Figure 3.

Simultaneous content and lipid release using TIRFM. v-SUVs contained 1 mol % DiD lipid dye and encapsulated 10 or 50 mM soluble content marker SRB. DiD and SRB were excited simultaneously using 638 nm and 561 nm laser lines, respectively. The emission was split and filtered to observe DiD (top, blue trace) and SRB (lower, red trace) fluorescence signals simultaneously projected onto an EMCCD detector. Total intensities from a region 20 pixels × 20 pixels (5.3 μm × 5.3 μm) is plotted for both the lipid (upper, blue trace) and content (lower, red trace) signals for a representative event. Snapshots from the lipid (top sequence, blue) and content (bottom sequence, red) signals are shown in inverted false color. When docking was clearly visible in the lipid channel, the content channel was still dim, because SRB was encapsulated at self-quenching concentrations (1). In the same frame where the lipid signals start to increase, announcing lipid mixing, the content signals also increase (dashed vertical line) due to dilution and dequenching of encapsulated SRB as some molecules escape through the pore. Once lipid transfer is complete (shortly after the maximum in the upper blue trace), the intensity in the lipid channel decreases due to photobleaching, as in Fig. 2. The SRB signals remain stable after lipid release (but bleach slowly (3)), indicating that the pore resealed after partial release of contents. In this example, initial lipid and content release occurred with comparable kinetics (2) within a few frames (each 18.3 ms apart). In other cases, release was markedly slower (see Fig. S7). To see this figure in color, go online.