Fig. 2. Typical example of FRET between donor GM1 and acceptor Gb3 molecules. (D1) Donor Cy3-CTB-GM1 image before acceptor photobleaching. (A1) Acceptor Cy5-Stx1B-Gb3 image before photobleaching. (A2) Acceptor image after photobleaching. (D2) Donor image after acceptor photobleaching. Energy transfer can be detected from the increase in donor fluorescence (difference between D2 and D1 intensity fluorescence). In this experiment, the cells were labeled with donor- and acceptor-conjugated CTB and Stx1B at D:A=1:3 ratio. Colors reflect the fluorescence intensity of 8-bit images from lowest (black, 0 gray levels) to highest (red, 255 gray levels). The increase in D2 vs D1 fluorescence intensity is due to FRET. Note that neighboring cells, which are black on the D1 image because CTB intensity was below threshold level due to lower GM1 expression or due to a difference in cell thickness, appeared in blue color on the D2 image due to FRET. (B) Donor fluorescence intensity Cy3-CTB before and after (C) the bleaching period (30 seconds) in the absence of acceptor Cy5-Stx1B. Note that no donor bleaching or FRET occurred in that time period in the absence of acceptor.
