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

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

doi:10.1117/12.274324

23 May 1997 Fluorescence resonance energy transfer (FRET) imaging of a single living cell using green fluorescent protein

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

Author Affiliations +

Proceedings Volume 2983, Functional Imaging and Optical Manipulation of Living Cells; (1997) https://doi.org/10.1117/12.274324
Event: BiOS '97, Part of Photonics West, 1997, San Jose, CA, United States

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.

Citation Download Citation

Ammasi Periasamy, Steve A. Kay, and Richard N. Day "Fluorescence resonance energy transfer (FRET) imaging of a single living cell using green fluorescent protein", Proc. SPIE 2983, Functional Imaging and Optical Manipulation of Living Cells, (23 May 1997); https://doi.org/10.1117/12.274324

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