FLIM-FRET microscopy to visualize transcription factor interactions in the nucleus of the living cell

Richard N. Day, Ignacio A. Demarco, Ty C. Voss, Ye Chen, Ammasi Periasamy

Research output: Contribution to journalConference article

2 Scopus citations

Abstract

Wide-field fluorescence microscopy was used to monitor the co-localization of the homeodomain (HD) transcription factor Pit-1 and the basic-leucine zipper protein CCAAT/enhancer binding protein alpha (C/EBPα), each labeled with fluorescent proteins (FP) in the living cell nucleus. Fluorescence resonance energy transfer (FRET) microscopy was used to resolve the angstrom-scale spatial relationships of these expressed proteins, and the effect of a Pit-1 point mutation on the interaction with C/EBPα was characterized. Two-photon excitation fluorescence lifetime imaging microscopy (2p-FLIM) was then used as an independent method to detect these protein interactions. The excited-state lifetime for the cyan FP (CFP) labeling C/EBPα was determined, and the measurements were repeated in cells co-expressing yellow FP (YFP) labeled-proteins. The CFP lifetime was decreased in the presence of the YFP acceptor, which is consistent with donor quenching by FRET. This was verified by acceptor photobleaching, which caused a shift in the donor lifetime to that similar to the donor alone. However, a significant limitation of this technique was demonstrated by the observation that high-energy 2p-excitation resulted in CFP photobleaching and a parallel decrease in its excited-state lifetime. The key question is whether the sensitivity of this imaging approach will be sufficient to acquire significant data from living cells expressing physiological levels of the labeled proteins.

Original languageEnglish (US)
Pages (from-to)36-43
Number of pages8
JournalProceedings of SPIE - The International Society for Optical Engineering
Volume5323
DOIs
StatePublished - Oct 27 2004
Externally publishedYes
EventProgress in Biomedical Optics and Imaging - Multiphoton Microscopy in the Biomedical Sciences IV - San Jose, CA, United States
Duration: Jan 25 2004Jan 27 2004

Keywords

  • Colocalization
  • Fluorescence lifetime imaging microscopy (FLIM)
  • Fluorescence resonance energy transfer (FRET) microscopy
  • Green fluorescent protein (GFP)
  • Transcription factors
  • Two-photon (2p) excitation

ASJC Scopus subject areas

  • Electronic, Optical and Magnetic Materials
  • Condensed Matter Physics
  • Computer Science Applications
  • Applied Mathematics
  • Electrical and Electronic Engineering

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