Changes in intensity of DiO and PBd22-PEO14 -TMR dyes in hybrid GUV membranes was monitored with a Zeiss LSM880 + Airyscan inverted confocal microscope on the same day of GUV formation. The microscope tile scanning option with a 2% intensity laser beam (488 nm for DiO excitation and 561 nm for PBd22-PEO14 -TMR excitation) pinhole aperture of 0.7 µm was used to scan large areas of the sample and facilitate the acquisition of statistical data. The images were analysed using FIJI software (National Institutes of Health, Bethesda, MD).