Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Protocol
  • Published:

Investigating protein-protein interactions in living cells using fluorescence lifetime imaging microscopy

Nature Protocols volume 6, pages 1324–1340 (2011)Cite this article

Subjects

Abstract

Fluorescence lifetime imaging microscopy (FLIM) is now routinely used for dynamic measurements of signaling events inside living cells, including detection of protein-protein interactions. An understanding of the basic physics of fluorescence lifetime measurements is required to use this technique. In this protocol, we describe both the time-correlated single photon counting and the frequency-domain methods for FLIM data acquisition and analysis. We describe calibration of both FLIM systems, and demonstrate how they are used to measure the quenched donor fluorescence lifetime that results from Förster resonance energy transfer (FRET). We then show how the FLIM-FRET methods are used to detect the dimerization of the transcription factor CCAAT/enhancer binding protein-α in live mouse pituitary cell nuclei. Notably, the factors required for accurate determination and reproducibility of lifetime measurements are described. With either method, the entire protocol including specimen preparation, imaging and data analysis takes ∼2 d.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: TCSPC FLIM setup.
Figure 2: Frequency-domain (FD) FLIM setup.
Figure 3: Photobleaching affects the accuracy of FLIM measurements.
Figure 4: TCSPC FLIM data representation of the FRET standard.
Figure 5: FD FLIM data representation of the FRET standard.
Figure 6: Localization of dimerized C/EBPα-bZip in living cell nucleus using TCSPC FLIM-FRET microscopy.
Figure 7: Investigation of the dimerization of C/EBPα-bZip in living cell nucleus using FD FLIM-FRET microscopy.

Similar content being viewed by others

References

  1. Lakowicz, J.R. Principles of Fluorescence Spectroscopy (Springer, 2006).

  2. Clegg, R.M. Fluorescence lifetime-resolved imaging: what, why, how—a prologue. In FLIM Microscopy in Biology and Medicine (eds. Periasamy, A. & Clegg, R.M.) 3–34 (CRC Press, 2009).

  3. Phipson, T.L. Phosphorescence or the Emission of Light by Minerals, Plants and Animals. (L. Reeve & Co, 1870).

  4. Venetta, B.D. Microscope phase fluorometer for determining the fluorescence lifetimes of fluorochromes. Rev. Sci. Instrum. 30, 450–457 (1959).

    Article  CAS  Google Scholar 

  5. Periasamy, A. & Clegg, R.M. FLIM applications in the biomedical sciences. In FLIM Microscopy in Biology and Medicine (eds. Periasamy, A. & Clegg, R.M.) 385–400 (CRC Press, 2009).

  6. Agronskaia, A.V., Tertoolen, L. & Gerritsen, H.C. High frame rate fluorescence lifetime imaging. J. Phys. D. 36, 1655–1662 (2003).

    Article  Google Scholar 

  7. Appelblom, H. et al. Homogeneous TR-FRET high-throughput screening assay for calcium-dependent multimerization of sorcin. J. Biomol. Screen 12, 842–848 (2007).

    Article  CAS  PubMed  Google Scholar 

  8. Vishwasrao, H.D., Heikal, A.A., Kasischke, K.A. & Webb, W.W. Conformational dependence of intracellular NADH on metabolic state revealed by associated fluorescence anisotropy. J. Biol. Chem. 280, 25119–25126 (2005).

    Article  CAS  PubMed  Google Scholar 

  9. Wang, H.W. et al. Differentiation of apoptosis from necrosis by dynamic changes of reduced nicotinamide adenine dinucleotide fluorescence lifetime in live cells. J. Biomed. Opt. 13, 054011 (2008).

    Article  PubMed  Google Scholar 

  10. Konig, K., Schneckenburger, H. & Hibst, R. Time-gated in vivo autofluorescence imaging of dental caries. Cell. Mol. Biol. (Noisy-le-grand) 45, 233–239 (1999).

    CAS  Google Scholar 

  11. Ehlers, A., Riemann, I., Stark, M. & Konig, K. Multiphoton fluorescence lifetime imaging of human hair. Microsc. Res. Tech. 70, 154–161 (2007).

    Article  PubMed  Google Scholar 

  12. Uchugonova, A. & Konig, K. Two-photon autofluorescence and second-harmonic imaging of adult stem cells. J. Biomed. Opt. 13, 054068 (2008).

    Article  PubMed  Google Scholar 

  13. Berezovska, O. et al. Familial Alzheimer's disease presenilin 1 mutations cause alterations in the conformation of presenilin and interactions with amyloid precursor protein. J. Neurosci. 25, 3009–3017 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Eckert, H., Petrášek, Z. & Kemnitz, K. Application of novel low-intensity nonscanning fluorescence lifetime imaging microscopy for monitoring excited state dynamics in individual chloroplasts and living cells of photosynthetic organisms. Proc. SPIE 6372, 637207 (2006).

