ASTM E3143-18

5.1.1 Fig. 1 demonstrates that liposomes may become distorted and are difficult to measure and analyze when they are air-dried, while the same sample is clearly easier to analyze when the specimen is near-instantly preserved by vitrification.

5.1.2 Cryo-TEM involves applying a small volume of sample to a specially prepared holey, ultra-thin or continuous carbon grid suspended in cryo-TEM plunger over a cup of liquid ethane cooled in a container filled with liquid nitrogen 5.1.2.1 Thick Ice—The vitrified ice thickness is often determined by the sample or the cryo-TEM procedure itself. Large liposomes, defined to include larger structure and sizes with respect to this standard, are generally associated with thicker ice, while smaller liposomes (structure and sizes) are associated with thinner ice. Generally, thick ice occurs when either excess water forms a thicker ice layer or samples containing larger liposomes are fully covered with water making the ice thicker around the specimen. Thicker ice tends to block the electron beam either completely or partially which compromises image quality.

5.1.2.2 Larger liposomes (structure and sizes) are preferentially lost by sample preparation.Larger liposomes are more difficult to image for two reasons. The first is the cryo-TEM procedure itself. This procedure requires the use of filter paper to blot away excess aqueous solution from the EM grid just prior to vitrification. The larger liposomes suspended within the sample preferentially wash away from the grid and into the filter paper, ending up in the filter paper. This is perhaps because the larger liposomes provide a larger surface areas that expose them to relatively larger forces during the rapid flow of the water to the filter paper. This makes them difficult to find and measure in electron micrographs when their relative concentration in the specimen is low-meaning that few are left behind after blotting. The second reason is that larger liposomes that are left behind on the EM grid, are often embedded in thicker ice that is too thick for the electron beam to either penetrate or if it does, causes the images to be too low in quality to provide adequate signal for image processing.

5.1.2.3 Liposomal Distortion—Because liposomes are essentially loose membrane bounded fluid compartments, freezing them within a layer of vitrified ice that is thinner than their diameter may cause the surface tension on both sides of the specimen to compress some of the liposomes leading to various levels of flattening distortions. Accurate size measurements of such distorted liposomes would require volumetric measurements of all the liposomes within a field of view through a three-dimensional analysis using electron tomography.