Fluorescence resonance energy transfer (FRET) imaging of a single living cell using green fluorescent protein

Ammasi Periasamy, Steve A. Kay, Richard N. Day

Research output: Contribution to journalConference article

7 Scopus citations

Abstract

Fluorescence resonance energy transfer (FRET) imaging microscopy is a unique tool to visualize the spatiotemporal dynamics of protein interactions in living cells. Genetic vectors that encode protein fusions with green fluorescent protein (GFP) provide a method for imaging protein localization in living cells. We used FRET to study dimerization of the pituitary-specific transcription factor Pit-1 fused to GFP and BFP. A fusion protein containing GFP separated from BFP by 29 amino acids served as a positive control for FRET. Transcriptional activity of the GFP- and BFP-Pit-1 fusion proteins was demonstrated by their ability to activate the prolactin gene promoter. Using optimized excitation and emission filters, cells expressing the fluorescently-tagged Pit-1 proteins were imaged with a back- thinned, back-illuminated CCD chip that has about 50% quantum efficiency in the blue range. 2D FRET images acquired at the focal plane demonstrated Pit-1 protein associations in the nucleus of living cells. The significance of 2- and 3-D energy transfer imaging from these living cells is discussed.

Original languageEnglish (US)
Pages (from-to)58-66
Number of pages9
JournalProceedings of SPIE - The International Society for Optical Engineering
Volume2983
DOIs
StatePublished - Dec 1 1997
Externally publishedYes
EventFunctional Imaging and Optical Manipulation of Living Cells - San Jose, CA, United States
Duration: Feb 10 1997Feb 10 1997

Keywords

  • Blue fluorescent protein
  • CCD camera
  • Green fluorescent protein
  • Huorescence resonance energy transfer (FRET)
  • Microscopy imaging
  • Pit-1 protein and dimerization

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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