Confocal Microscopy of Membrane Samples

Spectral imaging of the different membrane samples was performed on a Zeiss LSM 780 confocal microscope equipped with a 32-channel GaAsP detector array. Laser light at 405 and 488 nm was selected for fluorescence excitation of C-Laurdan and Di-4-ANEPPDHQ, respectively. The lambda detection range was set between 415 and 691 nm for C-Laurdan, and between 495 and 691 nm for Di-4-ANEPPDHQ (Figure S1). The wavelengths 415 and 691 nm were the ultimate limits of our detector. Despite the fact that wavelength intervals of down to 4 nm could be chosen for the individual detection channels, we have set these intervals to 8.9 nm, which allowed the simultaneous coverage of the whole spectrum with the 32 detection channels (Figure S1). The images were saved in .lsm file format and then analyzed by using a custom plug-in compatible with Fiji/ImageJ, as described further on.