Introduction | How to do FRET | Filters | Example Images

FLIM-FRET imaging of GHFT1-5 cells expressing BFP- and RFP-C/EBP. Time-resolved donor images were acquired in the presence (AD0, AD1, AD2, AD3, AD4) and absence (DD0, DD1) of the acceptor, and then processed. In the absence of the acceptor, one obtains donor (BFP-C/EBP) two-dimensional distribution of a single component lifetime image (D = 3.7 ns, at position '-c') and there is no 2nd component. But, in the presence of the acceptor (BFP-RFP-C/EBP), the processed images provide two-dimensional distributions of double component lifetime images (DA1 = 2.1 ns and DA2 = 0.78 ns, at position '-c'), indicating the occurrence of protein dimerization (DA1 < D) and distance distribution. Intensity FRET image (A/D = F) using DVFRET system were also acquired and processed for the same cell in a different system configuration, as explained in the text. As shown, the intensity FRET image (F) does not provide any detailed information regarding the energy transfer process. *DA1 is the same image DA1. Adapted from Periasamy et al., 2001, Methods in Cellular Imaging, edited by A. Periasamy, Oxford University Press, New York.