2.3. Preparation of Liposomes

Lipid vesicles were prepared by the natural swelling method [19]. Lipids, Chol, menthol, and Rho-DHPE fluorescent probes were dissolved in a 2:1 v/v (chloroform/methanol) solution, to give concentrations of 2 mM for lipids, Chol, and menthol, and 0.1 mM for Rho-DHPE. Lipids, Chol, and menthol were mixed at the desired composition to give a total volume of 20 μL. Rho-DHPE (2 μL) was further added. The organic solvent was evaporated under a flow of nitrogen gas and the lipids were further dried in a vacuum desiccator for 3 h. The film was hydrated with 200 µL Milli-Q water at 37 °C for one hour and then kept overnight at room temperature (21.7 ± 1.7 °C). The final lipid concentration was 0.2 mM and the Rho-DHPE concentration was 1 μM respectively. The prepared lipid compositions for microscopic observation were DOPC/DPPC/Chol = 50:50:0, 40:40:20, and 35:35:30 as control systems without menthol. When we add 10 mol % menthol to lipid compositions, we fixed DOPC:DPPC:Chol = 1:1:0, 2:2:1, and 7:7:6, that is DOPC/DPPC/Chol/d- or l-menthol = 45:45:0:10, 36:36:18:10, and 31.5:31.5:27:10.