Two-photon excitation fluorescence microscopy

Annu Rev Biomed Eng. 2000;2:399-429. doi: 10.1146/annurev.bioeng.2.1.399.


Two-photon fluorescence microscopy is one of the most important recent inventions in biological imaging. This technology enables noninvasive study of biological specimens in three dimensions with submicrometer resolution. Two-photon excitation of fluorophores results from the simultaneous absorption of two photons. This excitation process has a number of unique advantages, such as reduced specimen photodamage and enhanced penetration depth. It also produces higher-contrast images and is a novel method to trigger localized photochemical reactions. Two-photon microscopy continues to find an increasing number of applications in biology and medicine.

Publication types

  • Historical Article
  • 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.
  • Review

MeSH terms

  • Animals
  • Biomedical Engineering
  • Fluorescent Dyes
  • History, 20th Century
  • Humans
  • Image Processing, Computer-Assisted
  • Microscopy, Confocal
  • Microscopy, Fluorescence / history
  • Microscopy, Fluorescence / instrumentation
  • Microscopy, Fluorescence / methods*
  • Microscopy, Video
  • Optics and Photonics / instrumentation
  • Photons


  • Fluorescent Dyes