1、Designation: E3143 18Standard 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 last revi
2、sion. 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 re-cording images of a suspension of liposomes with a cryo-transmission elec
3、tron microscope (cryo-TEM) for the purposeof evaluating their shape, size distribution and lamellarity forquality assessment. The sample is vitrified in liquid ethane ontospecially prepared holey, ultra-thin, or continuous carbon TEMgrids, and imaged in a cryo-holder placed in a cryo-TEM.1.2 The val
4、ues stated in SI units are to be regarded asstandard. No other units of measurement are included in thisstandard.1.3 This standard does not purport to address all of thesafety concerns, if any, associated with its use. It is theresponsibility of the user of this standard to establish appro-priate sa
5、fety, health, and environmental practices and deter-mine the applicability of regulatory limitations prior to use.1.4 This international standard was developed in accor-dance with internationally recognized principles on standard-ization established in the Decision on Principles for theDevelopment o
6、f International Standards, Guides and Recom-mendations issued by the World Trade Organization TechnicalBarriers to Trade (TBT) Committee.2. Referenced Documents2.1 ASTM Standards:2E1617 Practice for Reporting Particle Size CharacterizationData2.2 ISO Standards:313322-1 Particle Size Analysis Image A
7、nalysis Methods Static Image Analysis Methods3. Terminology3.1 Definitions:3.1.1 anti-contaminator, na specially designed devicebuilt into the column of a cryo-transmission electron micro-scope designed to pull contamination produced by outgassingwithin the column away from the frozen specimen durin
8、gimaging. The device is cooled with liquid nitrogen to atemperature below that of the specimen creating a coldersurface for contamination to build on.3.1.2 carbon evaporator, na device used to evaporatecarbon in a high vacuum chamber generally by applyingcurrent through two carbon rods pressed again
9、st one another,with one rod being sharpened in order to provide resistance tothe current; this causes the rods to heat up and evaporatecarbon. The same device can also be used as a glow dischargedevice (3.1.15) in order to glow discharge surfaces.3.1.3 continuous carbon grid, na copper electron micr
10、o-scope grid coated with a self-supporting layer of continuouscarbon over the square mesh of the grid.3.1.4 copper electron microscope grid, ncommonly re-ferred to as an “EM grid,” a thin 3-mm diameter copper foildisk, usually manufactured with a pattern of square holescalled a mesh through which im
11、aging is conducted in anelectron microscope. The number, pattern, and shape of theholes can vary depending on imaging conditions and samplerequirements.3.1.5 cryo-grid storage device, nany device used to storecryo-EM grids indefinitely in a liquid nitrogen dewar.3.1.6 cryo-TEM, nalso referred to sim
12、ply as cryo-EM, orcryo electron microscopy, the process of imaging frozenvitrified samples using a cryo-transmission electron micro-scope.3.1.7 cryo-TEM holder, na liquid nitrogen refrigerateddevice specifically designed to hold and maintain a preparedgrid containing a frozen, hydrated, vitrified sp
13、ecimen in acryo-TEM while imaging is conducted.3.1.8 cryo-TEM plunger, na device used to vitrify asample onto a holey, ultra-thin, or continuous carbon grid forcryo-TEM by plunging the grid containing a thin layer ofaqueous sample into an ethane slush or other cryogen(s). There1This practice is unde
14、r the jurisdiction of ASTM Committee E56 on Nanotech-nology and is the direct responsibility of Subcommittee E56.02 on Physical andChemical Characterization.Current edition approved Feb. 1, 2018. Published February 2018. DOI: 10.1520/E1343-18.2For referenced ASTM standards, visit the ASTM website, w
15、ww.astm.org, orcontact ASTM Customer Service at serviceastm.org. For Annual Book of ASTMStandards volume information, refer to the standards Document Summary page onthe ASTM website.3Available from International Organization for Standardization (ISO), ISOCentral Secretariat, BIBC II, Chemin de Bland
16、onnet 8, CP 401, 1214 Vernier,Geneva, Switzerland, http:/www.iso.org.Copyright ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United StatesThis international standard was developed in accordance with internationally recognized principles on standardization
17、established in the Decision on Principles for theDevelopment of International Standards, Guides and Recommendations issued by the World Trade Organization Technical Barriers to Trade (TBT) Committee.1are many types of plunger designs both homemade andcommercial. The simplest is the homemade guilloti
18、ne type (seeFig. 3(B), 8.3).3.1.9 cryo-transmission electron microscope, na speciallydesigned transmission electron microscope (see 3.1.25) ca-pable of imaging frozen vitrified specimens under low electrondose conditions in order to prevent beam-induced specimendamage.3.1.10 electron micrograph, nth
19、e individual image re-corded by the electron microscope as data are collected.3.1.11 electron tomography, nthe process of using imagesderived from an electron microscope in which the images wererecorded of a sample through an incremental series of tiltangles that are then used with image analysis so
20、ftware toreconstruct the structure 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 orpincer used to pick up electron microscope grids.3.1.14 glow discharge, na plasma generated by passing anelectric
21、 current through a low-pressure gas environment withina chamber, usually a bell jar and with respect to this standard,is used to clean and statically charge the carbon surface coatingof copper electron microscope grids in order to make themhydrophilic so that they will wet during sample application.
