Significance and Use
5.1 Cryo-TEM is a technique used to record high resolution images of samples that are frozen and embedded in a thin layer of vitrified, amorphous ice (2-5). Because vitrification occurs so rapidly, the resultant specimen is almost instantly frozen, yielding a very accurate representation of the specimen at the moment of freezing, without the distortions typically associated with air drying delicate wet samples. Once frozen, images of the specimen are recorded at low temperature using a specially designed electron microscope equipped with a cryo-holder capable of operating under low dose conditions in order to prevent beam induced structural damage to the specimen. The cryo-TEM technique is the consensus choice to directly observe, analyze and accurately measure liposomes suspended in aqueous solutions. Fig. 1 illustrates this by comparing an electron micrograph from an air-dried negatively stained liposomal preparation with an electron micrograph of the same solution imaged by cryo-TEM.
FIG. 1 Left—An Electron Micrograph of an Air-Dried Liposomal Preparation that has been Negatively Stained with 2 % Uranyl Acetate for Contrast; Right—An Electron Micrograph of the Same Liposomal Preparation Prepared as a Frozen Vitrified Specimen for Cryo-TEM
NOTE 1: Both images are shown to the same scale; scale bar is 200 nm.
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 liposomal preparation 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 a cryo-TEM plunger over a cup of liquid ethane cooled in a container filled with liquid nitrogen (2, 3). These grids can be purchased or prepared in the laboratory using a carbon evaporator with glow discharge capabilities. Once the sample has wet the surface of the grid, and sufficient time allowed for the solution to equilibrate with regard to liposome spreading over the grid surface, the excess is wicked off (blotted) with filter paper and the grid plunged into the liquid ethane, vitrifying the sample. Once frozen, the sample is maintained at a liquid nitrogen temperature while it is imaged in a cryo-TEM operating under low electron dose conditions. There are several limitations associated with implementing this technique to analyze liposomes:
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 practice, 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 sample. 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 during sample preparation.Larger liposomes, defined to include larger structures and sizes with respect to this practice, 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 have 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, results in images that are 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.
Scope
1.1 This practice covers procedures for vitrifying and recording images of a suspension of liposomes with a cryo-transmission electron microscope (cryo-TEM) for the purpose of evaluating their shape, size distribution and lamellarity for quality assessment. The sample is vitrified in liquid ethane onto specially prepared holey, ultra-thin, or continuous carbon TEM grids, and imaged in a cryo-holder placed in a cryo-TEM.
1.2 The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard.
1.3 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety, health, and environmental practices and determine the applicability of regulatory limitations prior to use.
1.4 This international standard was developed in accordance with internationally recognized principles on standardization established in the Decision on Principles for the Development of International Standards, Guides and Recommendations issued by the World Trade Organization Technical Barriers to Trade (TBT) Committee.
