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Quantitative Imaging Of the Green Fluorescent Proteins

Published online by Cambridge University Press:  02 July 2020

David W. Piston ,
George H. Patterson  and
Susan M. Knobel
David W. Piston
Affiliation:
Department of Molecular Physiology and Biophysics Vanderbilt University, Nashville, TN37232
George H. Patterson
Affiliation:
Department of Molecular Physiology and Biophysics Vanderbilt University, Nashville, TN37232
Susan M. Knobel
Affiliation:
Department of Molecular Physiology and Biophysics Vanderbilt University, Nashville, TN37232
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Extract

The cloning and expression of GFP in heterologous systems introduced a fantastic tool for studying specific gene expression and protein localization inside living cells. However, one aspect of GFP that has not been exploited to its full potential is its use as a quantitative imaging tool. To determine its quantitative usefulness, we have addressed five points that are important in GFP imaging: detectable signal over background, photostability, pH stability of the molecule, temperature dependence of chromophore formation, and estimation and normalization of GFP levels.

To determine the quantitative limits of GFP in cells, several GFP versions (wtGFP, αGFP (F99S/M153T/V163A), S65T, EGFP (F64L/S65T), and a blue-shifted variant, EBFP (F64L/S65T/Y66H/Y145F)) were compared by imaging of GFP expressing cells or by spectroscopic measurements of purified proteins. When imaged, the GFP signals are contaminated by the naturally occurring background autofluorescence, but improved detection can be achieved for each green GFP by combination of confocal microscopy using 488 nm excitation, a rapid cut-on dichroic mirror, and a narrow bandpass emission filter (Figure l).

Type
Detection and Application of Green (and other Colored) Fluorescent Proteins
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. 1004 - 1005
Copyright
Copyright © Microscopy Society of America

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References

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5. These studies were supported by grants from the Beckman Foundation Young Investigator Program and the Whitaker Foundation Biomedical Engineering Research Program to DWP. During part of this work, GHP was an NIH trainee (GM08320). Confocal microscopy was performed at the Cell Imaging Shared Resource, supported by the Vanderbilt Cancer Center (CA68485) and Diabetes Research and Training Center (DK20593).Google Scholar