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. 2012 Jun 27;303(7):C728–C742. doi: 10.1152/ajpcell.00448.2011

Fig. 3.

Fig. 3.

Colocalization and FRET imaging between CFP-Plin2 and NBD-PC. CFP-Plin2-overexpressing cells were labeled with NBD-PC to determine colocalization and FRET efficiency between the fluorescently labeled Plin2 and lipid. The extent of colocalization (A) was imaged and delineated graphically as a pixel fluorogram (B) where CFP-labeled cells (red) stained with NBD-PC (green) were combined to yield yellow to orange in areas where both probes colocalized. The extent of FRET efficiency was imaged from the following: donor emission image of CFP-Plin2 colabeled with NBD-PC before acceptor (NBD) photobleaching (C); donor emission image of CFP-Plin2 colabeled with NBD-PC after acceptor photobleaching (D); acceptor emission image of NBD-PC before photobleaching overlaid with a FRET pseudo-colored FRET image (E); and acceptor emission image of NBD-PC after photobleaching showing residual emission (F). Cells were imaged and processed as described in materials and methods. FRET efficiency images were generated by subtracting the donor (CFP) emission before acceptor (NBD) photobleaching from the donor (CFP) emission after acceptor photobleaching. The resultant image was divided by the image of donor emission after photobleaching and multiplied by 100 to show the grayscale FRET efficiencies. A FRET overlay was created and pseudo-colored to visualize regions of higher and lower FRET as shown by the inset color scale. See Glossary for definitions of abbreviations.