3D reconstruction of high-resolution STED microscope images

Microsc Res Tech. 2008 Sep;71(9):644-50. doi: 10.1002/jemt.20602.

Abstract

Tackling biological problems often involves the imaging and localization of cellular structures on the nanometer scale. Although optical super-resolution below 100 nm can be readily attained with stimulated emission depletion (STED) and photoswitching microscopy methods, attaining an axial resolution <100 nm with focused light generally required the use of two lenses in a 4Pi configuration or exceptionally bright photochromic fluorophores. Here, we describe a simple technical solution for 3D nanoscopy of fixed samples: biological specimens are fluorescently labeled, embedded in a polymer resin, cut into thin sections, and then imaged via STED microscopy with nanoscale resolution. This approach allows a 3D image reconstruction with a resolution <80 nm in all directions using available state-of-the art STED microscopes.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Cellular Structures / ultrastructure*
  • Image Processing, Computer-Assisted*
  • Imaging, Three-Dimensional
  • Microscopy, Fluorescence / instrumentation*
  • Microscopy, Fluorescence / methods*
  • Microspheres
  • Photons