RESOLFT nanoscopy with photoswitchable organic fluorophores

Sci Rep. 2015 Dec 7:5:17804. doi: 10.1038/srep17804.

Abstract

Far-field optical nanoscopy has been widely used to image small objects with sub-diffraction-limit spatial resolution. Particularly, reversible saturable optical fluorescence transition (RESOLFT) nanoscopy with photoswitchable fluorescent proteins is a powerful method for super-resolution imaging of living cells with low light intensity. Here we demonstrate for the first time the implementation of RESOLFT nanoscopy for a biological system using organic fluorophores, which are smaller in size and easier to be chemically modified. With a covalently-linked dye pair of Cy3 and Alexa647 to label subcellular structures in fixed cells and by optimizing the imaging buffer and optical parameters, our RESOLFT nanoscopy achieved a spatial resolution of ~74 nm in the focal plane. This method provides a powerful alternative for low light intensity RESOLFT nanoscopy, which enables biological imaging with small organic probes at nanoscale resolution.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Buffers
  • Carbocyanines / chemistry
  • Dimerization
  • Fluorescence
  • Fluorescent Dyes / chemistry*
  • Humans
  • Imaging, Three-Dimensional
  • Light*
  • Male
  • Mice
  • Molecular Probes / chemistry
  • NIH 3T3 Cells
  • Nanotechnology / methods*
  • Organic Chemicals / chemistry*
  • Subcellular Fractions / metabolism

Substances

  • Alexa Fluor 647
  • Buffers
  • Carbocyanines
  • Fluorescent Dyes
  • Molecular Probes
  • Organic Chemicals
  • cyanine dye 3