The image from the PBd22-PEO14 -TMR channel was duplicated to create the mask. First this duplicate was adjust for brightness and contrast, and a threshold applied. Then a mask was created to ensure only vesicles greater than > 10 µm were measured using the automatic particle analysis tool. This size was chosen to prevent selection of non-vesicular aggregates. The mask created regions of interest around selected vesicles of a particular size. These regions of interest were then applied to the split channel images with intensity contributions from each dye to retrieve their intensity values. The intensity values from each dye image are normalised to the mean. Then the relative intensity ratio, R, between the intensity contributions from DiO, IDiO , and PBd22-PEO14 -TMR, IPBd22-PEO14 -TMR, was calculated using: