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

Nat Methods. 2015 Mar;12(3):244-50, 3 p following 250. doi: 10.1038/nmeth.3256. Epub 2015 Jan 19.

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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Azetidines / chemistry
  • Chemistry Techniques, Synthetic
  • Coumarins / chemistry
  • Fluorescein / chemistry
  • Fluorescent Dyes / analysis
  • Fluorescent Dyes / chemical synthesis
  • Fluorescent Dyes / chemistry*
  • HeLa Cells
  • Humans
  • Microscopy, Ultraviolet / methods*
  • Models, Molecular
  • Molecular Imaging / methods*
  • Quantum Theory
  • Rhodamines / chemistry
  • Spectrometry, Fluorescence
  • Spectrophotometry, Ultraviolet / methods
  • Structure-Activity Relationship

Substances

  • Azetidines
  • Coumarins
  • Fluorescent Dyes
  • Rhodamines
  • azetidine
  • coumarin
  • Fluorescein