Hostname: page-component-cd9895bd7-mkpzs Total loading time: 0 Render date: 2024-12-26T15:00:51.608Z Has data issue: false hasContentIssue false
  • English
  • Français

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
Get access

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

Access options

Get access to the full version of this content by using one of the access options below. (Log in options will check for institutional or personal access. Content may require purchase if you do not have access.)

Footnotes

†

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

References

Axelrod, D. (1981). Cell-substrate contacts illuminated by total internal reflection fluorescence. J Cell Biol 89(1), 141–145.Google Scholar
Axelrod, D. (2001). Total internal reflection fluorescence microscopy in cell biology. Traffic 2, 764–774.Google Scholar
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), 2307–2320.CrossRefGoogle ScholarPubMed
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. Google Scholar
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), 470–480.CrossRefGoogle ScholarPubMed
Boyer, S.B. & Slesinger, P.A. (2010). Probing novel GPCR interactions using a combination of FRET and TIRF. Comm Integrative Biol 3(4), 343–346.Google Scholar
Buning, R. & van Noort, J. (2010). Single-pair FRET experiments on nucleosome conformational dynamics. Biochimie 92(12), 1729–1740.Google Scholar
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), 3873–3882.Google Scholar
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. Google Scholar
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), 45–50.Google Scholar
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), 29188–29194.Google Scholar
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), 7322–7336.Google Scholar
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), 3102–3111.Google Scholar
Hoppe, A.D. (2007). Quantitative FRET microscopy of live cells. In Imaging Cellular and Molecular Biological Functions, Shorte, S.L. & Frischknecht, F. (Eds.), pp. 157–180. Heidelberg, Germany: Springer.CrossRefGoogle Scholar
Hoppe, A., Christensen, K. & Swanson, J.A. (2002). Fluorescence resonance energy transfer-based stoichiometry in living cells. Biophys J 83(6), 3652–3664.Google Scholar
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), 400–418.Google Scholar
Hoppe, A.D. & Swanson, J.A. (2004). Cdc42, Rac1, and Rac2 display distinct patterns of activation during phagocytosis. Mol Biol Cell 15(8), 3509–3519.Google Scholar
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. 3–36. New York: Cold Spring Harbor.Google Scholar
Jouvenet, N., Bieniasz, P.D. & Simon, S.M. (2008). Imaging the biogenesis of individual HIV-1 virions in live cells. Nature 454(7201), 236–240.Google Scholar
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. CrossRefGoogle ScholarPubMed
Kotowski, S.J., Hopf, F.W., Seif, T., Bonci, A. & von Zastrow, M. (2011). Endocytosis promotes rapid dopaminergic signaling. Neuron 71(2), 278–290.CrossRefGoogle ScholarPubMed
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), 1311–1320.Google Scholar
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), 1394–1406.Google Scholar
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. Google Scholar
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), 642–647.Google Scholar
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), 3621–3628.Google Scholar
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), 442–450.Google Scholar
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), 2160–2167.CrossRefGoogle ScholarPubMed
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), 445–449.Google Scholar
Roy, R., Hohng, S. & Ha, T. (2008). A practical guide to single-molecule FRET. Nat Methods 5(6), 507–516.Google Scholar
Sekar, R.B. & Periasamy, A. (2003). Fluorescence resonance energy transfer (FRET) microscopy imaging of live cell protein localizations. J Cell Biol 160(5), 629–633.Google Scholar
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), 367–379.Google Scholar
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), 2517–2529.Google Scholar
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), 18495–18504.Google Scholar
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), 559–571.Google Scholar
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), 986–1000.Google Scholar
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), 3250–3261.Google Scholar
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), 913–916.Google Scholar
Zal, T. & Gascoigne, N.R. (2004). Photobleaching-corrected FRET efficiency imaging of live cells. Biophys J 86(6), 3923–3939.Google Scholar
Supplementary material: PDF

Jia Lin & Adam D. Hoppe Supplementary Material

Appendix

Download Jia Lin & Adam D. Hoppe Supplementary Material(PDF)
PDF 710.8 KB