Introduction | How to do FRET | Filters | Example Images

Localization of BFP- and RFP-C/EBP protein expressed in mouse 3T3 cells using 2p-FRET microscopy. The doubly expressed cells (BFP-RFP-C/EBP) were excited by 740 nm and the donor (A) and acceptor (B) images of proteins localized in the nucleus of a single living cell were acquired by single scan (slow scan). As explained in the text, the bleed-through (or cross talk) correction was implemented and then ratioed to obtain a FRET image is shown in C. The respective histograms are shown below the images (D, E, F). The higher gray level intensity distribution in Figure C compared to E indicates the importance of bleed-through correction and ratioing of the corrected D and A (A/D) images to localize the proteins. (Donor-blue color, Uncorrected FRET-pink color, and Corrected FRET-red color dots) Adapted from A. Periasamy, Microscopy and Microanalysis, In Press, 2001.