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Two-Photon Laser Scanning Confocal Microscopy

Published online by Cambridge University Press:  02 July 2020

P.C. Cheng ,
S.J. Pan ,
A. Shih ,
W.S. Liou ,
M.S. Park ,
T. Watson ,
J. Bhawalkar  and
P. Prasard
P.C. Cheng
Affiliation:
Advanced Microscopy and Imaging Laboratory, Dept. of Electrical and Computer Engineering, State Univ. of New York, Buffalo, NY14260, USA
S.J. Pan
Affiliation:
Advanced Microscopy and Imaging Laboratory, Dept. of Electrical and Computer Engineering, State Univ. of New York, Buffalo, NY14260, USA
A. Shih
Affiliation:
Advanced Microscopy and Imaging Laboratory, Dept. of Electrical and Computer Engineering, State Univ. of New York, Buffalo, NY14260, USA
W.S. Liou
Affiliation:
Advanced Microscopy and Imaging Laboratory, Dept. of Electrical and Computer Engineering, State Univ. of New York, Buffalo, NY14260, USA
M.S. Park
Affiliation:
Advanced Microscopy and Imaging Laboratory, Dept. of Electrical and Computer Engineering, State Univ. of New York, Buffalo, NY14260, USA
T. Watson
Affiliation:
Dept. of Conservative Dentisty, Guy Hopital, London, SEI 9RT, UK
J. Bhawalkar
Affiliation:
Dept. of Chemistry, State University of New York at Buffalo, Buffalo, NY, 14260USA
P. Prasard
Affiliation:
Dept. of Chemistry, State University of New York at Buffalo, Buffalo, NY, 14260USA
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Extract

Two-photon fluorescence microscopy has become an important research tool in both biological and material sciences. The technique uses long wavelength, typically in the near IR, as the excitation light to obtain shorter wavelength fluorescence (e.g. visible light). Because of the low linear absorption coefficient of most biological and polymeric specimens, this technique allows deeper penetration of the excitation beam, achieving optical sectioning to a depth of 250μm or more into the specimen. As a result of the quadratic dependency of the two-photon induced fluorescence to the excitation intensity, the fluorescent emission and photobleaching are limited to the vicinity of focal spot. This capability of addressing a specimen’s 3D space allows exciting possibilities in biological researches, such as 3D photobleaching recovery experiment.

Two-photon confocal fluorescence microscopy is ideal for the study of thick biological and material specimen in 3D. For example, Figure 1 shows a three-dimensional isosurface rendered image of a vascular bundle from a maize stem.

Type
Optical Microanalysis
Information
Microscopy and Microanalysis , Volume 3 , Issue S2: Proceedings: Microscopy & Microanalysis '97, Microscopy Society of America 55th Annual Meeting, Microbeam Analysis Society 31st Annual Meeting, Histochemical Society 48th Annual Meeting, Cleveland, Ohio, August 10-14, 1997 , August 1997 , pp. 847 - 848
Copyright
Copyright © Microscopy Society of America 1997

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References

1. Denk, W.et al., Science 248, 73–76 (1990)CrossRefGoogle Scholar

2. Cheng, P.C.et al., Scanning 18, 3, 148 (1996)Google Scholar

3. Pan, S.J.et al., Scanning, 19 (Sup.), 1997 (in press)Google Scholar