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Quantitative Immunocytochemistry of Proteins Using 2- Photon Microscopy and Digital Image Analysis.

Published online by Cambridge University Press:  02 July 2020

H. Wan ,
C. Soeller ,
D.R. Garrod ,
C. Robinson  and
M.B. Cannell
H. Wan
Affiliation:
Department of Pharmacology & Clinical Pharmacology, St George's Hospital Medical School, London, SW17 0RE, UK
C. Soeller
Affiliation:
Department of Pharmacology & Clinical Pharmacology, St George's Hospital Medical School, London, SW17 0RE, UK Department of Physiology, University of Auckland, Auckland 1, NZ.
D.R. Garrod
Affiliation:
School of Biological Sciences, University of Manchester, Manchester, Ml3 9PT, UK
C. Robinson
Affiliation:
Department of Pharmacology & Clinical Pharmacology, St George's Hospital Medical School, London, SW17 0RE, UK
M.B. Cannell
Affiliation:
Department of Pharmacology & Clinical Pharmacology, St George's Hospital Medical School, London, SW17 0RE, UK Department of Physiology, University of Auckland, Auckland 1, NZ.
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Extract

The two photon microscope provides optical sectioning of fluorescent specimens with a resolution comparable to that obtained in confocal microscopy (see refs 2,3). However, the excited volume in 2-photon microscopy is limited to the focal volume (unlike conventional fluorescence microscopy where excitation occurs throughout the specimen). This means that photodamage is limited to the plane of section being examined. Thus, the light emitted from each point in the specimen depends on the amount of fluorochrome present without the problem of prior illumination (of other planes within the specimen) reducing the photon yield so a better signal to noise ratio can be obtained when examination of multiple image planes is needed. Since 2-photon excitation spectra are wide and chromatic aberration is eliminated (because the emitted light does not have to be focused on a pinhole), it is possible to excite several fluorochromes simultaneously and map their positions with high accuracy.

Type
New Developments in Multi-Photon Excitation Microscopy
Information
Microscopy and Microanalysis , Volume 4 , Issue S2: Proceedings: Microscopy & Microanalysis '98, Microscopy Society of America 56th Annual Meeting, Microbeam Analysis Society 32nd Annual Meeting, Atlanta, Georgia July 12-16, 1998 , July 1998 , pp. 416 - 417
Copyright
Copyright © Microscopy Society of America

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References

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