Vesicle preparation and fusion chamber: Large unilamellar vesicles (compositions summarized in Figure ) were prepared by extrusion of a multilamellar dispersion through 100 nm polycarbonate filters. For content mixing experiments, self-quenching concentration (30 mM) of sulforhodamine B was included in the hydration buffer and the vesicles were separated from the free dye by size exclusion chromatography. All viral experiments were carried out using a simple microfluidic flow cell prepared from glass and polydimethylsiloxane (PDMS) by plasma-bonding. The glass surfaces of the flow cell's channels were passivated with co-polymers PLL-PEG/PLL-biotin-PEG. The vesicles were tethered to the passivated surface via NeutrAvidin, and excess vesicles were removed by flowing vesicle buffer through the channel.