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

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. Two-dimensional (2-D) FRET images acquired al 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.