FIG. 5.

Size estimation of virus-fusing endosomes. (A) Determination of probe dilution. PC liposomes were made with various concentrations of DiOC18 and R18. Forty liposomes with various sizes (diameter ≤ 1 μm), which were attached to coverslips, were examined at each concentration, and G/R values of the liposomes were measured with the confocal microscope. Dilution rates corresponding to peaks no. 1 to 3 (labeled with arrows) in Fig. 3 were read and are summarized in Table 1. (B) Electron microscopic (EM) images of negative-stained influenza virus. The diameters of virus particles were measured and are summarized below the photographs. One hundred virus particles were examined in each experiment. *, this value is used in Table 1. Bar = 100 nm. (C) EM images of endosome containing virus. HeLa cells were bound with labeled virus for 10 min at 4°C and then incubated at 37°C for 5 min (upper left) and 10 min (others). The endosomal membrane joins to the viral membrane at the point indicated by the arrowhead. The C-shaped structures appear to be endosomes immediately after fusion with the virus. Bar = 100 nm. The distribution of diameters of endosomes containing the virus is presented in a histogram. From EM images, the diameters of 70 endosomes, which obviously contained virus particle(s), were measured.