Two-photon laser scanning fluorescence microscopy
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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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 ]”
From Bioactive scaffolds for tissue engineering: A review of decellularized extracellular matrix applications and innovations (2025) · cites this paper as [121]
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).”
From Barriers in the brain: resolving dendritic spine morphology and compartmentalization (2014) · cites this paper as Denk et al., 1990
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“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)
- ORCID
- Denk, Winfried
- 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
About these checks
Registry, retraction, Cites and statistics results are computed, not judged, with one exception: a printed reference that no index lists is read once by a model before it counts as a citation error. Claims are extracted by a model and kept only when their quote appears verbatim in the text; each cited source is then checked, with a model’s help, for whether it says what the claim relies on. Those verdicts can be wrong — read the claim’s quote and the cited source before relying on one.
Statistics and arithmetic are recomputed, not judged.
Confidence bands are shown only for fields calibrated on our annotated test set. None is calibrated yet, so no band is shown.
What counts as a claim ›How we measure confidence ›How we check this ›
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