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Far-field Light Microscopy

  1. Christoph Cremer

Published Online: 15 AUG 2014

DOI: 10.1002/9780470015902.a0005922.pub2

eLS

eLS

How to Cite

Cremer, C. 2014. Far-field Light Microscopy. eLS. .

Author Information

  1. University Heidelberg, Heidelberg, Germany

Publication History

  1. Published Online: 15 AUG 2014

Abstract

Novel developments in optical technology and photophysics made it possible to radically overcome the diffraction limit (ca. 200 nm laterally, 600 nm along the optical axis) of conventional far-field fluorescence microscopy. Presently, three principal ‘nanoscopy’ families have been established: Nanoscopy based on highly focused laser beams; nanoscopy based on structured illumination excitation; and nanoscopy based on localisation microscopy approaches. With such ‘superresolution’ or ‘nanoscopy’ techniques, it has become possible to analyse biostructures with a substantially enhanced light optical resolution down to a few tens of nanometre in 3D, and a few nanometre in the object plane, corresponding to 1/100 of the exciting wavelength. These methods allow to study individual membrane complexes, cellular protein distributions, nuclear nanostructures, bacteria or individual viruses down to the molecular level; they open new perspectives to combine molecular and structural biology to unravel the basic mechanisms of life and their emergence from fundamental laws.

Key Concepts

  • Light microscopy of biostructures has reemerged as an essential tool of the life sciences.

  • Conventional light microscopy methods suffer from the limited resolution and thus prevent the direct study of nanostructural details.

  • Novel developments in optics and photophysics have made it possible to overcome the conventional resolution limits.

  • Present ‘superresolution’/‘nanoscopy’ techniques are based either on focused laser beam excitation; or on patterned/structured illumination schemes; or on homogeneous wide-field illumination.

  • In addition to advanced optical techniques, nanoscopy requires appropriate photostable/photoswitchable fluorochromes with specific spectral characteristics.

  • Far-field light microscopy based ‘nanoscopy’ techniques presently allow to discriminate (resolve) in a cellular environment individual fluorescent molecules down to a minimum distance of few nanometre.

  • The combination of the novel nanoscopy approaches with molecular biology methods is expected to open an avenue towards a substantially improved understanding of the basic mechanisms of life and its emergence from the basic laws of nature.

Keywords:

  • superresolution;
  • light microscopy;
  • light optical nanoscopy;
  • focused nanoscopy;
  • structured illumination microscopy;
  • localisation microscopy;
  • biological nanostructures;
  • 4Pi-, STED, SIM, SMI microscopy;
  • single molecule microscopy;
  • GSDIM;
  • PALM;
  • SPDM;
  • STORM;
  • photoswitching