1、Designation: E3143 18E3143 18aStandard Practice forPerforming Cryo-Transmission Electron Microscopy ofLiposomes1This standard is issued under the fixed designation E3143; the number immediately following the designation indicates the year oforiginal adoption or, in the case of revision, the year of
2、last revision. A number in parentheses indicates the year of last reapproval. Asuperscript epsilon () indicates an editorial change since the last revision or reapproval.1. Scope1.1 This practice covers procedures for vitrifying and recording images of a suspension of liposomes with a cryo-transmiss
3、ionelectron 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 ina cryo-holder placed in a cryo-TEM.1.
4、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 responsibilityof the user of this standard to establish app
5、ropriate safety, health, and environmental practices and determine the applicability ofregulatory limitations prior to use.1.4 This international standard was developed in accordance with internationally recognized principles on standardizationestablished in the Decision on Principles for the Develo
6、pment of International Standards, Guides and Recommendations issuedby the World Trade Organization Technical Barriers to Trade (TBT) Committee.2. Referenced Documents2.1 ASTM Standards:2E1617 Practice for Reporting Particle Size Characterization Data2.2 ISO Standards:313322-1 Particle Size Analysis
7、Image Analysis Methods Static Image Analysis Methods3. Terminology3.1 Definitions:3.1.1 anti-contaminator, na specially designed device built into the column of a cryo-transmission electron microscopedesigned to pull contamination produced by outgassing within the column away from the frozen specime
8、n during imaging. Thedevice is cooled with liquid nitrogen to a temperature below that of the specimen creating a colder surface for contamination tobuild on.3.1.2 carbon evaporator, na device used to evaporate carbon in a high vacuum chamber generally by applying current throughtwo carbon rods pres
9、sed against one another, with one rod being sharpened in order to provide resistance to the current; this causesthe rods to heat up and evaporate carbon. The same device can also be used as a glow discharge device (3.1.15) in order to glowdischarge surfaces.3.1.3 continuous carbon grid, na copper el
10、ectron microscope grid coated with a self-supporting layer of continuous carbonover the square mesh of the grid.3.1.4 copper electron microscope grid, ncommonly referred to as an “EM grid,” a thin 3-mm diameter copper foil disk,usually manufactured with a pattern of square holes called a meshthrough
11、 which imaging is conducted in an electron microscope.The number, pattern, and shape of the holes can vary depending on imaging conditions and sample requirements.1 This practice is under the jurisdiction of ASTM Committee E56 on Nanotechnology and is the direct responsibility of Subcommittee E56.02
12、 on Physical and ChemicalCharacterization.Current edition approved Feb. 1, 2018June 1, 2018. Published February 2018July 2018. Originally approved in 2018. Last previous edition approved in 2018 as E3143 18. DOI: 10.1520/E1343-18.10.1520/E1343-18A.2 For referencedASTM standards, visit theASTM websit
13、e, www.astm.org, or contactASTM Customer Service at serviceastm.org. For Annual Book of ASTM Standardsvolume information, refer to the standards Document Summary page on the ASTM website.3 Available from International Organization for Standardization (ISO), ISO Central Secretariat, BIBC II, Chemin d
14、e Blandonnet 8, CP 401, 1214 Vernier, Geneva,Switzerland, http:/www.iso.org.This document is not an ASTM standard and is intended only to provide the user of an ASTM standard an indication of what changes have been made to the previous version. Becauseit may not be technically possible to adequately
15、 depict all changes accurately, ASTM recommends that users consult prior editions as appropriate. In all cases only the current versionof the standard as published by ASTM is to be considered the official document.Copyright ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, P
16、A 19428-2959. United States13.1.5 cryo-grid storage device, nany device used to store cryo-EM grids indefinitely in a liquid nitrogen dewar.3.1.6 cryo-TEM, nalso referred to simply as cryo-EM, or cryo electron microscopy, the process of imaging frozen vitrifiedsamples using a cryo-transmission elect
17、ron microscope.3.1.7 cryo-TEM holder, na liquid nitrogen refrigerated device specifically designed to hold and maintain a prepared gridcontaining a frozen, hydrated, vitrified specimen in a cryo-TEM while imaging is conducted.3.1.8 cryo-TEM plunger, na device used to vitrify a sample onto a holey, u
