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

  • Jia Lin (a1) and Adam D. Hoppe (a1)

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.

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* Corresponding author. E-mail: adam.hoppe@sdstate.edu

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

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References

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Axelrod, D. (1981). Cell-substrate contacts illuminated by total internal reflection fluorescence. J Cell Biol 89(1), 141145.
Axelrod, D. (2001). Total internal reflection fluorescence microscopy in cell biology. Traffic 2, 764774.
Bal, M., Zaika, O., Martin, P. & Shapiro, M.S. (2008). Calmodulin binding to M-type K+ channels assayed by TIRF/FRET in living cells. J Physiol 586(9), 23072320.
Beemiller, P., Hoppe, A.D. & Swanson, J.A. (2006). A phosphatidylinositol-3-kinase-dependent signal transition regulates ARF1 and ARF6 during Fcgamma receptor-mediated phagocytosis. PLoS Biol 4(6), e162.
Beemiller, P., Zhang, Y., Mohan, S., Levinsohn, E., Gaeta, I., Hoppe, A.D. & Swanson, J.A. (2010). A Cdc42 activation cycle coordinated by PI 3-kinase during Fc receptor-mediated phagocytosis. Mol Biol Cell 21(3), 470480.
Boyer, S.B. & Slesinger, P.A. (2010). Probing novel GPCR interactions using a combination of FRET and TIRF. Comm Integrative Biol 3(4), 343346.
Buning, R. & van Noort, J. (2010). Single-pair FRET experiments on nucleosome conformational dynamics. Biochimie 92(12), 17291740.
Dovas, A., Gevrey, J.-C., Grossi, A., Park, H., Abou-Kheir, W. & Cox, D. (2009). Regulation of podosome dynamics by WASp phosphorylation: Implication in matrix degradation and chemotaxis in macrophages. J Cell Sci 122(pt 21), 38733882.
Fernandez-Trillo, J., Barros, F., Machin, A., Carretero, L., Dominguez, P. & Pena, P.d.I. (2011). Molecular determinants of interactions between the N-terminal domain and the transmembrane core that modulate hERG K+ channel gating. PLoS ONE 6(9), e24674.
Fiolka, R., Belyaev, Y., Ewers, H. & Stemmer, A. (2008). Even illumination in total internal reflection fluorescence microscopy using laser light. Microsc Res Techniq 71(1), 4550.
Griesbeck, O., Baird, G.S., Campbell, R.E., Zacharias, D.A. & Tsien, R.Y. (2001). Reducing the environmental sensitivity of yellow fluorescent protein. Mechanism and applications. J Biol Chem 276(31), 2918829194.
Hogue, I.B., Hoppe, A. & Ono, A. (2009). Quantitative fluorescence resonance energy transfer microscopy analysis of the human immunodeficiency virus type 1 Gag-Gag interaction: Relative contributions of the CA and NC domains and membrane binding. J Virology 83(14), 73227336.
Holden, S.J., Uphoff, S., Hohlbein, J., Yadin, D., Le Reste, L., Britton, O.J. & Kapanidis, A.N. (2010). Defining the limits of single-molecule FRET resolution in TIRF microscopy. Biophys J 99(9), 31023111.
Hoppe, A.D. (2007). Quantitative FRET microscopy of live cells. In Imaging Cellular and Molecular Biological Functions, Shorte, S.L. & Frischknecht, F. (Eds.), pp. 157180. Heidelberg, Germany: Springer.
Hoppe, A., Christensen, K. & Swanson, J.A. (2002). Fluorescence resonance energy transfer-based stoichiometry in living cells. Biophys J 83(6), 36523664.
Hoppe, A.D., Shorte, S.L., Swanson, J.A. & Heintzmann, R. (2008). Three-dimensional FRET reconstruction microscopy for analysis of dynamic molecular interactions in live cells. Biophys J 95(1), 400418.
Hoppe, A.D. & Swanson, J.A. (2004). Cdc42, Rac1, and Rac2 display distinct patterns of activation during phagocytosis. Mol Biol Cell 15(8), 35093519.
Joo, C. & Ha, T. (2007). Single-molecule FRET with total internal reflection microscopy. In Single-Molecule Techniques: A Laboratory Manual, Selvin, P.R. & Ha, T. (Eds.), pp. 336. New York: Cold Spring Harbor.
