1990/04/06 by Winfried Denk, James H. Strickler, Watt W. Webb · 15 citations
Engineering · Biochemistry, Genetics and Molecular Biology · Materials Science · #Nonlinear Optical Materials Studies #Advanced Fluorescence Microscopy Techniques #Photochromic and Fluorescence Chemistry
paper · doi:10.1126/science.2321027
openalex publication_date 1990/04/06 · openalex created_date 2025/10/10 · openalex updated_date 2026/07/30
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.