We hypothesized that super-localization microscopy techniques can be used to distinguish between the above two fusion scenarios since such techniques are widely used to pinpoint the centroid of a fluorescent particle with a lateral resolution of only a few tens of nanometers or less . Although both the viruses (28) and vesicles (Figure ) have variable morphologies and sizes, given that the dimensions of the fused volume (150-250 nm) is approximately at or below the diffraction limit, we assumed that the precise geometry of the latter will not have any significant effect on the super-localization procedure. In the case of a large fusion pore, the dyebound fluorescent vRNPs (content) will be evenly distributed within the fused volume and the centroids of the signals from the membrane dye and the content dye should more or less overlap, therefore leading to a small Euclidean distance between them (Scenario 1, Figure ). In the case of formation of transient or small pores, the nucleic acid-binding dye (QuantiFluor), being a relatively small molecule (approximately 600 Da) (Materials and Methods), will transfer to the virus side and bind there to the vRNPs remain and turn on. The centroids of the signals from the membrane dye and the content dye should be farther apart, therefore leading to a larger Euclidean distance between them (Scenario 2, Figure ). Throughout the text and illustrations, the Euclidean distances between the centroids of the membrane (561 nm excitation, referred to as magenta) and content (488 nm excitation, referred to as green) channels are abbreviated as dMG.