Selection of a suitable nucleic acid-binding dye for content transfer experiments. Initially we screened the dyes (Table , Figure ) based on the following criteria: (i) good aqueous solubility to allow vesicle formation with sufficiently high encapsulated dye concentration; (ii) fluorescence properties not significantly altered between pH 7.5 and 5; (iii) impermeability to lipid membranes; (iv) minimal binding to lipid membranes ; (v) efficient binding to vRNPs leading to high signal above background when bound to viral RNA; and (vi) photostability. In a typical screening experiment, we prepared unilamellar vesicles (ULVs) composed of the lipids POPC:DOPE:Cholesterol:GD1a:Biotin-DPPE:Texas Red-DHPE (37.4:20:40:2:0.5:0.1 by molar ratio) and encapsulated each dye by extrusion through polycarbonate membrane filters of 100 nm pore size followed by size exclusion chromatography. The vesicles were found to have a narrow size dispersity (polydispersity index = 0.061) from dynamic light scattering (DLS) measurements (Figure ). Cryo-TEM imaging of the vesicles revealed that they are overwhelmingly unilamellar (Figure ). We targeted a bulk concentration of 10-100 µM for a given dye to ensure encapsulation of tens of molecules in a single vesicle. For all viral fusion experiments, we used simple microfluidic flow cells prepared by plasma-bonding polydimethylsiloxane (PDMS) blocks to clean glass cover slips and connecting them to a syringe pump for flowing various solutions (Figure ). We passivated the glass surface of the flow cell with block copolymers PLL-g-PEG/PLL-g-PEG-biotin to prevent any non-specific binding of vesicles and/or virus particles and attached the vesicles encapsulating a particular dye via interaction with NeutrAvidin (Figure ). Unbound vesicles were removed by washing with a pH 7.4 buffer (vesicle buffer). Next, H3N2 influenza virus particles (strain X-31, A/Aichi/68) were added to the same flow cell and allowed to incubate for about 10 min. Excess virus particles were removed by washing, and then the pH was rapidly dropped by flowing in pH 5.1 buffer (fusion buffer). Simultaneous with the pH drop, a continuous video stream of the slide was acquired. Bright spots were generated against minimal background if a dye bound to vRNPs (Figure ). Among all dyes tested, we observed that the RNA-specific dye QuantiFluor (Promega) offered the best combination of properties necessary for detection of viral contents (Figure ). We further tested the binding of QuantiFluor with vRNPs present in a detergent-lysed virus sample and found that the fluorescence of QuantiFluor was significantly turned on in the viral lysate (Figure ). The fluorescence was practically unchanged when the lysate was digested with proteinase K suggesting that NPs do not hinder binding of QuantiFluor to the viral RNA. A few members of the SYBR family of dyes also showed fluorescence turn on in the microscopy assay but they presented some limitations. The signals from SYBR Green I and SYBR Green II diminished rapidly under the standard imaging conditions due to photobleaching (Figure ). The signal from SYBR Gold was relatively stable; however, the level of signal above background was significantly weaker as compared to QuantiFluor (Figure ). SYBR Safe, a dye used in gel staining, did not lead to any fluorescence turn on. We tested the dye EvaGreen, a bis-intercalating dimeric acridine orange dye which is commonly used to detect dsDNA but were unable to detect any fluorescence from the microscopy experiments. We tested the performance of the dye in viral lysates and found that when the lysate is digested with proteinase K, a much larger turn-on is observed (Figure ). This result suggests that EvaGreen cannot bind sufficiently to the viral RNA complexed with proteins. We found that another bis-intercalating dye DiYO-1 was suitable for content transfer assay, consistent with the results described previously in an influenza viral genome exposure assay . We further observed that DiYO-1 produced a stable signal under our imaging conditions (Figure ), albeit we had to use a starting dye concentration of 100 µM to encapsulate sufficient dye molecules inside target vesicles to achieve appreciable signal levels as compared to 10 µM described by the previous authors. The SYTO family of dyes was found to be completely unsuitable for content transfer experiments. Most of these dyes tended to form aggregates during vesicle preparation due to apparent hydrophobicity, and we were unable to detect any turn-on behavior in the microscopy experiments. a molecular ion peak detected by MS (Figure ); b sampler kit; c free dye in PBS, N.A. Not available. In the case of SYTO TM 11-16, the excitation/emission wavelengths correspond to dye bound to DNA/RNA; d reference 24. "Yes" and "No" in the extreme right column denotes whether a particular dye is suitable for content transfer experiment or not.