    Article  Google Scholar 

  15. Petrasek, Z., Eckert, H.J. & Kemnitz, K. Wide-field photon counting fluorescence lifetime imaging microscopy: application to photosynthesizing systems. Photosynth Res. 102, 157–168 (2009).

    Article  CAS  PubMed  Google Scholar 

  16. Wouters, F.S. & Bastiaens, P.I. Fluorescence lifetime imaging of receptor tyrosine kinase activity in cells. Curr. Biol. 9, 1127–1130 (1999).

    Article  CAS  PubMed  Google Scholar 

  17. Chen, Y., Mills, J.D. & Periasamy, A. Protein localization in living cells and tissues using FRET and FLIM. Differentiation 71, 528–541 (2003).

    Article  CAS  PubMed  Google Scholar 

  18. Krishnan, R.V., Masuda, A., Centonze, V.E. & Herman, B. Quantitative imaging of protein-protein interactions by multiphoton fluorescence lifetime imaging microscopy using a streak camera. J. Biomed. Opt. 8, 362–367 (2003).

    Article  CAS  PubMed  Google Scholar 

  19. Biskup, C., Zimmer, T. & Benndorf, K. FRET between cardiac Na+ channel subunits measured with a confocal microscope and a streak camera. Nat. Biotechnol. 22, 220–224 (2004).

    Article  CAS  PubMed  Google Scholar 

  20. Chen, Y. & Periasamy, A. Characterization of two-photon excitation fluorescence lifetime imaging microscopy for protein localization. Microsc. Res. Tech. 63, 72–80 (2004).

    Article  CAS  PubMed  Google Scholar 

  21. Biener, E. et al. Quantitative FRET imaging of leptin receptor oligomerization kinetics in single cells. Biol. Cell. 97, 905–919 (2005).

    Article  CAS  PubMed  Google Scholar 

  22. Wallrabe, H. & Periasamy, A. Imaging protein molecules using FRET and FLIM microscopy. Curr. Opin. Biotechnol. 16, 19–27 (2005).

    Article  CAS  PubMed  Google Scholar 

  23. Periasamy, A. & Day, R.N. Molecular Imaging: FRET Microscopy and Spectroscopy (Oxford University Press, 2005).

  24. Forde, T.S. & Hanley, Q.S. Spectrally resolved frequency domain analysis of multi-fluorophore systems undergoing energy transfer. Appl. Spectrosc. 60, 1442–1452 (2006).

    Article  CAS  PubMed  Google Scholar 

  25. Biskup, C. et al. Multi-dimensional fluorescence lifetime and FRET measurements. Microsc. Res. Tech. 70, 442–451 (2007).

    Article  CAS  PubMed  Google Scholar 

  26. Periasamy, A. & Clegg, R.M. FLIM Microscopy in Biology and Medicine (CRC Press, 2009).

  27. Sun, Y., Wallrabe, H., Seo, S.A. & Periasamy, A. FRET microscopy in 2010: The legacy of Theodor Forster on the 100th anniversary of his birth. Chemphyschem 12, 462–474 (2011).

    Article  CAS  PubMed  Google Scholar 

  28. Förster, T. Delocalized excitation and excitation transfer. In In Modern Quantum Chemistry (ed. Sinanoglu, O.) 93–137 (Academic Press, 1965).

  29. Clegg, R.M. Fluorescence resonance energy transfer. In Fluorescence Imaging Spectroscopy and Microscopy (eds. Wang, X.F. & Herman, B.) 179–251 (John Wiley & Sons, 1996).

  30. Jares-Erijman, E.A. & Jovin, T.M. FRET imaging. Nat. Biotechnol. 21, 1387–1395 (2003).

    Article  CAS  PubMed  Google Scholar 

  31. Sekar, R.B. & Periasamy, A. Fluorescence resonance energy transfer (FRET) microscopy imaging of live cell protein localizations. J. Cell Biol. 160, 629–633 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Vogel, S.S., Thaler, C. & Koushik, S.V. Fanciful FRET. Sci. STKE 2006, re2 (2006).

    PubMed  Google Scholar 

  33. Piston, D.W. & Kremers, G.J. Fluorescent protein FRET: the good, the bad and the ugly. Trends Biochem. Sci. 32, 407–414 (2007).

