Characterization of QuantiFluor. QuantiFluor ® (Promega) is a commercially available dye whose structure and concentration are currently proprietary. We carried out a few characterizations of the dye to roughly estimate the concentration. 500 µL of deep orange colored DMSO solution obtained from Promega was mixed with 5 mL of Milli-Q water, lyophilized, and the measured mass of the orange residue was approximately 0.30 mg. This residue was dissolved in 500 µL Milli-Q water, aliquoted into smaller volumes and stored at -20 °C. The aqueous solution is stable at room temperature for several weeks when kept in dark. The turn-on properties of the dye with RNA remained unchanged through the lyophilization/re-dissolving process. We found that the residue could be re-dissolved in Milli-Q water at concentrations up to 10 times that of the original stock. Additionally, we found that the residue was soluble in methanol, ethanol, and chloroform. However, the dye degraded (color faded and turned yellowish) within a week when a CDCl3 solution of the dye was accidentally left in an NMR tube at room temperature. A UV-Vis spectrum of the dye in Milli-Q water revealed an absorption maximum at 483 nm (Figure ). The dye solution was run through HPLC (Figure ), and a single peak was identified. Mass spectra of the aqueous solution of the dye were taken and a strong peak at m/z = 291.7 and a weaker peak at m/z = 582.2 were detected (Figure ). We assign the peak at m/z = 582.2 to be from [M + ] species and the peak at m/z = 291.7 to be from [M+H + ] species. We assume that there is at least one counter-anion permanently associated with the molecule and the molecular weight may range between approximately 620-660 Da. Given that 0.3 mg of dye residue was present in 0.5 mL solution, we estimate that the concentration of the stock solution of the dye is approximately 0.9-0.95 mM. ). Experimental setup. In a freshly prepared microfluidic flow cell (Figure ), the channels (glass surfaces) are passivated with PLL-PEG/PLL-biotin-PEG. The vesicles encapsulating QuantiFluor (or another nucleic acid-binding dye) are tethered with the passivated surface via NeutrAvidin. Excess vesicles are removed by rinsing the channel with vesicle buffer. Following this, virus particles are added to the flow cell and allowed to bind to the vesicles for ~10 min. Unbound virus particles are removed by rinsing the channel with vesicle buffer. pH was dropped by flowing in fusion buffer while a continuous video stream was acquired for 1200 frames at 3.47 frames per second. The wait time between lowering of pH and appearance of a bright spot (corresponding to a fusion event) were calculated using custom-written MATLAB scripts (details below). The wait times are plotted into cumulative distribution functions (CDF). At neutral pH or at slightly acidic pH (6.4), we did not observe any fluorescence turn on events.