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

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

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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.

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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.

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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.

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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

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  1. Yuansheng Sun
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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.

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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)

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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

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