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Optical Sectioning in Fluorescence Microscopy by Confocal and 2-Photon Molecular Excitation Techniques

Published online by Cambridge University Press:  14 March 2018

M.B. Cannell  and
C. Soeller
M.B. Cannell
Affiliation:
St. George's Hospital Medical School, Cranmer Terrace, LondonSW17 ORE
C. Soeller
Affiliation:
St. George's Hospital Medical School, Cranmer Terrace, LondonSW17 ORE

Extract

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Fluorescence microscopy has proved to be an invaluable tool for biomedical science since it is possible to visualise small quantities of labeled materials (such as intracellular ions and proteins) in both fixed and living cells, However, the conventional wide field fluorescence microscope suffers from the disadvantage that objects outside the focal plane also fluoresce (in response to the excitation light) and this leads to a marked loss of contrast for objects in the focal plane, This is especially a problem when the fluorescent probe is distributed throughout the thickness of the cell and the cell is thicker than about 1 µm. The confocal microscope overcomes this problem by illuminating the preparation with a point source of excitation light and limiting the collection of light with a pinhole that is confocal with the illumination source.

Type
Research Article
Information
Microscopy Today , Volume 5 , Issue 8 , October 1997 , pp. 12 - 15
Copyright
Copyright © Microscopy Society of America 1997

References

Cannell, M. B., Cheng, H., and Lederer, W.J., 1995, The control of calcium release in heart muscle. Science 268:1045–1050 Google Scholar
Cheng, H., Lederer, W. J., and Cannell, M. B.. 1993. Calcium sparks: Elementary events underlying excitation-contraction coupling in heart muscle. Science 262:740–744.Google Scholar
Cheng, H., Lederer, W. J., and Cannell, M. B.. 1994. Propagation of excitation-contraction coupling into ventricular celts. Pflugere Archii.. 428:415–417.Google Scholar
Denk, W., Strickler, J. H., and Webb, W. W.. 1990. Two-photon laserscanntng fluorescence microscopy. Science 248:73–76.Google Scholar
López-Lopez, J. R., Shacklock, P. S., Balke, C. W., and Wier, W. G.. 1995. Local calcium transients triggered by single L-type calcium channel currents in cardiac cells. Science 263:1042–1045.Google Scholar
Gómez, A.M., Valdivia, H.H., Cheng, H., Lederer, M., Santana, L.F., Cannell, M.B., McCune, S.A., Altshuld, R.A. & Lederer, W.J. (1997) Defective excitation-contraction coupling in experimental cardiac hypertrophy and heart failure. Science 276:800–806.Google Scholar
Pawley, J.B. [ed.]. 1995, Handbook of biological confocal microscopy. 2nd ed. Plenum Press, N.Y. Google Scholar
Soeller, C, and Cannell, M. B. 1996. Construction of a two-photon microscope and optimisation of illumination pulse duration. Pflugers Arch. 432:555–561.Google Scholar
Sommer, J. R. and Waugh, R. A.. 1976. The ultrastnicture of the mammalian cardiac cell - with special reference on the tubular membrane systems. Am, J. Pathology 82:192–232.Google Scholar
Xu, C, and Webb, W. W.. 1996. Measurement of two-photon excitation crass sections of molecular fluorophores with data from 690 to 1050 nm. J. Opt. Soc. Am. B. 13:481–491.Google Scholar