Molecules and methods for super-resolution imaging

Methods Enzymol. 2010;475:27-59. doi: 10.1016/S0076-6879(10)75002-3.

Abstract

By looking at a fluorescently labeled structure one molecule at a time, it is possible to side-step the optical diffraction limit and obtain "super-resolution" images of small nanostructures. In the Moerner Lab, we seek to develop both molecules and methods to extend super-resolution fluorescence imaging. Methodologies and protocols for designing and characterizing fluorophores with switchable fluorescence required for super-resolution imaging are reported. These fluorophores include azido-DCDHF molecules, covalently linked Cy3-Cy5 dimers, and also the first example of a photoswitchable fluorescent protein, enhanced yellow fluorescent protein (EYFP). The imaging of protein superstructures in living Caulobacter crescentus bacteria is used as an example of the power of super-resolution imaging by single-molecule photoswitching to extract information beyond the diffraction limit. Finally, a new method is described for obtaining three-dimensional super-resolution information using a double-helix point-spread function.

Publication types

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

MeSH terms

  • Animals
  • Azides / chemistry
  • Bacteria / ultrastructure
  • Fluorescent Dyes* / chemistry
  • Humans
  • Imaging, Three-Dimensional
  • Microscopy, Fluorescence / methods*
  • Molecular Structure
  • Photochemistry
  • Photons*

Substances

  • Azides
  • Fluorescent Dyes