During an experiment, the sample is excited at both 460 and 403 nm with a short delay (300 ms) between both excitations. After recording each samples emission intensity at 510 nm after each excitation, which some machines perform automatically, the software provides an I460/I403 ratio. Note: the wavelengths values should be predefined before starting the experiment in 'the method'. After each experiment is concluded (by adding detergent to lyse the vesicles), one dataset is obtained, which contains timestamps and the I460/I403 ratios. Conducting several assays during the day and over several days ensures the accuracy of the data. Then, average ratios can be normalised using Equation (2).