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Quantitative Imaging of Metabolism by Two-Photon Excitation Microscopy

Published online by Cambridge University Press:  02 July 2020

David W. Piston ,
Susan Knobel  and
George Patterson
David W. Piston
Affiliation:
Department of Molecular Physiology and Biophysics, Vanderbilt University, Nashville, TN
Susan Knobel
Affiliation:
Department of Molecular Physiology and Biophysics, Vanderbilt University, Nashville, TN
George Patterson
Affiliation:
Department of Molecular Physiology and Biophysics, Vanderbilt University, Nashville, TN
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Extract

Two-photon excitation microscopy provides attractive advantages over confocal microscopy for three-dimensionally resolved fluorescence imaging. Because of the intensity-squared dependence of the two-photon absorption, the excitation is limited to the focal volume. This inherent localization minimizes photobleaching and photodamage - the ultimate limiting factors in fluorescence microscopy of living cells. One of the most powerful applications of two-photon excitation microscopy is imaging from the naturally occurring reduced pyridine nucleotides (NAD(P)H). NAD(P)H is a useful indicator of cellular metabolism, but it is not a “good“ fluorophore (it has a small absorption cross-section and a low quantum yield). Two-photon excitation of NAD(P)H yields minimal photodamage, thus allowing time-resolved threedimensional metabolic mapping of cellular redox state. We have used two-photon excitation microscopy to examine glucose metabolism in pancreatic and muscle cells. As glucose is metabolized by these cells, intermediate metabolism results in an increase in the reduced-tooxidized NAD(P)H/NAD(P)+ ratio, and a concomitant increase in autofluorescence.

Type
Novel Approaches to Microscopy Of Living Cells
Information
Microscopy and Microanalysis , Volume 5 , Issue S2: Proceedings: Microscopy & Microanalysis '99, Microscopy Society of America 57th Annual Meeting, Microbeam Analysis Society 33rd Annual Meeting, Portland, Oregon August 1-5, 1999 , August 1999 , pp. 1048 - 1049
Copyright
Copyright © Microscopy Society of America

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References

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6. Experiments described were performed in collaboration with Jim May and Richard Whitesell. Support for this work is from the NIH (DK53434), Vanderbilt Diabetes Research and Training Center, and Vanderbilt Cancer Center.Google Scholar