18、ltra-thin, or continuous carbon grid for cryo-TEMby plunging the grid containing a thin layer of aqueous sample into an ethane slush or other cryogen(s). There are many types ofplunger designs both homemade and commercial. The simplest is the homemade guillotine type (see Fig. 3(B), 8.3).3.1.9 cryo-
19、transmission electron microscope, na specially designed transmission electron microscope (see 3.1.25) capable ofimaging frozen vitrified specimens under low electron dose conditions in order to prevent beam-induced specimen damage.3.1.10 electron micrograph, nthe individual image recorded by the ele
20、ctron microscope as data are collected.3.1.11 electron tomography, nthe process of using images derived from an electron microscope in which the images wererecorded of a sample through an incremental series of tilt angles that are then used with image analysis software to reconstruct thestructure of
21、 the specimen in 3D.3.1.12 ethane, nchemical formula C2H6. A colorless, odorless flammable gas.3.1.13 forceps, na very fine pointed pair of tweezers or pincer used to pick up electron microscope grids.3.1.14 glow discharge, na plasma generated by passing an electric current through a low-pressure ga
22、s environment within achamber, usually a bell jar and with respect to this standard, is used to clean and statically charge the carbon surface coating ofcopper electron microscope grids in order to make them hydrophilic so that they will wet during sample application.3.1.15 glow discharge device, na
23、 device, such as a carbon evaporator (see 3.1.2), designed to generate a glow dischargeplasma.3.1.16 hang time, vthe amount of time after blotting of the sample from the prepared grid, prior to plunging the grid into theliquid ethane for vitrification.3.1.17 holey carbon grid, na copper electron mic
24、roscope grid specially prepared for cryo-TEM consisting of a thin electronsemi-transparent carbon film containing small holes that is suspended over the larger mesh of square holes of the copper electronmicroscope grid (see Fig. 3(A), 8.2.1).3.1.18 image analysis, nthe process of analyzing digital i
25、mages with computer software for the purpose of extractingmeaningful information from the data, for example, a size distribution.NOTE 1Both images are shown to the same scale; scale bar is 200 nm.FIG. 1 LeftAn Electron Micrograph of an Air-Dried Liposomal Preparation that has been Negatively Stained
26、 with 2 % Uranyl Acetatefor Contrast; RightAn Electron Micrograph of the Same Liposomal Preparation Prepared as a Frozen Vitrified Specimen for Cryo-TEME3143 18a23.1.19 liposomes, nmicrovesicles composed of a bilayer and/or a concentric series of multiple bilayers separated by aqueouscompartments fo
27、rmed by amphipathic molecules such as phospholipids that enclose a central aqueous compartment.Liposome Drug Products (1)43.1.20 liquid ethane, nliquefied ethane made by cooling gaseous ethane to the liquid state.3.1.21 liquid nitrogen, na cryogen, is the liquid state of nitrogen that boils at 196C
28、and is commonly used for extremecooling. Poses a freezing and suffocation hazard requiring caution when used.3.1.22 sample, na small volume of a preparation of liposomes suspended in an aqueous solution.3.1.23 specimen, na cryo-TEM grid onto which a sample has been applied and vitrified. The specime
29、n will be placed in acryo-TEM holder and imaged by cryo-TEM.3.1.24 structure, nthe 3D shape, arrangement, composition, and construction of any element that has physical x,y, and zdimensions.4 The boldface numbers in parentheses refer to a list of references at the end of this standard.FIG. 2 The Thr
30、ee Different Types of Carbon Film on EM Grids That May be Used to Conduct Cryo-TEM of LiposomesFIG. 3 (A) A Holey Carbon EM Grid with an Expanded View of Holey Carbon Film Shown to the Right; (B) Guillotine-Style Cryo-TEMPlunger Showing the Forceps Aligned with the Ethane Cup, and the Liquid Nitroge
31、n ReservoirE3143 18a33.1.25 transmission electron microscope, na microscope that employs an electron beam and a series of electro-magneticlenses to illuminate transmit through very thin samples and then image these samples to extremely high resolutions and highmagnifications.3.1.26 ultra-thin carbon
32、 grid, na holey carbon grid additionally coated with a very thin and fragile layer of carbon that issupported by the thicker more sturdy holey carbon film.3.1.27 vitrification, na process of vitrifying, that is, freezing a sample so rapidly that ice crystals do not have sufficient timeto form around