22、3.1.15 glow discharge device, na device, such as a carbonevaporator (see 3.1.2), designed to generate a glow dischargeplasma.3.1.16 hang time, vthe amount of time after blotting of thesample from the prepared grid, prior to plunging the grid intothe liquid ethane for vitrification.3.1.17 holey carbo
23、n grid, na copper electron microscopegrid specially prepared for cryo-TEM consisting of a thinelectron semi-transparent carbon film containing small holesthat is suspended over the larger mesh of square holes of thecopper electron microscope grid (see Fig. 3(A), 8.2.1).3.1.18 image analysis, nthe pr
24、ocess of analyzing digitalimages with computer software for the purpose of extractingmeaningful information from the data, for example, a sizedistribution.3.1.19 liposomes, nmicrovesicles composed of a bilayerand/or a concentric series of multiple bilayers separated byaqueous compartments formed by
25、amphipathic molecules suchas phospholipids that enclose a central aqueous compartment.Liposome Drug Products (1)43.1.20 liquid ethane, nliquefied ethane made by coolinggaseous ethane to the liquid state.3.1.21 liquid nitrogen, na cryogen, is the liquid state ofnitrogen that boils at 196C and is comm
26、only used forextreme cooling. Poses a freezing and suffocation hazardrequiring caution when used.3.1.22 sample, na small volume of a preparation ofliposomes suspended in an aqueous solution.3.1.23 specimen, na cryo-TEM grid onto which a samplehas been applied and vitrified. The specimen will be plac
27、ed ina cryo-TEM holder and imaged by cryo-TEM.3.1.24 structure, nthe 3D shape, arrangement,composition, and construction of any element that has physicalx, y, and z dimensions.3.1.25 transmission electron microscope, na microscopethat employs an electron beam and a series of electro-magneticlenses t
28、o illuminate transmit through very thin samples andthen image these samples to extremely high resolutions andhigh magnifications.4The boldface numbers in parentheses refer to a list of references at the end ofthis standard.NOTE 1Both images are shown to the same scale; scale bar is 200 nm.FIG. 1 Lef
29、tAn 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 1823.1.26 ultra-thin carbon grid, na holey carbo
30、n grid addi-tionally coated with a very thin and fragile layer of carbon thatis supported by the thicker more sturdy holey carbon film.3.1.27 vitrification, na process of vitrifying, that is, freez-ing a sample so rapidly that ice crystals do not have sufficienttime to form around the sample. The re
31、sulting specimen is saidto be vitrified, essentially embedded in amorphous glass likeice.4. Summary of Practice4.1 In this practice, cryo-TEM of liposomes is conducted byapplying a small volume (sample) of a preparation of lipo-somes suspended in an aqueous solution to a freshly glowdischarged holey
32、, or continuous carbon TEM grid, or a freshlyprepared ultra-thin carbon EM grid, vitrifying the sample inliquid ethane and placing the resulting specimen into a cryo-TEM for imaging. Once recorded, images of individual lipo-somes in each electron micrograph can be analyzed usingimage analysis softwa
33、re in order to determine shape character-istics and size distributions. Analyzing many images will yieldstatistically valid values that can then be used to evaluate theproduct quality. Specific image analysis procedures are appli-cation dependent and are not addressed in this practice.5. Significanc
34、e and Use5.1 Cryo-TEM is a technique used to record high resolutionimages of samples that are frozen and embedded in a thin layerof vitrified, amorphous ice (2-5). Because vitrification occursso rapidly, the resultant specimen is almost instantly frozen,yielding a very accurate representation of the
35、 specimen at theFIG. 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-Style Cryo-TEMPlunger Showing the Forceps Aligned with the Etha
36、ne Cup, and the Liquid Nitrogen ReservoirE3143 183moment of freezing, without the distortions typically associ-ated with air drying delicate wet samples. Once frozen, imagesof the specimen are recorded at low temperature using aspecially designed electron microscope equipped with a cryo-holder capab
