A schematic diagram outlining the procedure for measurement of dMG values is illustrated in Figure . Briefly, images are taken in magenta and green channels at various locations of the flow cell before fusion. The pH was then dropped to induce fusion and after 30 min, images were obtained in 2 channels at identical positions to identify the locations of the turned-on green spots (QuantiFluor-RNA). We needed to precisely locate the centroids of the membrane and content signals to reliably measure the distances between their centroids (Figure ). However, we had to address an important issue prior to carrying out distance measurements. While acquiring multichannel fluorescence images, the images acquired in different color channels are misaligned due to chromatic aberration . Even though advanced objective lenses correct this aberration for most practical purposes, it can still limit super-localization of diffraction-limited point sources . Additionally, chromatic aberration is not uniform across the field-of-view (FOV) . The spectral channels need to be registered to reliably interpret the extent of co-localization between signals in two channels as outlined in Figure . Channel registration was accomplished by imaging multiply labeled 100 nm TetraSpeck TM beads in magenta (561 nm excitation) and green (488 nm excitation) emission channels, with the latter being the reference channel. The centroids of the point spread functions of the spots were super-localized using the FIJI plugin ThunderSTORM by 2D Gaussian fitting . The chromatic calibration table containing the displacement vectors across the FOV were calculated using the FIJI plugin Detection of Molecules (DoM) which uses continuous smooth B-spline grid registration . The coordinates of the spots in the magenta channel were corrected by vector transformation using the chromatic calibration table (Figure ). A distortion map generated with DoM illustrates the variability of the chromatic shift across the FOV (Figure ). Following this, dMG values were calculated using ThunderSTORM. It was found that, in uncorrected calibration set images, dMG values of approximately 0-140 nm were measured with a mean of 76.1±1.4 nm (Figure ). In the corrected images, dMG ranged only over 0-50 nm with a mean of 8.9±0.4 nm (Figure ).