ASTM E3143-18b
(Practice)Standard Practice for Performing Cryo-Transmission Electron Microscopy of Liposomes
Standard Practice for Performing Cryo-Transmission Electron Microscopy of Liposomes
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 o...
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.
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Designation: E3143 − 18b
Standard Practice for
Performing Cryo-Transmission Electron Microscopy of
1
Liposomes
This standard is issued under the fixed designation E3143; the number immediately following the designation indicates the year of
original adoption or, in the case of revision, the year of last revision. A number in parentheses indicates the year of last reapproval. A
superscript epsilon (´) indicates an editorial change since the last revision or reapproval.
1. Scope 3. Terminology
1.1 This practice covers procedures for vitrifying and re- 3.1 Definitions:
cording images of a suspension of liposomes with a cryo-
3.1.1 anti-contaminator, n—a specially designed device
transmission electron microscope (cryo-TEM) for the purpose
built into the column of a cryo-transmission electron micro-
of evaluating their shape, size distribution and lamellarity for
scope designed to pull contamination produced by outgassing
quality assessment. The sample is vitrified in liquid ethane onto
within the column away from the frozen specimen during
specially prepared holey, ultra-thin, or continuous carbon TEM
imaging. The device is cooled with liquid nitrogen to a
grids, and imaged in a cryo-holder placed in a cryo-TEM.
temperature below that of the specimen creating a colder
surface for contamination to build on.
1.2 The values stated in SI units are to be regarded as
standard. No other units of measurement are included in this
3.1.2 carbon evaporator, n—a device used to evaporate
standard.
carbon in a high vacuum chamber generally by applying
current through two carbon rods pressed against one another,
1.3 This standard does not purport to address all of the
with one rod being sharpened in order to provide resistance to
safety concerns, if any, associated with its use. It is the
the current; this causes the rods to heat up and evaporate
responsibility of the user of this standard to establish appro-
carbon. The same device can also be used as a glow discharge
priate safety, health, and environmental practices and deter-
device (3.1.15) in order to glow discharge surfaces.
mine the applicability of regulatory limitations prior to use.
1.4 This international standard was developed in accor-
3.1.3 continuous carbon grid, n—a copper electron micro-
dance with internationally recognized principles on standard-
scope grid coated with a self-supporting layer of continuous
ization established in the Decision on Principles for the
carbon over the square mesh of the grid.
Development of International Standards, Guides and Recom-
3.1.4 copper electron microscope grid, n—commonly re-
mendations issued by the World Trade Organization Technical
ferred to as an “EM grid,” a thin 3-mm diameter copper foil
Barriers to Trade (TBT) Committee.
disk, usually manufactured with a pattern of square holes
called a ‘mesh’ through which imaging is conducted in an
2. Referenced Documents
electron microscope. The number, pattern, and shape of the
2
2.1 ASTM Standards:
holes can vary depending on imaging conditions and sample
E456 Terminology Relating to Quality and Statistics
requirements.
3
2.2 ISO Standards:
3.1.5 cryo-grid storage device, n—any device used to store
13322-1 Particle Size Analysis – Image Analysis Methods –
cryo-EM grids indefinitely in a liquid nitrogen dewar.
Static Image Analysis Methods
3.1.6 cryo-TEM, n—also referred to simply as cryo-EM, or
cryo electron microscopy, the process of imaging frozen
hydrated, vitrified nanomaterial using a cryo-transmission
1
This practice is under the jurisdiction of ASTM Committee E56 on Nanotech-
electron microscope.
nology and is the direct responsibility of Subcommittee E56.02 on Physical and
Chemical Characterization.
3.1.7 cryo-TEM holder, n—a liquid nitrogen refrigerated
Current edition approved Nov. 15, 2018. Published January 2019. Originally
device specifically designed to hold and maintain a prepared
approved in 2018. Last previous edition approved in 2018 as E3143 – 18a. DOI:
10.1520/E1343-18B.
grid containing a frozen, hydrated, vitrified specimen in a
2
For referenced ASTM standards, visit the ASTM website, www.astm.org, or
cryo-TEM while imaging is conducted.
contact ASTM Customer Service at service@astm.org. For Annual Book of ASTM
Standards volume information, refer to the standard’s Document Summary page on
3.1.8 cryo-TEM plunger, n—a device used to vitrify a
the ASTM website.
sample onto a holey, ultra-thin, or continuous carbon grid for
3
Available from International Organization for Standardization (ISO), ISO
cryo-TEM by plunging the grid containing a thin layer of
Central Secretariat, BIBC II, Chemin de Blandonnet 8, CP 401, 1214 Vernier,
Geneva, Switzerland, http://www.iso.org. aqueous sample into an ethane slush or other cryogen(s). There
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, We
...
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. Because
it 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 version
of the standard as published by ASTM is to be considered the official document.
Designation: E3143 − 18a E3143 − 18b
Standard Practice for
Performing Cryo-Transmission Electron Microscopy of
1
Liposomes
This standard is issued under the fixed designation E3143; the number immediately following the designation indicates the year of
original adoption or, in the case of revision, the year of last revision. A number in parentheses indicates the year of last reapproval. A
superscript epsilon (´) indicates an editorial change since the last revision or reapproval.
1. 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.
2. Referenced Documents
2
2.1 ASTM Standards:
E1617E456 Practice for Reporting Particle Size Characterization DataTerminology Relating to Quality and Statistics
3
2.2 ISO Standards:
13322-1 Particle Size Analysis – Image Analysis Methods – Static Image Analysis Methods
3. Terminology
3.1 Definitions:
3.1.1 anti-contaminator, n—a specially designed device built into the column of a cryo-transmission electron microscope
designed to pull contamination produced by outgassing within the column away from the frozen specimen during imaging. The
device is cooled with liquid nitrogen to a temperature below that of the specimen creating a colder surface for contamination to
build on.
3.1.2 carbon evaporator, n—a device used to evaporate carbon in a high vacuum chamber generally by applying current through
two carbon rods pressed against one another, with one rod being sharpened in order to provide resistance to the current; this causes
the 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 glow
discharge surfaces.
3.1.3 continuous carbon grid, n—a copper electron microscope grid coated with a self-supporting layer of continuous carbon
over the square mesh of the grid.
3.1.4 copper electron microscope grid, n—commonly referred to as an “EM grid,” a thin 3-mm diameter copper foil disk,
usually manufactured with a pattern of square holes called a ‘mesh’ through 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 on Physical and Chemical
Characterization.
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/E1343-18B.
2
For referenced ASTM standards, visit the ASTM website, www.astm.org, or contact ASTM Customer Service at service@astm.org. For Annual Book of ASTM Standards
volume information, refer to the standard’s Document Summary page on the ASTM website.
3
Available from International Organization for Standardization (ISO), ISO Central Secretariat, BIBC II, Chemin de Blandonnet 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 States
1
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E3143 − 18b
3.1.5 cryo-grid storage device, n—any device used to store cryo-EM grids indefinitely in a liquid nitrogen dewar.
3.1.6 cryo-TEM, n—also referred to simply as cryo-EM, or cryo electron microscopy, the process of imaging frozen hydrated,
vitrified samplesnanomaterial us
...
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