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A general method to improve fluorophores for live-cell and single-molecule microscopy
Open accessAbstract
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.
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Metadata
- Authors
- Grimm, J. B., English, B. P., Chen, J., Slaughter, J. P., Zhang, Z., Revyakin, A., Patel, R., Macklin, J. J., Normanno, D., Singer, R. H., Lionnet, T., & Lavis, L. D.
- Venue
- Nature Methods
- Published
- 2015-01-19
- Volume
- 12
- Issue
- 3
- Pages
- 244-250
- ISSN
- 1548-7091 (ISSN-L)
- 1548-7105
- OpenAlex
- W2129788878
- Cited by
- 1,501
- References
- 2
- FWCI
- 65.53
- Type
- journal article
- Language
- en
- Open access
- green
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