ASTM E1881-12(2020)

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.
Designation: E1881 − 12 (Reapproved 2020)
Standard Guide for
Cell Culture Analysis with SIMS
This standard is issued under the fixed designation E1881; 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 4. Summary of Guide
1.1 This guide provides the Secondary Ion Mass Spectrom-
4.1 This guide describes a cryogenic freeze-fracture method
etry (SIMS) analyst with a cryogenic method for analyzing
of sample preparation for cell culture specimens for SIMS
individual tissue culture cells growing in vitro. This guide is
analysis. In brief, cell cultures are grown on a conducting
suitable for frozen-hydrated and frozen-freeze-dried sample
substrate, such as silicon. When cells reach about 80 %
types. Included are procedures for correlating optical, laser
confluency, they are fast frozen and fractured by using a
scanning confocal and secondary electron microscopies to
sandwich method (1). This allows freeze-fixation of cellular
complement SIMS analysis.
contents and removal of the EF-leaflet of the apical plasma
membrane. Since this kind of fracture occurs in groups of cells
1.2 This guide is not suitable for cell cultures that do not
attach to the substrate. growing together, fractured cells are easily recognized for
optical, SEM and SIMS imaging.
1.3 This guide is not suitable for any plastic embedded cell
culture specimens.
4.2 By correlative laser scanning confocal microscopy and
SIMS, the same frozen freeze-dried cell can be analyzed for
1.4 This standard does not purport to address all of the
safety concerns, if any, associated with its use. It is the organelle localization in relation to elemental content (2).
responsibility of the user of this standard to establish appro-
priate safety, health, and environmental practices and deter- 5. Significance and Use
mine the applicability of regulatory limitations prior to use.
5.1 The presence of cell growth medium complicates a
1.5 This international standard was developed in accor-
direct analysis of cells with SIMS. Attempts to wash out the
dance with internationally recognized principles on standard-
nutrient medium results in the exposure of cells to unphysi-
ization established in the Decision on Principles for the
ological reagents that may also alter their chemical composi-
Development of International Standards, Guides and Recom-
tion. This obstacle is overcome by using a sandwich freeze-
mendations issued by the World Trade Organization Technical
fracture method (1). This cryogenic method has provided a
Barriers to Trade (TBT) Committee.
uniquewayofsamplingindividualcellsintheirnativestatefor
2. Referenced Documents
SIMS analysis.
2.1 ASTM Standards:
5.2 The procedure described here has been successfully
+ +
E673 Terminology Relating to SurfaceAnalysis (Withdrawn
used for imaging Na and K ion transport (3), calcium
2012)
alterations in stimulated cells (4,5), and localization of thera-
peutic drugs and isotopically labeled molecules in single cells
3. Terminology
(6). The frozen freeze-dried cells prepared according to this
3.1 Definitions:
method have been checked for SIMS matrix effects (7). Ion
3.1.1 SeeTerminology E673 for definitions of terms used in
imagequantificationhasalsobeenachievedinthissampletype
SIMS.
(8).
5.3 The procedure described here is amenable to a wide
This guide is under the jurisdiction of ASTM Committee E42 on Surface
variety of cell cultures and provides a way for studying the
Analysis and is the direct responsibility of Subcommittee E42.06 on SIMS.
Current edition approved Dec. 1, 2020. Published December 2020. Originally
response of individual cells for chemical alterations in the state
approved in 1997. Last previous edition approved in 2012 as E1881 – 12. DOI:
of health and disease and localization of isotopically-labeled
10.1520/E1881-12R20.
molecules and theraputic drugs in cell culture models.
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.
The last approved version of this historical standard is referenced on The boldface numbers in parentheses refer to a list of references at the end of
www.astm.org. this guide.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
E1881 − 12 (2020)
6. Apparatus or dorsal cells surfaces, and randomly scattered individual
cross fractured cells where the fracture plane has passed
6.1 This guide can be used for the analysis of cell cultures
through the cytoplasm and/or nucleus (10). In a group of cells
with virtually any SIMS instrument.
fractured at the dorsal cell surface the apical plasma membrane
6.2 A cold stage in the SIMS instrument is needed to
fracture removes the extracellular nutrient medium and the
analyze frozen-hydrated specimens (9).
EF-leaflet of the plasma membrane on the top silicon piece (1,
10).Thefracturedcellsonthesiliconsubstratecanbeanalyzed
7. Procedure
frozen-hydrated or after freeze-drying with SIMS imaging
7.1 Cells are grown on silicon wafer pieces (approximately
techniques.
1cm area) of any shape.Alternatively, high purity germanium
7.1.2 Depending on the need of a particular SIMS analysis,
wafer pieces are used for cell growth for studies involving the
the freeze-dried cells may be analyzed directly or gold coated
use of Ca stable isotope. These substrates are nontoxic to
to enhance electrical conductivity.
cells and have been used for growing various cell lines (1,2,8).
7.1.3 For correlative optical, SEM and SIMS, fractured
Sterilize the silicon or germanium pieces prior to cell seeding.
freeze-dried cells can be imaged with a reflected light micro-
After the cells reach about 80 % confluency, replace the
scope or SEM prior to SIMS analysis (11).
nutrient growth medium with new medium containing 11 µm
7.1.4 For organelle localization in relation to SIMS isotope
polystyrene beads (approximately 50 000 beads per 100 mm
images, a correlative laser scanning confocal microscopy and
plastic dish, see Ref (1) for details on size of the beads). These
SIMS approach has been developed (2). This approach relies
beads act as spacers during the sandwich-fracture technique. It
on labeling the organelles with specific fluorescent markers in
takes approximately 30 min for the beads to settle down on the
live cells and then mapping the organelle localization in 3-D
substrate.After beads settle down on the substrate the cells can
with a laser scanning confocal microscope in a fractured
...