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Localizing Protein–Protein Interactions in Living Cells Using Fluorescence Lifetime Imaging Microscopy

  • Yuansheng Sun
  • Ammasi Periasamy
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1251)

Abstract

In the past decade, advances in fluorescence lifetime imaging have extensively applied in the life sciences, from fundamental biological investigations to advanced clinical diagnosis. Fluorescence lifetime imaging microscopy (FLIM) is now routinely used in the biological sciences to monitor dynamic signaling events inside living cells, e.g., Protein–Protein interactions. In this chapter, we describe the calibration of both time-correlated single-photon counting (TCSPC) and frequency domain (FD) FLIM systems and the acquisition and analysis of FLIM-FRET data for investigating Protein–Protein interactions in living cells.

Key words

Fluorescence lifetime imaging microscopy (FLIM) Förster resonance energy transfer (FRET) FLIM-FRET Time-domain FLIM Time-correlated single-photon counting (TCSPC) FLIM Frequency-domain FLIM Protein–protein interactions 

Notes

Acknowledgments

The authors acknowledge funding from the University of Virginia, National Heart, Lung, and Blood Institute (NHLBI) PO1HL101871 and National Center for Research Resources NCRR-NIH RR027409. The authors thank Ms. Kay Christopher (Biology, University of Virginia) for preparing the samples, Dr. Steven Vogel (NIH/NIAAA) for providing the FRET-standard constructs, and Dr. Richard Day (Indiana University School of Medicine) for providing the C/EBPα-bZip constructs.

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Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  1. 1.W.M. Keck Center for Cellular Imaging, BiologyUniversity of VirginiaCharlottesvilleUSA

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