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Proceedings Paper

Fluorescence resonance energy transfer (FRET) imaging of a single living cell using green fluorescent protein
Author(s): Ammasi Periasamy; Steve A. Kay; Richard N. Day
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Paper 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.

Paper Details

Date Published: 23 May 1997
PDF: 9 pages
Proc. SPIE 2983, Functional Imaging and Optical Manipulation of Living Cells, (23 May 1997); doi: 10.1117/12.274324
Show Author Affiliations
Ammasi Periasamy, Univ. of Virginia (United States)
Steve A. Kay, Scripps Research Institute (United States)
Richard N. Day, Univ. of Virginia (United States)

Published in SPIE Proceedings Vol. 2983:
Functional Imaging and Optical Manipulation of Living Cells
Daniel L. Farkas; Bruce J. Tromberg, Editor(s)

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