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Uniform Total Internal Reflection Fluorescence Illumination Enables Live Cell Fluorescence Resonance Energy Transfer Microscopy

Published online by Cambridge University Press:  11 March 2013

Jia Lin  and
Adam D. Hoppe
Jia Lin
Affiliation:
Department of Chemistry and Biochemistry, South Dakota State University, Brookings, SD 57007, USA
Adam D. Hoppe*
Affiliation:
Department of Chemistry and Biochemistry, South Dakota State University, Brookings, SD 57007, USA
*
*Corresponding author. E-mail: adam.hoppe@sdstate.edu
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Abstract

Fluorescence resonance energy transfer (FRET) microscopy is a powerful technique to quantify dynamic protein-protein interactions in live cells. Total internal reflection fluorescence (TIRF) microscopy can selectively excite molecules within about 150 nm of the glass-cell interface. Recently, these two approaches were combined to enable high-resolution FRET imaging on the adherent surface of living cells. Here, we show that interference fringing of the coherent laser excitation used in TIRF creates lateral heterogeneities that impair quantitative TIRF-FRET measurements. We overcome this limitation by using a two-dimensional scan head to rotate laser beams for donor and acceptor excitation around the back focal plane of a high numerical aperture objective. By setting different radii for the circles traced out by each laser in the back focal plane, the penetration depth was corrected for different wavelengths. These modifications quell spatial variations in illumination and permit calibration for quantitative TIRF-FRET microscopy. The capability of TIRF-FRET was demonstrated by imaging assembled cyan and yellow fluorescent protein–tagged HIV-Gag molecules in single virions on the surfaces of living cells. These interactions are shown to be distinct from crowding of HIV-Gag in lipid rafts.

Keywords

TIRF-FRETTIRFFRETGFPpenetration depthinterference fringesHIV-Gag assembly
Type
Biological Applications
Information
Microscopy and Microanalysis , Volume 19 , Issue 2 , April 2013 , pp. 350 - 359
Copyright
Copyright © Microscopy Society of America 2013

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Footnotes

†

Current address: Department of Pathology, University of New Mexico, Albuquerque, NM 87131, USA

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