We next tested the performance of our distance measurement method by considering a few cases where the centers of magenta and green signals should co-localize (Figure ). First, we analyzed images of TetraSpeck beads separate from the images used for calibration. In the corrected images, we measured a narrow distribution of dMG with a mean of 11.0±0.2 nm. Next, we calculated the dMG for 50 nm, 100 nm, and 200 nm vesicles having membrane stained with Texas Red-DHPE and encapsulating Alexa Fluor 488. We obtained values of 29.2±0.5 nm, 25.7±0.8 nm and 20.7±0.7 nm respectively (Figure ). To test the performance with alternate green-emitting fluorescent dyes we measured dMG for Texas Red-DHPE labeled 100 nm vesicles encapsulating carboxyfluorescein and pyranine and obtained values of 28.2±0.6 nm and 28.9±0.4 nm, respectively (Figure ). Finally, we sought to test a dually labeled system which best represented the fluorescence signal level expected in an actual fusion experiment. We found that the RNA of intact influenza virus particles can be fluorescently labeled by incubating a virus suspension with a buffer containing QuantiFluor, similar to previously described methods for labeling of viral genomes . We prepared virus particles having the lipid membrane labeled with Texas Red-DHPE and the RNA labeled with QuantiFluor. Dually labeled virus particles adsorbed to plasma-cleaned glass surface were imaged and a mean dMG value of 26.6±1.0 nm was measured (Figure ). We combined all dMG distributions from objects with fully co-localizing magenta and green signals into cumulative distribution functions and determined the dMG value below which 95% of the distances lie (Figure ). In the case of dually labeled virus particles, 95% of dMG values were below 58 nm which may be chosen as the limiting value below which two signals may be interpreted as fully overlapping. Therefore, we arbitrarily chose 58 nm as the cut-off value to distinguish between symmetric (<58 nm) and non-symmetric (>58 nm) content distribution obtained in the viral fusion experiments as will be described in the next sections.