Skip to main content
. 2003 Sep;85(3):1826–1838. doi: 10.1016/S0006-3495(03)74611-6

FIGURE 3.

FIGURE 3

FIGURE 3

PCFM measurement of single TMR/Cy5-gramicidin heterodimer ion channels. Green and red arrows mark the timing of the toggled excitation at 514 nm and 632 nm, respectively. (a) Colocalization of two gramicidin monomers labeled with two different dyes, TMR and Cy5. Single-channel current trajectory recorded at 100-mV polarization potential, with arrows indicating the point of exposure of two consecutively recorded frames. The first frame was taken at 514-nm excitation (with the OG550 long-pass filter), indicating single-molecule fluorescence of TMR. The second frame was taken at 632-nm excitation, indicating single-molecule fluorescence of Cy5. From a 2-D Gaussian fitting, the molecules were found to be within 30 nm of each other (the minimal positional accuracy allowed by the measurement system). “C” and “O” denote the current corresponding to closed and open states, respectively. (b) spFRET within a pair of gramicidin monomers labeled with two different dyes, TMR and Cy5. Single-channel current trajectory (upper panel) with four arrows indicating the exposure of four consecutively acquired frames (lower panel). Frames 1 and 3 were taken at 514-nm excitation with a red-emission long-pass filter (RG645). Frames 2 and 4 were taken at 632-nm excitation with a red-emission long-pass filter (RG645). Frame 1 indicates that FRET occurred between the TMR/Cy5 spFRET pair when the gramicidin channel was at an open state. Frame 2 verifies that a single Cy5 molecule was present. Frame 3 indicates that no spFRET occurred when the channel was at a closed state. Frame 4 verifies that the Cy5 was still present. Subsequent images taken without the red long-pass filter (RG645) did show colocalization as shown in a. (c) FRET efficiency distribution of “channel open” TMR/Cy5 heterodimers (shaded bars) with mean of 0.59, width of 0.21, and “channel closed” (open bars) with a maximum value of 0.57. An asterisk is inserted to indicate the 70% of channels showing no appreciable FRET (below 0.3) in the channel-closed state (or 30% of channels in the channel-open state).