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

Nature methods (2015-01-20)
Jonathan B Grimm, Brian P English, Jiji Chen, Joel P Slaughter, Zhengjian Zhang, Andrey Revyakin, Ronak Patel, John J Macklin, Davide Normanno, Robert H Singer, Timothée Lionnet, Luke D Lavis
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.

MATERIALS
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Product Description

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