    Article  CAS  PubMed  Google Scholar 

  34. Gadella, T.W.J. FRET and FLIM Techniques (Elsevier, 2009).

  35. Periasamy, A., Elangovan, M., Elliott, E. & Brautigan, D.L. Fluorescence lifetime imaging (FLIM) of green fluorescent fusion proteins in living cells. Methods Mol. Biol. 183, 89–100 (2002).

    CAS  PubMed  Google Scholar 

  36. Periasamy, A. Methods In Cellular Imaging (Oxford University Press, 2001).

  37. Wallrabe, H., Elangovan, M., Burchard, A., Periasamy, A. & Barroso, M. Confocal FRET microscopy to measure clustering of ligand-receptor complexes in endocytic membranes. Biophys. J. 85, 559–571 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Demarco, I.A., Periasamy, A., Booker, C.F. & Day, R.N. Monitoring dynamic protein interactions with photoquenching FRET. Nat. Methods 3, 519–524 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Chen, Y., Mauldin, J.P., Day, R.N. & Periasamy, A. Characterization of spectral FRET imaging microscopy for monitoring nuclear protein interactions. J. Microsc. 228, 139–152 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Li, H., Li, H.F., Felder, R.A., Periasamy, A. & Jose, P.A. Rab4 and Rab11 coordinately regulate the recycling of angiotensin II type I receptor as demonstrated by fluorescence resonance energy transfer microscopy. J. Biomed. Opt. 13, 031206 (2008).

    Article  PubMed  Google Scholar 

  41. Day, R.N., Booker, C.F. & Periasamy, A. Characterization of an improved donor fluorescent protein for Forster resonance energy transfer microscopy. J. Biomed. Opt. 13, 031203 (2008).

    Article  PubMed  Google Scholar 

  42. Periasamy, A., Wallrabe, H., Chen, Y. & Barroso, M. Chapter 22: Quantitation of protein-protein interactions: confocal FRET microscopy. Methods Cell Biol. 89, 569–598 (2008).

    Article  CAS  PubMed  Google Scholar 

  43. Sun, Y. et al. Characterization of an orange acceptor fluorescent protein for sensitized spectral fluorescence resonance energy transfer microscopy using a white-light laser. J. Biomed. Opt. 14, 054009 (2009).

    Article  PubMed  PubMed Central  Google Scholar 

  44. Li, H. et al. Actin cytoskeleton-dependent Rab GTPase-regulated angiotensin type I receptor lysosomal degradation studied by fluorescence lifetime imaging microscopy. J. Biomed. Opt. 15, 056003 (2010).

    Article  PubMed  PubMed Central  Google Scholar 

  45. Day, R.N., Periasamy, A. & Schaufele, F. Fluorescence resonance energy transfer microscopy of localized protein interactions in the living cell nucleus. Methods 25, 4–18 (2001).

    Article  CAS  PubMed  Google Scholar 

  46. Gratton, E., Breusegem, S., Sutin, J., Ruan, Q. & Barry, N. Fluorescence lifetime imaging for the two-photon microscope: time-domain and frequency-domain methods. J. Biomed. Opt. 8, 381–390 (2003).

    Article  PubMed  Google Scholar 

  47. Colyer, R.A., Lee, C. & Gratton, E. A novel fluorescence lifetime imaging system that optimizes photon efficiency. Microsc. Res. Tech. 71, 201–213 (2008).

    Article  PubMed  Google Scholar 

  48. Elangovan, M., Day, R.N. & Periasamy, A. Nanosecond fluorescence resonance energy transfer-fluorescence lifetime imaging microscopy to localize the protein interactions in a single living cell. J. Microsc. 205, 3–14 (2002).

    Article  CAS  PubMed  Google Scholar 

  49. Buranachai, C., Kamiyama, D., Chiba, A., Williams, B.D. & Clegg, R.M. Rapid frequency-domain FLIM spinning disk confocal microscope: lifetime resolution, image improvement and wavelet analysis. J. Fluoresc. 18, 929–942 (2008).

    Article  CAS  PubMed  Google Scholar 

  50. Gerritsen, H.C., Asselbergs, M.A., Agronskaia, A.V. & Van Sark, W.G. Fluorescence lifetime imaging in scanning microscopes: acquisition speed, photon economy and lifetime resolution. J. Microsc. 206, 218–224 (2002).

    Article  CAS  PubMed  Google Scholar 

  51. Grant, D.M. et al. High speed optically sectioned fluorescence lifetime imaging permits study of live cell signaling events. Opt. Express 15, 15656–15673 (2007).

