Breaking the diffraction barrier in fluorescence microscopy by optical shelving

Phys Rev Lett. 2007 May 25;98(21):218103. doi: 10.1103/PhysRevLett.98.218103. Epub 2007 May 24.

Abstract

We report the breaking of the diffraction resolution barrier in far-field fluorescence microscopy by transiently shelving the fluorophore in a metastable dark state. Using a relatively modest light intensity of several kW/cm(2) in a focal distribution featuring a local zero, we confine the fluorescence emission to a spot whose diameter is a fraction of the wavelength of light. Nanoscale far-field optical resolution down to 50 nm is demonstrated by imaging microtubules in a mammalian cell and proteins on the plasma membrane of a neuron. The presence of dark states in virtually any fluorescent molecule opens up a new venue for far-field microscopy with resolution that is no longer limited by diffraction.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Cell Line
  • Fluorescent Dyes / chemistry
  • Humans
  • Microscopy, Fluorescence / methods*
  • Optics and Photonics*
  • Synaptosomal-Associated Protein 25 / chemistry

Substances

  • Fluorescent Dyes
  • Synaptosomal-Associated Protein 25