Spectral imaging of the different GUV compositions was performed using the Zeiss LSM880 + Airyscan inverted confocal microscope 32-channel GaAsP detector array. Laurdan is excited at 405 nm using a 4% laser intensity. The lambda detection range was set between 412 and 555 nm with intervals of 8.9 nm, allowing simultaneous coverage of the whole emission spectrum. The confocal pinhole aperture was set to 3.1 µm and, to increase the scan speed, single GUVs were maximally magnified to allow the whole vesicle to be imaged. The images of resolution 512 x 512 pixels were saved in 'lsm' file format and filtered with a Gaussian blur filter before being analysed to find the general polarisation value of each pixel using a custom plug-in compatible with Fiji, found at . By recording the whole emission spectrum for each image pixel, the spatial heterogeneity in GP values can be accurately observed. The whole emission spectrum of Laurdan for each image pixel is recorded and a Gaussian distribution was fitted to the emission spectrum captured for each pixel. From this, the GP value for each pixel was obtained by comparing the fluorescence signal intensities at liquid disordered or liquid ordered emission wavelengths. For Laurdan, emission at wavelength 444 nm (blue-shifted) indicates an ordered membrane environment, while emission at 488 nm (redshifted) indicates a disordered membrane environment :