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

Yuansheng Sun, Richard Day, Ammasi Periasamy

Research output: Contribution to journalArticle

110 Citations (Scopus)

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 FÃ ¶rster 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-Î ± 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.

Original languageEnglish
Pages (from-to)1324-1340
Number of pages17
JournalNature Protocols
Volume6
Issue number9
DOIs
StatePublished - Sep 2011

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Optical Imaging
Microscopy
Microscopic examination
Fluorescence
Cells
Imaging techniques
Energy Transfer
Proteins
CCAAT-Enhancer-Binding Proteins
Energy transfer
Physics
Dimerization
Cell Nucleus
Photons
Calibration
Specimen preparation
Transcription Factors
Data acquisition

ASJC Scopus subject areas

  • Biochemistry, Genetics and Molecular Biology(all)

Cite this

Investigating protein-protein interactions in living cells using fluorescence lifetime imaging microscopy. / Sun, Yuansheng; Day, Richard; Periasamy, Ammasi.

In: Nature Protocols, Vol. 6, No. 9, 09.2011, p. 1324-1340.

Research output: Contribution to journalArticle

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