During membrane fusion, efficient release of viral contents is decided by factors such as the sizes of the fusion pores . The vRNPs of influenza are large structures measuring tens of nm (Figure ), so it is not obvious that they will pass through the fusion pore even though small dye molecules may. The diameter of the fusion pore has been measured using cryogenic electron tomography (cryo-ET) and is estimated to be ~15 nm at initial stages of fusion . Therefore, for transfer of vRNPs to the target vesicle, the fusion pore must expand to several tens of nanometers. Also, the vRNPs must dissociate fully from the M1 layer to freely distribute within the fused volume. Previous cryo-ET studies indeed revealed that two kinds of fused volumes may be obtained: (1) the virus and vesicle fused into a larger compartment and the viral contents were evenly distributed; (2) the virus and vesicle compartments were connected through a narrow stalklike pore but the vRNPs were not transferred from the virus side. A. In the case where the vRNP contents are symmetrically distributed within the fusion volume, the centroids of the membrane dye (magenta) and content (green) channels overlap. Alternatively, if the viral contents are nonsymmetrically distributed within the fusion volume, i.e. the QuantiFluor passed through a small fusion pore but the vRNP did not, the centroids of the membrane (magenta) and content (green) channels are spaced farther apart. B. Workflow of measurement of Euclidean distances between centroids of membrane (magenta) and content (green) dyes (dMG) using super-localization and chromatic aberration correction procedures.