1.1 本规程涵盖了用冷冻透射电子显微镜（cryo-TEM）对脂质体悬浮液进行玻璃化和记录图像的程序，目的是评估其形状、尺寸分布和薄片度，以进行质量评估。将样品在液态乙烷中玻璃化到专门制备的多孔、超薄或连续碳TEM格栅上，并在放置在低温TEM中的低温支架中成像。 1.2 以国际单位制表示的数值应视为标准。本标准中不包括其他计量单位。 1.3 本标准并非旨在解决与其使用相关的所有安全问题（如有）。本标准的使用者有责任在使用前制定适当的安全、健康和环境实践，并确定监管限制的适用性。 1.4 本国际标准是根据世界贸易组织技术性贸易壁垒委员会发布的《关于制定国际标准、指南和建议的原则的决定》中确立的国际公认的标准化原则制定的。 ===意义和用途====== 5.1 Cryo-TEM是一种用于记录冷冻并嵌入玻璃化无定形冰薄层中的样品的高分辨率图像的技术 ( 2- 5. ) 由于玻璃化发生得如此之快，所得到的样品几乎立即被冷冻，从而在冷冻时产生样品的非常准确的表示，而没有通常与空气干燥细腻潮湿样品相关的失真。冷冻后，使用专门设计的电子显微镜在低温下记录标本的图像- 能够在低剂量条件下操作的支架，以防止梁对试样造成结构损伤。冷冻透射电镜技术是直接观察、分析和准确测量悬浮在水溶液中的脂质体的普遍选择。 图1 通过将来自空气干燥的负染色脂质体制剂的电子显微照片与通过冷冻TEM成像的相同溶液的电子显微图进行比较来说明这一点。 图1 左图——已用2%乙酸铀酰进行阴性染色以进行对比的风干脂质体制剂的电子显微图； 右图——作为冷冻玻璃化样品制备的用于冷冻TEM的相同脂质体制剂的电子显微图 注1: 两个图像都以相同的比例显示；比例尺为200nm。 5.1.1 图1 表明脂质体在空气干燥时可能会变形，难以测量和分析，而当样品几乎立即通过玻璃化保存时，同样的脂质体制剂显然更容易分析。 5.1.2 低温TEM包括将小体积的样品应用于专门制备的多孔、超- 薄的或连续的碳格栅悬浮在低温TEM柱塞中，置于一杯在充满液氮的容器中冷却的液态乙烷上 ( 2. ， 3. ) 这些格栅可以在实验室中使用具有辉光放电能力的碳蒸发器购买或制备。一旦样品润湿了格栅的表面，并且有足够的时间使溶液相对于脂质体在格栅表面上的扩散达到平衡，用滤纸将过量的脂质体吸出（吸干），并将格栅浸入液体乙烷中，使样品玻璃化。 一旦冷冻，样品保持在液氮温度，同时在低电子剂量条件下操作的低温TEM中成像。实施该技术来分析脂质体有几个局限性: 5.1.2.1 厚厚的冰-- 玻璃化冰的厚度通常由样品或冷冻TEM程序本身确定。相对于这种做法，大的脂质体被定义为包括较大的结构和尺寸，通常与较厚的冰有关，而较小的脂质体（结构和尺寸）与较薄的冰有关。 通常，当过量的水形成较厚的冰层，或者含有较大脂质体的样品被水完全覆盖，使样品周围的冰变厚时，就会出现厚冰。较厚的冰往往会完全或部分阻挡电子束，从而损害图像质量。 5.1.2.2 较大的脂质体（结构和大小）在样品制备过程中优先损失。 由于两个原因，更大的脂质体更难成像，相对于这种实践，更大脂质体被定义为包括更大的结构和尺寸。 第一个是低温TEM程序本身。该程序要求在玻璃化之前使用滤纸从EM格栅中吸走多余的水溶液。悬浮在样品中的较大脂质体优先从格栅中洗去并进入滤纸，最终进入滤纸。这可能是因为较大的脂质体具有较大的表面积，在水快速流动到滤纸的过程中使它们暴露于相对较大的力。当它们在样本中的相对浓度较低时，这使得它们很难在电子显微照片中找到和测量，这意味着在印迹后几乎没有留下。 第二个原因是留在EM网格上的较大脂质体通常嵌入较厚的冰中，该冰太厚，电子束无法穿透，或者如果穿透，则导致图像质量太低，无法为图像处理提供足够的信号。 5.1.2.3 脂质体畸变-- 由于脂质体本质上是以膜为界的松散流体隔室，将其冷冻在一层比其直径薄的玻璃化冰中可能会导致样品两侧的表面张力压缩一些脂质体，导致不同程度的扁平变形。 这种扭曲脂质体的精确尺寸测量将需要通过使用电子断层扫描的三维分析在视场内对所有脂质体进行体积测量。

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. ====== 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.