33、 the sample. The resulting specimen is said to be vitrified, essentially embedded in amorphous glass like ice.4. Summary of Practice4.1 In this practice, cryo-TEM of liposomes is conducted by applying a small volume (sample) of a preparation of liposomessuspended in an aqueous solution to a freshly
34、glow discharged holey, or continuous carbon TEM grid, or a freshly preparedultra-thin carbon EM grid, vitrifying the sample in liquid ethane and placing the resulting specimen into a cryo-TEM for imaging.Once recorded, images of individual liposomes in each electron micrograph can be analyzed using
35、image analysis software in orderto determine shape characteristics and size distributions. Analyzing many images will yield statistically valid values that can thenbe used to evaluate the product quality. Specific image analysis procedures are application dependent and are not addressed in thispract
36、ice.5. Significance and Use5.1 Cryo-TEM is a technique used to record high resolution images of samples that are frozen and embedded in a thin layerof vitrified, amorphous ice (2-5). Because vitrification occurs so rapidly, the resultant specimen is almost instantly frozen, yieldinga very accurate r
37、epresentation of the specimen at the moment of freezing, without the distortions typically associated with airdrying delicate wet samples. Once frozen, images of the specimen are recorded at low temperature using a specially designedelectron microscope equipped with a cryo-holder capable of operatin
38、g under low dose conditions in order to prevent beam inducedstructural damage to the specimen. The cryo-TEM technique is the consensus choice to directly observe, analyze and accuratelymeasure liposomes suspended in aqueous solutions. Fig. 1 illustrates this by comparing an electron micrograph from
39、an air-driednegatively stained liposomal preparation with an electron micrograph of the same solution imaged by cryo-TEM.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 w
40、hen 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 gridsuspended in cryo-TEM plunger over a cup of liquid ethane cooled in a container filled with liquid nitrogen (2, 3
41、). These gridscan be purchased or prepared in the laboratory using a carbon evaporator with glow discharge capabilities. Once the sample haswet the surface of the grid, and sufficient time allowed for the solution to equilibrate with regard to liposome spreading over thegrid surface, the excess is w
42、icked off (blotted) with filter paper and the grid plunged into the liquid ethane, vitrifying the sampleto form a specimen. Once frozen, the specimen is maintained at a liquid nitrogen temperature while it is imaged in a cryo-TEMoperating under low electron dose conditions. There are several limitat
43、ions associated with implementing this technique to analyzeliposomes:5.1.2.1 Thick IceThe vitrified ice thickness is often determined by the sample or the cryo-TEM procedure itself. Largeliposomes, defined to include larger structure and sizes with respect to this standard, are generally associated
44、with thicker ice, whilesmaller liposomes (structure and sizes) are associated with thinner ice. Generally, thick ice occurs when either excess water formsa thicker ice layer or samples containing larger liposomes are fully covered with water making the ice thicker around the specimen.Thicker ice ten
45、ds 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 difficultto image for two reasons. The first is the cryo-TEM procedure itself. This pro
46、cedure requires the use of filter paper to blot awayexcess aqueous solution from the EM grid just prior to vitrification. The larger liposomes suspended within the samplepreferentially wash away from the grid and into the filter paper, ending up in the filter paper. This is perhaps because the large
47、rliposomes provide a larger surface areas that expose them to relatively larger forces during the rapid flow of the water to the filterpaper. This makes them difficult to find and measure in electron micrographs when their relative concentration in the specimen islow-meaning that few are left behind
48、 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 betoo low in quality to provide adequate signal for image processing.5.1.2
49、.3 Liposomal DistortionBecause liposomes are essentially loose membrane bounded fluid compartments, freezing themwithin a layer of vitrified ice that is thinner than their diameter may cause the surface tension on both sides of the specimen tocompress some of the liposomes leading to various levels of flattening distortions. Accurate size measurements of such distortedliposomes would require volumetric measurements of all the liposomes within a field of view through a three-dimensional analysisusing electron tomography.E3143 18a46. Rea