    Article  CAS  PubMed  Google Scholar 

  52. McGinty, J. et al. Fluorescence lifetime optical projection tomography. J. Biophotonics 1, 390–394 (2008).

    Article  CAS  PubMed  Google Scholar 

  53. De Beule, P. et al. Rapid hyperspectral fluorescence lifetime imaging. Microsc. Res. Tech. 70, 481–484 (2007).

    Article  PubMed  Google Scholar 

  54. Boens, N. et al. Fluorescence lifetime standards for time and frequency domain fluorescence spectroscopy. Anal. Chem. 79, 2137–2149 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  55. Sharman, K.K., Periasamy, A., Asworth, H. & Demas, J.N. Error analysis of the rapid lifetime determination method for double-exponential decays and new windowing schemes. Anal. Chem. 71947–71952.

  56. Hanley, Q.S. & Clayton, A.H. AB-plot assisted determination of fluorophore mixtures in a fluorescence lifetime microscope using spectra or quenchers. J. Microsc. 218, 62–67 (2005).

    Article  CAS  PubMed  Google Scholar 

  57. Redford, G.I. & Clegg, R.M. Polar plot representation for frequency-domain analysis of fluorescence lifetimes. J. Fluoresc. 15, 805–815 (2005).

    Article  CAS  PubMed  Google Scholar 

  58. Digman, M.A., Caiolfa, V.R., Zamai, M. & Gratton, E. The phasor approach to fluorescence lifetime imaging analysis. Biophys. J. 94, L14–6 (2008).

    Article  CAS  PubMed  Google Scholar 

  59. Verveer, P.J., Squire, A. & Bastiaens, P.I. Global analysis of fluorescence lifetime imaging microscopy data. Biophys. J. 78, 2127–2137 (2000).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  60. Pelet, S., Previte, M.J., Laiho, L.H. & So, P.T. A fast global fitting algorithm for fluorescence lifetime imaging microscopy based on image segmentation. Biophys. J. 87, 2807–2817 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  61. Koushik, S.V., Chen, H., Thaler, C., Puhl, H.L., III. & Vogel, S.S. Cerulean, Venus, and VenusY67C FRET reference standards. Biophys. J. 91, L99–L101 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  62. Periasamy, A., Siadat-Pajouh, M., Wodnicki, P., Wang, X.F. & Herman, B. Time-gated fluorescence microscopy for clinical imaging. Microscopy Anal. 19–21 (1995).

  63. Lundin, K., Blomberg, K., Nordstrom, T. & Lindqvist, C. Development of a time-resolved fluorescence resonance energy transfer assay (cell TR-FRET) for protein detection on intact cells. Anal. Biochem. 299, 92–97 (2001).

    Article  CAS  PubMed  Google Scholar 

  64. Zhou, V. et al. A time-resolved fluorescence resonance energy transfer-based HTS assay and a surface plasmon resonance-based binding assay for heat shock protein 90 inhibitors. Anal. Biochem. 331, 349–357 (2004).

    Article  CAS  PubMed  Google Scholar 

  65. Xu, Z. et al. Development of high-throughput TR-FRET and AlphaScreen assays for identification of potent inhibitors of PDK1. J. Biomol. Screen. 14, 1257–1262 (2009).

    Article  CAS  PubMed  Google Scholar 

  66. Vogel, S.S., Thaler, C., Blank, P.S. & Koushik, S.V. Time-resolved fluorescence anisotropy. In FLIM Microscopy in Biology and Medicine (eds. Periasamy, A. & Clegg, R.M.) 245–288 (CRC Press, 2009).

  67. Becker, W. et al. Fluorescence lifetime imaging by time-correlated single-photon counting. Microsc. Res. Tech. 63, 58–66 (2004).

    Article  CAS  PubMed  Google Scholar 

  68. Stiel, H., Teuchner, K., Paul, A., Leupold, D. & Kochevar, I.E. Quantitative comparison of excited state properties and intensity-dependent photosensitization by rose bengal. J. Photochem. Photobiol. B. 33, 245–254 (1996).

    Article  CAS  PubMed  Google Scholar 

  69. Periasamy, A. et al. Time-resolved fluorescence lifetime imaging microscopy using a picosecond pulsed tunable dye laser system. Rev. Sci. Instrum. 67, 3722–3731 (1996).

    Article  CAS  Google Scholar 

  70. Becker, W. Recording the instrument response function of a multiphoton FLIM system. Application Notes (2007).

  71. Rizzo, M.A., Springer, G.H., Granada, B. & Piston, D.W. An improved cyan fluorescent protein variant useful for FRET. Nat. Biotechnol. 22, 445–449 (2004).

    Article  CAS  PubMed  Google Scholar 

  72. Ryder, A.G., Glynn, T.J., Przyjalgowski, M. & Szczupak, B. A compact violet diode laser-based fluorescence lifetime microscope. Journal of Fluorescence 12, 177–180 (2002).

