We next tested a thioflavin T-derived dye ThT-NE which was previously described to bind to secondary structures of viral RNA inside cells . However, no fluorescence turn on was observed with the microscopy-based viral fusion assay with ThT-NE. Finally, we tested a recently developed fluorogenic indolizine dye which was described to emit red fluorescence upon binding to cellular RNA ; however, we found that no fluorescence turn on was observed when we carried out a viral fusion assay (Figure ). We were unable to observe any appreciable fluorescence enhancement above background in viral lysates as well even after proteinase K treatment (Figure ). In the subsequent experiments described in this paper, we used QuantiFluor as the sole content transfer dye. Unfortunately, the structure and concentration of QuantiFluor is not provided by the supplier, so we made a crude analysis to approximately determine a concentration of the working stock solution (Materials and Methods and Figure ). In a typical screening experiment using the configuration shown in Figure , vesicles stained with Texas Red-DHPE and encapsulating a nucleic acid-binding dye were imaged by excitation with 561 nm (membrane: magenta) and 488 nm (content: green) light before and after pH drop (i.e., 7.4 → 5.1). Appearance of a new green spot co-localizing with a magenta spot (marked in yellow squares) indicated that the dye was capable of binding to vRNPs upon membrane fusion. Scale bar: 3 µm. B. Fluorescence emission spectra of QuantiFluor added to influenza viral lysate showing significant turn on as compared to dye only (dissolved in lysis buffer).