Fig. 4.

Multi-dimensional TCSPC analysis of intramolecular FRET between tandem ECFP and EYFP chromophores in HEK293 cells (CY24; the two fluorescent proteins are separated by 24 amino acids). (a,b) The non-descanned TPE intensity images for CY24-expressing HEK293 cells, in the 435-458 nm and the LP530 nm channels, respectively. (c) A FLIM map for the donor, 435-458 nm channel. (d) The normalized fluorescence decay data for ECFP alone (black filled triangles) and CY24 (open circles). ECFP data were fit to a bi-exponential curve as described (black line; see text and Table 1); FRET data were also fit to a bi-exponential curve (red line). These data were plotted from three-binned TCSPC pixels. (e) The fluorescence lifetime vs. pixel frequency distribution for HEK 293 cells expressing CY24, showing a major peak centred around the $\bar{\tau }$ lifetime mean of ∼1300 ps, with a minor peak at ∼1400-1500 ps (see text). (f-j) The respective intensity images, $\bar{\tau }$ FLIM map and frequency distribution for the same cell with a region of interest photo-bleached in the acceptor, EYFP (514 nm laser line excitation) channel. Two FLIM maps are shown (h,i), each indicating that the donor lifetime has increased in the region where the acceptor was photo-bleached. This was emphasized particularly by applying discrete colours to the FLIM map (i). In these FLIM images, colour indicates the τ lifetime, and brightness indicates photon count. Images were made using a Zeiss Plan NeoFLUAR 1.4 NA 63× oil-immersion objective lens.