1. A general method to improve fluorophores for live-cell and single-molecule microscopy.
- Author
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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, and Lavis, Luke D
- Subjects
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FLUOROPHORES , *FLUORESCENCE microscopy , *BIOMOLECULE analysis , *LIGANDS (Biochemistry) , *FLUORESCENT proteins , *CELL permeability - 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. [ABSTRACT FROM AUTHOR]
- Published
- 2015
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