1、Designation: E3143 18aE3143 18bStandard 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-transmis
3、sionelectron 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 ap
5、propriate 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 Devel
6、opment of International Standards, Guides and Recommendations issuedby the World Trade Organization Technical Barriers to Trade (TBT) Committee.2. Referenced Documents2.1 ASTM Standards:2E1617E456 Practice for Reporting Particle Size Characterization DataTerminology Relating to Quality and Statistic
7、s2.2 ISO Standards:313322-1 Particle Size Analysis 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 outgas
8、sing within the column away from the frozen specimen 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 genera
9、lly by applying current throughtwo carbon rods pressed 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
10、surfaces.3.1.3 continuous carbon grid, na copper electron 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
11、with a pattern of square holes called a meshthrough 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
12、is the direct responsibility of Subcommittee E56.02 on Physical and ChemicalCharacterization.Current edition approved June 1, 2018Nov. 15, 2018. Published July 2018January 2019. Originally approved in 2018. Last previous edition approved in 2018 as E3143 18.18a. DOI: 10.1520/E1343-18A.10.1520/E1343-
13、18B.2 For referencedASTM standards, visit theASTM website, 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 Standardi
14、zation (ISO), ISO Central Secretariat, BIBC II, Chemin de 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.
15、 Becauseit may not be technically possible to adequately 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,
16、100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 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
17、 frozen hydrated,vitrified samplesnanomaterial using a cryo-transmission electron 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 conduc
18、ted.3.1.8 cryo-TEM plunger, na device used to vitrify a sample onto a holey, ultra-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.
19、 The simplest is the homemade guillotine type (see Fig. 3(B), 8.3).3.1.9 cryo-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 specime
20、n damage.3.1.10 electron micrograph, nthe individual image recorded by the electron 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
21、that are then used with image analysis software to reconstruct thestructure of 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
22、, na plasma generated by passing an electric current through a low-pressure gas environment within achamber, usually a bell jar and with respect to this standard,practice, is used to clean and statically charge the carbon surfacecoating of copper electron microscope grids in order to make them hydro
23、philic so that they will wet during sample application.3.1.15 glow discharge device, na 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 i
24、nto theliquid ethane for vitrification.3.1.17 holey carbon grid, na copper electron microscope grid specially prepared used for cryo-TEM consisting of a thinelectron semi-transparent carbon film containing small holes that is suspended over the larger mesh of square holes of the copperelectron micro
25、scope grid (see Fig. 3(A), 8.2.1).3.1.18 image analysis, nthe process of analyzing digital images 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 LeftA
26、n Electron Micrograph of an Air-Dried Liposomal Preparation that has been Negatively Stained with 2 % Uranyl Acetatefor Contrast; RightAn Electron Micrograph of the Same Liposomal Preparation Prepared as a Frozen Vitrified Specimen for Cryo-TEME3143 18b23.1.19 liposomes, nmicrovesicles composed of a
27、 bilayer and/or a concentric series of multiple bilayers separated by aqueouscompartments formed 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 s
28、tate.3.1.21 liquid nitrogen, na cryogen, is the liquid state of nitrogen that boils at 196C 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.3.1.2
29、3 specimen, na cryo-TEM grid onto which a sample has been applied and vitrified. The specimen 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 num
30、bers in parentheses refer to a list of references at the end of this standard.FIG. 2 The Three 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-S
31、tyle Cryo-TEMPlunger Showing the Forceps Aligned with the Ethane Cup, and the Liquid Nitrogen ReservoirE3143 18b33.1.25 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
32、 these samples to extremely high resolutions and highmagnifications.3.1.26 ultra-thin carbon 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
33、is, freezing a sample so rapidly that ice crystals do not have sufficient timeto form around the sample. The resulting specimensample is said to be vitrified, essentially embedded in amorphous glass like ice.4. Summary of Practice4.1 In this practice, cryo-TEM imaging of liposomes is conducted by ap
34、plying a small volume (sample) of a preparation ofliposomes suspended in an aqueous solution to a freshly glow discharged TEM grid (for example, holey, or continuous carbonTEM grid, or a freshly prepared ultra-thin carbon EM grid, grid), then vitrifying the sample in liquid ethane and placing theres
35、ulting specimen into a cryo-TEM for imaging. Once recorded, images of individual liposomes in each electron micrograph canbe analyzed using image analysis software in order to determine shape characteristics and size distributions. Analyzing manyimages will yield statistically valid values that can
36、then be used to evaluate the product quality. Specific image analysis proceduresare application dependent and are not addressed in this practice.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 vitrifie
37、d, amorphous ice (2-5). Because vitrification occurs so rapidly, the resultant specimen is almost instantly frozen, yieldinga very accurate representation of the specimen at the moment of freezing, without the distortions typically associated with airdrying delicate wet samples. Once frozen, images
38、of the specimen are recorded at low temperature using a specially designedelectron microscope equipped with a cryo-holder capable of operating under low dose conditions in order to prevent beam inducedstructural damage to the specimen. The cryo-TEM technique is the consensus choice to directly obser
39、ve, analyze and accuratelymeasure liposomes suspended in aqueous solutions. Fig. 1 illustrates this by comparing an electron micrograph from 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 liposom
40、es may become distorted and are difficult to measure and analyze when they are air-dried,while the same sample liposomal preparation is clearly easier to analyze when the specimen is near-instantly preserved byvitrification.5.1.2 Cryo-TEM involves applying a small volume of sample to a specially pre
41、pared holey, ultra-thin or continuous carbon gridsuspended in a cryo-TEM plunger over a cup of liquid ethane cooled in a container filled with liquid nitrogen (2, 3). These gridscan be purchased or prepared in the laboratory using a carbon evaporator with glow discharge capabilities. Once the sample
42、 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 wicked off (blotted) with filter paper and the grid plunged into the liquid ethane, vitrifying the sampleto form a specimen. sample. Once
43、 frozen, the specimensample is maintained at a liquid nitrogen temperature while it is imagedin a cryo-TEM operating under low electron dose conditions. There are several limitations associated with implementing thistechnique to analyze liposomes:5.1.2.1 Thick IceThe vitrified ice thickness is often
44、 determined by the sample or the cryo-TEM procedure itself. Largeliposomes, defined to include larger structure and sizes with respect to this standard,practice, are generally associated with thickerice, while smaller liposomes (structure and sizes) are associated with thinner ice. Generally, thick
45、ice occurs when either excesswater forms a thicker ice layer or samples containing larger liposomes are fully covered with water making the ice thicker aroundthe specimen.sample.Thicker ice tends to block the electron beam either completely or partially which compromises image quality.5.1.2.2 Larger
46、 liposomes (structure and sizes) are preferentially lost byduring sample preparation. Larger liposomes liposomes,defined to include larger structures and sizes with respect to this practice, are more difficult to image for two reasons. The first isthe cryo-TEM procedure itself. This procedure requir
47、es the use of filter paper to blot away excess aqueous solution from the EMgrid just prior to vitrification. The larger liposomes suspended within the sample preferentially wash away from the grid and intothe filter paper, ending up in the filter paper. This is perhaps because the larger liposomes p
48、rovide a have larger surface areas thatexpose them to relatively larger forces during the rapid flow of the water to the filter paper. This makes them difficult to find andmeasure in electron micrographs when their relative concentration in the specimen is low-meaning low, meaning that few are leftb
49、ehind after blotting. The second reason is that larger liposomes that are left behind on the EM grid, are often embedded in thickerice that is too thick for the electron beam to either penetrate or, if it does, causes the images to beresults in images that are toolow in quality to provide adequate signal for image processing.5.1.2.3 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