Jouvenet, N., Bieniasz, P.D. & Simon, S.M. (2008). Imaging the biogenesis of individual HIV-1 virions in live cells. Nature 454(7201), 236240.
Jouvenet, N., Neil, S.J.D., Bess, C., Johnson, M.C., Virgen, C.A., Simon, S.M. & Bieniasz, P.D. (2006). Plasma membrane is the site of productive HIV-1 particle assembly. PLoS Biol 4(12), e435.
Kotowski, S.J., Hopf, F.W., Seif, T., Bonci, A. & von Zastrow, M. (2011). Endocytosis promotes rapid dopaminergic signaling. Neuron 71(2), 278290.
Lam, A.D., Ismail, S., Wu, R., Yizhar, O., Passmore, D.R., Ernst, S.A. & Stuenkel, E.L. (2010). Mapping dynamic protein interactions to insulin secretory granule behavior with TIRF-FRET. Biophys J 99(4), 13111320.
Mattheyses, A.L., Atkinson, C.E. & Simon, S.M. (2011). Imaging single endocytic events reveals diversity in clathrin, dynamin and vesicle dynamics. Traffic 12(10), 13941406.
Mattheyses, A.L. & Axelrod, D. (2006). Direct measurement of the evanescent field profile produced by objective-based total internal reflection fluorescence. J Biomed Optics 11(1), 014006.
Mattheyses, A.L., Shaw, K. & Axelrod, D. (2006). Effective elimination of laser interference fringing in fluorescence microscopy by spinning azimuthal incidence angle. Microsc Res Techniq 69(8), 642647.
Mattheyses, A.L., Simon, S.M. & Rappoport, J.Z. (2010). Imaging with total internal reflection fluorescence microscopy for the cell biologist. J Cell Sci 123(21), 36213628.
Paar, C., Paster, W., Stockinger, H., Schutz, G.J., Sonnleitner, M. & Sonnleitner, A. (2008). High throughput FRET screening of the plasma membrane based on TIRFM. Cytom Part A 73(5), 442450.
Paster, W., Paar, C., Eckerstorfer, P., Jakober, A., Drbal, K., Schutz, G.J., Sonnleitner, A. & Stockinger, H. (2009). Genetically encoded Forster resonance energy transfer sensors for the conformation of the Src family kinase Lck. J Immunol 182(4), 21602167.
Rizzo, M.A., Springer, G.H., Granada, B. & Piston, D.W. (2004). An improved cyan fluorescent protein variant useful for FRET. Nat Biotechnol 22(4), 445449.
Roy, R., Hohng, S. & Ha, T. (2008). A practical guide to single-molecule FRET. Nat Methods 5(6), 507516.
Sekar, R.B. & Periasamy, A. (2003). Fluorescence resonance energy transfer (FRET) microscopy imaging of live cell protein localizations. J Cell Biol 160(5), 629633.
Sohn, H.W., Tolar, P. & Pierce, S.K. (2008). Membrane heterogeneities in the formation of B cell receptor-Lyn kinase microclusters and the immune synapse. J Cell Biol 182(2), 367379.
van Rheenen, J., Langeslag, M. & Jalink, K. (2004). Correcting confocal acquisition to optimize imaging of fluorescence resonance energy transfer by sensitized emission. Biophys J 86(4), 25172529.
van' t Hoff, M., de Sars, V. & Oheim, M. (2008). A programmable light engine for quantitative single molecule TIRF and HILO imaging. Opt Exp 16(22), 1849518504.
Wallrabe, H., Elangovan, M., Burchard, A., Periasamy, A. & Barroso, M. (2003). Confocal FRET microscopy to measure clustering of ligand-receptor complexes in endocytic membranes. Biophys J 85(1), 559571.
Wlodarczyk, J., Woehler, A., Kobe, F., Ponimaskin, E., Zeug, A. & Neher, E. (2008). Analysis of FRET signals in the presence of free donors and acceptors. Biophys J 94(3), 9861000.
Yoshida, S., Hoppe, A.D., Araki, N. & Swanson, J.A. (2009). Sequential signaling in plasma-membrane domains during macropinosome formation in macrophages. J Cell Sci 122(pt 18), 32503261.
Zacharias, D.A., Violin, J.D., Newton, A.C. & Tsien, R.Y. (2002). Partitioning of lipid-modified monomeric GFPs into membrane microdomains of live cells. Science 296(5569), 913916.
Zal, T. & Gascoigne, N.R. (2004). Photobleaching-corrected FRET efficiency imaging of live cells. Biophys J 86(6), 39233939.

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

  • Jia Lin (a1) and Adam D. Hoppe (a1)

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