Cited by 9,488Advanced Fluorescence Microscopy TechniquesNonlinear Optical Materials StudiesLaser-Matter Interactions and Applications

Two-photon laser scanning fluorescence microscopy

Denk, Winfried, Strickler, James H., Webb, Watt W.·1990·Science

Abstract

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.

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Crossref · 10.1126/science.2321027

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  • Multiphoton NIR femtosecond laser scanning microscopy acts as a novel optical tool for non-confocal 3D imaging of endogenous fluorophores and ECM structures like collagen and elastin.

    “Multiphoton NIR femtosecond laser scanning microscopy acts as a novel optical tool for non‐confocal 3D imaging of endogenous fluorophores and ECM structures like collagen and elastin.[ 121 , 122 ]”
  • Two-photon microscopy overcomes limitations of confocal microscopy through the use of a pulsed infrared light source that excites fluorophores by the combined energy of two photons arriving on the sample nearly simultaneously.

    “Two-photon microscopy overcomes both of these limitations through the use of a pulsed infrared light source that excites fluorophores by the combined energy of two photons arriving on the sample nearly simultaneously (Denk et al., 1990; Svoboda et al., 1996).”

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Authors
Denk, W., Strickler, J. H., & Webb, W. W.
Affiliations
Cornell University (US)
Venue
Science
Published
1990-04-06
Volume
248
Issue
4951
Pages
73-76
ISSN
  • 0036-8075 (ISSN-L)
  • 1095-9203
OpenAlex
W2061464631
Cited by
9,488
References
0
Citations by year
2026: 178 · 2025: 237 · 2024: 283 · 2023: 303 · 2022: 288 · 2021: 326 · 2020: 337 · 2019: 413 · 2018: 358 · 2017: 390 · 2016: 403 · 2015: 389 · 2014: 406 · 2013: 426 · 2012: 451
FWCI
16.91
Type
journal article
Language
en
Open access
closed
Locations
  • Science (publishedVersion)
  • PubMed (publishedVersion)
Funders
National Center for Research Resources (RR04224, P41RR004224)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Biophysics › Advanced Fluorescence Microscopy Techniques
  • Physical Sciences › Engineering › Biomedical Engineering › Nonlinear Optical Materials Studies
  • Physical Sciences › Physics and Astronomy › Atomic and Molecular Physics, and Optics › Laser-Matter Interactions and Applications
Keywords
two-photon excitation, three-dimensional resolution, subpicosecond laser pulses, photobleaching, photochemistry, living cells
MeSH terms
Animals, Cell Line, Chromosomes, Fluorescent Dyes, Kidney, Lasers, Microscopy, Fluorescence, Photochemistry, Radiation, Swine, Ultraviolet Rays

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