    Article  CAS  Google Scholar 

  73. Thaler, C., Koushik, S.V., Blank, P.S. & Vogel, S.S. Quantitative multiphoton spectral imaging and its use for measuring resonance energy transfer. Biophys. J. 89, 2736–2749 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  74. Nagai, T. et al. A variant of yellow fluorescent protein with fast and efficient maturation for cell-biological applications. Nat. Biotechnol. 20, 87–90 (2002).

    Article  CAS  PubMed  Google Scholar 

  75. Tang, Q.Q. & Lane, M.D. Activation and centromeric localization of CCAAT/enhancer-binding proteins during the mitotic clonal expansion of adipocyte differentiation. Genes Dev. 13, 2231–2241 (1999).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  76. Enwright, J.F. III., Kawecki-Crook, M.A., Voss, T.C., Schaufele, F. & Day, R.N. A PIT-1 homeodomain mutant blocks the intranuclear recruitment of the CCAAT/enhancer binding protein alpha required for prolactin gene transcription. Mol. Endocrinol. 17, 209–222 (2003).

    Article  CAS  PubMed  Google Scholar 

  77. Lew, D. et al. GHF-1-promoter-targeted immortalization of a somatotrophic progenitor cell results in dwarfism in transgenic mice. Genes Dev. 7, 683–693 (1993).

    Article  CAS  PubMed  Google Scholar 

  78. Landschulz, W.H., Johnson, P.F., Adashi, E.Y., Graves, B.J. & McKnight, S.L. Isolation of a recombinant copy of the gene encoding C/EBP. Genes Dev. 2, 786–800 (1988).

    Article  CAS  PubMed  Google Scholar 

  79. Chen, Y. & Periasamy, A. Time-correlated single photon counting (TCSPC) FLIM-FRET microscopy for protein localization. In Molecular Imaging: FRET Microscopy and Spectroscopy (eds. Periasamy, A. & Day, R.N.) 239–259 (Oxford University Press, 2005).

  80. Sun, Y. & Periasamy, A. Additional correction for energy transfer efficiency calculation in filter-based Forster resonance energy transfer microscopy for more accurate results. J. Biomed. Opt. 15, 020513 (2010).

    Article  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

We acknowledge funding from the University of Virginia, National Center for Research Resources NCRR-NIH RR027409 and National Heart, Lung, and Blood Institute (NHLBI) P01HL101871 (A.P.), and National Institutes of Health (NIH) grants 2RO1 DK43701 and 3RO1 DK43701-15S1 (R.N.D.). We also thank K. Christopher (Biology, University of Virginia) and N. Hays (Indiana University School of Medicine) for preparing the samples, S. Vogel (NIH/NIAAA) for providing the FRET standard constructs, and M. Davidson (National High Magnetic Field Laboratory of Florida State University) for providing the mVenus C1 vector. We express sincere thanks to W. Becker (Becker & Hickl) and B. Barbieri (ISS) for their valuable feedback.

Author information

Authors and Affiliations

  1. Departments of Biology and Biomedical Engineering, W.M. Keck Center for Cellular Imaging, University of Virginia, Charlottesville, Virginia, USA

    Yuansheng Sun & Ammasi Periasamy

  2. Department of Cellular and Integrative Physiology, Indiana University School of Medicine, Indianapolis, Indiana, USA

    Richard N Day

Authors
  1. Yuansheng Sun
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Richard N Day
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Ammasi Periasamy
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

All authors contributed equally to the paper. A.P. is one of the pioneers in FLIM imaging technology development for biological applications.

Corresponding author

Correspondence to Ammasi Periasamy.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Data 1

SI_1: Complete sequence for the C5V plasmid (TXT 5 kb)

Supplementary Data 2

SI_2: Complete sequence for the C5A plasmid (TXT 5 kb)

Supplementary Data 3

SI_3: Complete sequence for the mCerulean CI plasmid (TXT 4 kb)

Supplementary Data 4

SI_4: Complete sequence for the invenus CI plasmid (TXT 4 kb)

Supplementary Data 5

SI_5: Complete sequence for the cerulean-B-zip construct (TXT 5 kb)

Supplementary Data 6

SI_6: Complete sequence for the Venus-B-zip construct (TXT 5 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Sun, Y., Day, R. & Periasamy, A. Investigating protein-protein interactions in living cells using fluorescence lifetime imaging microscopy. Nat Protoc 6, 1324–1340 (2011). https://doi.org/10.1038/nprot.2011.364

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nprot.2011.364

This article is cited by

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing