Two-photon laser scanning fluorescence microscopy

Science. 1990 Apr 6;248(4951):73-6. doi: 10.1126/science.2321027.


Molecular excitation by the simultaneous absorption of two photons provides intrinsic three-dimensional resolution in laser scanning fluorescence microscopy. The excitation of fluorophores having single-photon absorption in the ultraviolet with a stream of strongly focused subpicosecond pulses of red laser light has made possible fluorescence images of living cells and other microscopic objects. The fluorescence emission increased quadratically with the excitation intensity so that fluorescence and photo-bleaching were confined to the vicinity of the focal plane as expected for cooperative two-photon excitation. This technique also provides unprecedented capabilities for three-dimensional, spatially resolved photochemistry, particularly photolytic release of caged effector molecules.

Publication types

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

MeSH terms

  • Animals
  • Cell Line
  • Chromosomes / ultrastructure
  • Fluorescent Dyes
  • Kidney / ultrastructure
  • Lasers*
  • Microscopy, Fluorescence / methods*
  • Photochemistry
  • Radiation*
  • Swine
  • Ultraviolet Rays


  • Fluorescent Dyes