37、le of operating under low dose conditions in orderto prevent beam induced structural damage to the specimen.The cryo-TEM technique is the consensus choice to directlyobserve, analyze and accurately measure liposomes suspendedin aqueous solutions. Fig. 1 illustrates this by comparing anelectron micro
38、graph from an air-dried negatively stained lipo-somal preparation with an electron micrograph of the samesolution imaged by cryo-TEM.5.1.1 Fig. 1 demonstrates that liposomes may become dis-torted and are difficult to measure and analyze when they areair-dried, while the same sample is clearly easier
39、 to analyzewhen the specimen is near-instantly preserved by vitrification.5.1.2 Cryo-TEM involves applying a small volume ofsample to a specially prepared holey, ultra-thin or continuouscarbon grid suspended in cryo-TEM plunger over a cup ofliquid ethane cooled in a container filled with liquid nitr
40、ogen(2, 3). These grids can be purchased or prepared in thelaboratory using a carbon evaporator with glow dischargecapabilities. Once the sample has wet the surface of the grid,and sufficient time allowed for the solution to equilibrate withregard to liposome spreading over the grid surface, the exc
41、essis wicked off (blotted) with filter paper and the grid plungedinto the liquid ethane, vitrifying the sample to form a specimen.Once frozen, the specimen is maintained at a liquid nitrogentemperature while it is imaged in a cryo-TEM operating underlow electron dose conditions. There are several li
42、mitationsassociated with implementing this technique to analyze lipo-somes:5.1.2.1 Thick IceThe vitrified ice thickness is often deter-mined by the sample or the cryo-TEM procedure itself. Largeliposomes, defined to include larger structure and sizes withrespect to this standard, are generally assoc
43、iated with thickerice, while smaller liposomes (structure and sizes) are associ-ated with thinner ice. Generally, thick ice occurs when eitherexcess water forms a thicker ice layer or samples containinglarger liposomes are fully covered with water making the icethicker around the specimen. Thicker i
44、ce tends to block theelectron beam either completely or partially which compro-mises image quality.5.1.2.2 Larger liposomes (structure and sizes) are preferen-tially lost by sample preparation. Larger liposomes are moredifficult to image for two reasons. The first is the cryo-TEMprocedure itself. Th
45、is procedure requires the use of filter paperto blot away excess aqueous solution from the EM grid justprior to vitrification. The larger liposomes suspended withinthe sample preferentially wash away from the grid and into thefilter paper, ending up in the filter paper. This is perhapsbecause the la
46、rger liposomes provide a larger surface areas thatexpose them to relatively larger forces during the rapid flow ofthe water to the filter paper. This makes them difficult to findand measure in electron micrographs when their relativeconcentration in the specimen is low-meaning that few are leftbehin
47、d after blotting. The second reason is that larger lipo-somes that are left behind on the EM grid, are often embeddedin thicker ice that is too thick for the electron beam to eitherpenetrate or if it does, causes the images to be too low inquality to provide adequate signal for image processing.5.1.
48、2.3 Liposomal DistortionBecause liposomes are es-sentially loose membrane bounded fluid compartments, freez-ing them within a layer of vitrified ice that is thinner than theirdiameter may cause the surface tension on both sides of thespecimen to compress some of the liposomes leading to variouslevel
49、s of flattening distortions. Accurate size measurements ofsuch distorted liposomes would require volumetric measure-ments of all the liposomes within a field of view through athree-dimensional analysis using electron tomography.6. Reagents and Equipment6.1 Purified preparation of liposomes suspended in anaqueous solution.6.2 Cryo-TEM plunger, commercial or homemade.6.3 Ethane gas, research or higher purity grade.6.4 2 to 20-L pipette.6.5 Pipette tips.6.6 Four brass or copper rods, approximately 5 mm (diam-eter) 12 cm.6.7 Liquid nitrogen.6
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