Images were obtained with a microscope (DM IRE2; Leica) equipped with photon-multiplier tubes and acquisition software (Leica). Laurdan fluorescence was excited at 800 nm with a multiphoton laser system (Verdi/Mira 900; Coherent). Laurdan intensity images were recorded simultaneously and emissions were in the range of 400–460 and 470–530 nm (Gaus et al., 2003 (link)). Microscopy calibrations were performed as described previously (Gaus et al., 2003 (link)). For confocal microscopy a helium-neon laser was used to excite Cy3 (Ex: 543 nm; Em: 550–620 nm) and Cy5 (Ex: 633 nm; Em 650–720 nm) with appropriate cut-off filters and pinhole widths. For fixed cells, a 100× oil objective, NA 1.4, was used; for live cell, a 63× water objective, NA 1.3, was used, and images were recorded at RT.