*2.6. Liposome Imaging by Cryo-Electron Microscopy (Cryo-EM) and Fluorescent Microscopy*

For cryo-EM imaging, the retentates from the centrifugal filtration were diluted with 500 µL of MilliQ water, loaded on holey carbon-coated grids (Lacey, Tedpella, Redding, CA, USA) and vitrified in liquid ethane at −180 ◦C using a vitrobot (FEI Company, Eindhoven, Netherlands). The frozen grids were transferred to a Talos Electron microscope (FEI, Hillsboro, OR, USA) and imaged at cryogenic temperatures using a Gatan 626 cryo-holder (Gatan, Pleasanton, CA, USA). The electron micrographs were recorded at an accelerating voltage of 200 V using a low-dose system (30 e−/Å<sup>2</sup> ) and keeping the sample at −175 ◦C. Defocus values were −2 to −3 µm. Micrographs were recorded on a 4K × 4K Ceta CMOS camera.

For fluorescent imaging, formulations loaded with the 1,10 -dioctadecyl-3,3,30 ,30 tetramethylindocarbocyanine perchlorate (DiI) as a model hydrophobic substance were used. The samples were dispersed in a high concentration, suitable for imaging, as follows. A 20 mg aliquot of the sample was added to 50 mL of the solvent and the dispersion was shaken in a water bath at 37 ◦C. A droplet of the sample was visualized on a microscope slide with a cover glass using an Olympus CKX41 microscope (Olympus Ltd., Tokyo, Japan) at an excitation wavelength of 549 nm and an emission wavelength of 565 nm.
