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

36 Scopus citations

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

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology

Fingerprint Dive into the research topics of 'Monitoring protein interactions in living cells with fluorescence lifetime imaging microscopy'. Together they form a unique fingerprint.

  • Cite this