title: A general method to improve fluorophores for live-cell and single-molecule microscopy.
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.
authors.affiliation: Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, Virginia, USA.
authors.forename: Jonathan B
authors.initials: JB
authors.lastname: Grimm
authors.affiliation: Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, Virginia, USA.
authors.forename: Brian P
authors.initials: BP
authors.lastname: English
authors.affiliation: Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, Virginia, USA.
authors.forename: Jiji
authors.initials: J
authors.lastname: Chen
authors.affiliation: Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, Virginia, USA.
authors.forename: Joel P
authors.initials: JP
authors.lastname: Slaughter
authors.affiliation: Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, Virginia, USA.
authors.forename: Zhengjian
authors.initials: Z
authors.lastname: Zhang
authors.affiliation: 1] Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, Virginia, USA. [2] Department of Biochemistry, University of Leicester, Leicester, UK.
authors.forename: Andrey
authors.initials: A
authors.lastname: Revyakin
authors.affiliation: Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, Virginia, USA.
authors.forename: Ronak
authors.initials: R
authors.lastname: Patel
authors.affiliation: Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, Virginia, USA.
authors.forename: John J
authors.initials: JJ
authors.lastname: Macklin
authors.affiliation: 1] Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, Virginia, USA. [2] Laboratoire Physico-Chimie Curie, Institut Curie, Paris, France.
authors.forename: Davide
authors.identifier: 0000000347405542
authors.initials: D
authors.lastname: Normanno
authors.affiliation: 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.
authors.forename: Robert H
authors.initials: RH
authors.lastname: Singer
authors.affiliation: Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, Virginia, USA.
authors.forename: Timothée
authors.initials: T
authors.lastname: Lionnet
authors.affiliation: Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, Virginia, USA.
authors.forename: Luke D
authors.initials: LD
authors.lastname: Lavis
mesh_terms: D001384:Azetidines / Q000737:chemistry; D060326:Chemistry Techniques, Synthetic; D003374:Coumarins / Q000737:chemistry; D019793:Fluorescein / Q000737:chemistry; D005456:Fluorescent Dyes / Q000032:analysis / Q000138:chemical synthesis / Q000737:chemistry*; D006367:HeLa Cells; D006801:Humans; D008860:Microscopy, Ultraviolet / Q000379:methods*; D008958:Models, Molecular; D057054:Molecular Imaging / Q000379:methods*; D011789:Quantum Theory; D012235:Rhodamines / Q000737:chemistry; D013050:Spectrometry, Fluorescence; D013056:Spectrophotometry, Ultraviolet / Q000379:methods; D013329:Structure-Activity Relationship
chemical_list: D001384:Azetidines; D003374:Coumarins; D005456:Fluorescent Dyes; D012235:Rhodamines; C082735:azetidine; C030123:coumarin; D019793:Fluorescein