Two-photon excitation microscopy for the study of living cells and tissues
Open accessAbstract
Two-photon excitation microscopy is an alternative to confocal microscopy that provides advantages for three-dimensional and deep tissue imaging. This unit will describe the basic physical principles behind two-photon excitation and discuss the advantages and limitations of its use in laser-scanning microscopy. The principal advantages of two-photon microscopy are reduced phototoxicity, increased imaging depth, and the ability to initiate highly localized photochemistry in thick samples. Practical considerations for the application of two-photon microscopy will then be discussed, including recent technological advances. This unit will conclude with some recent applications of two-photon microscopy that highlight the key advantages over confocal microscopy and the types of experiments which would benefit most from its application.
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Confocal microscopy has limitations including photobleaching due to broad excitation light exposure and limited penetration depth caused by photon scattering in dense spheroid tissue.
“confocal microscopy has limitations, including photobleaching due to broad excitation light exposure and limited penetration depth caused by photon scattering in dense spheroid tissue.”
From Modeling Tumor Microenvironment Complexity In Vitro: Spheroids as Physiologically Relevant Tumor Models and Strategies for Their Analysis (2025) · cites this paper as [296]
The Introduction (page not provided) says confocal excitation causes photobleaching throughout a specimen and that scattering limits penetration depth. The Fundamental Depth Limit section explains the loss of signal in thick samples. The paper does not specifically address dense spheroid tissue, so it supports the general mechanisms but not their application to that particular sample type. (LLM, conf=0.84)
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References 115 · Cited by 311
- Authors
- Benninger, R. K. P., & Piston, D. W.
- Affiliations
- University of Colorado Anschutz (US)
- Vanderbilt University Medical Center (US)
- Venue
- Current Protocols in Cell Biology
- Publisher
- Wiley
- Published
- 2013-06
- Volume
- 59
- Issue
- 1
- Pages
- 4.11.1-4.11.24
- ISSN
- 1934-2500 (ISSN-L)
- 1934-2616
- PMC
- PMC4004770
- OpenAlex
- W1828367075
- Cited by
- 311
- References
- 115
- Citations by year
- 2026: 21 · 2025: 22 · 2024: 42 · 2023: 26 · 2022: 35 · 2021: 52 · 2020: 30 · 2019: 17 · 2018: 20 · 2017: 11 · 2016: 10 · 2015: 8 · 2014: 6 · 2012: 3
- FWCI
- 4.85
- Type
- journal article
- Language
- en
- Open access
- green
- www.ncbi.nlm.nih.gov
- Locations
- Current Protocols in Cell Biology (publishedVersion)
- PubMed (publishedVersion)
- PubMed Central (acceptedVersion)
- Funders
- National Institute of Diabetes and Digestive and Kidney Diseases (R00DK085145)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Biophysics › Advanced Fluorescence Microscopy Techniques
- Physical Sciences › Engineering › Biomedical Engineering › Nonlinear Optical Materials Studies
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Biophysics › Cell Image Analysis Techniques
- Keywords
- two-photon microscopy, confocal microscopy comparison, 3D imaging, deep tissue imaging, phototoxicity reduction, laser scanning microscopy, imaging depth
- MeSH terms
- Animals, Humans, Microscopy, Proteins, Imaging, Three-Dimensional, Optics and Photonics
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