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- Title
A general method to improve fluorophores for live-cell and single-molecule microscopy.
- Authors
Grimm, Jonathan B; English, Brian P; Chen, Jiji; Slaughter, Joel P; Zhang, Zhengjian; Revyakin, Andrey; Patel, Ronak; Macklin, John J; Normanno, Davide; Singer, Robert H; Lionnet, Timothée; Lavis, Luke D
- 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.
- Subjects
FLUOROPHORES; FLUORESCENCE microscopy; BIOMOLECULE analysis; LIGANDS (Biochemistry); FLUORESCENT proteins; CELL permeability
- Publication
Nature Methods, 2015, Vol 12, Issue 3, p244
- ISSN
1548-7091
- Publication type
Article
- DOI
10.1038/nmeth.3256