Monitoring protein interactions in living cells with fluorescence lifetime imaging microscopy

Yuansheng Sun, Nicole M. Hays, Ammasi Periasamy, Michael W. Davidson, Richard Day

Research output: Contribution to journalArticle

32 Citations (Scopus)

Abstract

Fluorescence lifetime imaging microscopy (FLIM) is now routinely used for dynamic measurements of signaling events inside single living cells, such as monitoring changes in intracellular ions and detecting protein-protein interactions. Here, we describe the digital frequency domain FLIM data acquisition and analysis. We describe the methods necessary to calibrate the FLIM system and demonstrate how they are used to measure the quenched donor fluorescence lifetime that results from Förster Resonance Energy Transfer (FRET). We show how the "FRET-standard" fusion proteins are used to validate the FLIM system for FRET measurements. We then show how FLIM-FRET can be used to detect the dimerization of the basic leucine zipper (B Zip) domain of the transcription factor CCAAT/enhancer binding protein α in the nuclei of living mouse pituitary cells. Importantly, the factors required for the accurate determination and reproducibility of lifetime measurements are described in detail.

Original languageEnglish
Pages (from-to)371-391
Number of pages21
JournalMethods in Enzymology
Volume504
DOIs
StatePublished - 2012

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Optical Imaging
Energy Transfer
Microscopy
Microscopic examination
Fluorescence
Cells
Imaging techniques
Energy transfer
Monitoring
Proteins
Basic-Leucine Zipper Transcription Factors
CCAAT-Enhancer-Binding Proteins
Dimerization
Leucine Zippers
Ions
Data acquisition
Transcription Factors
Fusion reactions

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology

Cite this

Monitoring protein interactions in living cells with fluorescence lifetime imaging microscopy. / Sun, Yuansheng; Hays, Nicole M.; Periasamy, Ammasi; Davidson, Michael W.; Day, Richard.

In: Methods in Enzymology, Vol. 504, 2012, p. 371-391.

Research output: Contribution to journalArticle

Sun, Yuansheng ; Hays, Nicole M. ; Periasamy, Ammasi ; Davidson, Michael W. ; Day, Richard. / Monitoring protein interactions in living cells with fluorescence lifetime imaging microscopy. In: Methods in Enzymology. 2012 ; Vol. 504. pp. 371-391.
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