We measured the dMG within a fusion volume between 100 nm vesicles and viruses and obtained a significantly broader distribution of dMG's ranging between 0-150 nm and a mean of 68.2±2.4 nm (Figure ). This value is significantly larger than that obtained for fusion of virus particles with 100 nm vesicles tethered to PLL-PEG surface (Figure ), which suggests that the tethering strategy in Figure indeed increases the probability of side-by-side fusion. To test the consistency of our methodology, we asked whether the range of measured dMG values increases if the size of the fused volume is increased. We carried out fusion between 200 nm vesicles and viruses, and indeed dMG values were spread over larger range (0-250 nm) with a significantly higher mean of 82.4±2.8 nm (Figure ) as compared to the case with 100 nm vesicles. Cumulative distribution function plots of distances between the centroids of membrane and content signals for -dually labeled influenza virus particles; fused volumes between 100 nm vesicles and influenza virus; and fused volumes between 100 nm vesicles and influenza virus pre-treated with nucleozin. The shaded region corresponds to distances less than 58 nm which is the value below which 95% of all the dMG's measured for dually labeled viruses belong.