] Laboratoire Physico-Chimie Curie, Institut Curie, Paris, France.
4
1] Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, Virginia, USA. [2] Department of Anatomy and Structural Biology, Albert Einstein College of Medicine, Bronx, New York, USA.

Abstract

Specific labeling of biomolecules with bright fluorophores is the keystone of fluorescence microscopy. Genetically encoded self-labeling tag proteins can be coupled to synthetic dyes inside living cells, resulting in brighter reporters than fluorescent proteins. Intracellular labeling using these techniques requires cell-permeable fluorescent ligands, however, limiting utility to a small number of classic fluorophores. Here we describe a simple structural modification that improves the brightness and photostability of dyes while preserving spectral properties and cell permeability. Inspired by molecular modeling, we replaced the N,N-dimethylamino substituents in tetramethylrhodamine with four-membered azetidine rings. This addition of two carbon atoms doubles the quantum efficiency and improves the photon yield of the dye in applications ranging from in vitro single-molecule measurements to super-resolution imaging. The novel substitution is generalizable, yielding a palette of chemical dyes with improved quantum efficiencies that spans the UV and visible range.

PMID:
25599551
PMCID:
PMC4344395
DOI:
10.1038/nmeth.3256
[Indexed for MEDLINE]
Free PMC Article
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