Figure 2.

Single molecule tracking in supported DOPC bilayers. (A) The upper row sketches the applied laser timing protocol, the lower row an example of an image sequence. After recording a prebleach image the according area was totally photobleached by applying a laser pulse for t_bleach∼250 ms. The photobleaching efficiency was controlled by recording an image immediately after the bleach pulse. After a recovery time of t_rec∼2000 ms, single fluorescently labeled lipids entering the field of view can be resolved as diffraction limited signals. By recording an image sequence with high time resolution (t_ill = 3 ms, t_delay = 2 ms) individual lipids can be tracked over multiple images for determination of their diffusion constant. Panel B shows the cumulative density function cdf of square displacements between consecutive images at t_lag = 10 ms for PGPE-Alexa647. The monoexponential behavior confirms the presence of just a single mobile fraction. (C) Exact values for diffusion constants D were calculated by plotting ⟨r²⟩ versus t_lag, and fitting with eq 1. A clear difference for D is observed by comparing PGPE-Alexa647 (dark gray line, D = 7.2 ± 0.22 μm²/s) and DHPE-Bodipy (gray line, D = 3.1 ± 0.09 μm²/s). Experiments were performed at 37 °C.