1、Designation: E1881 12Standard Guide forCell Culture Analysis with SIMS1This standard is issued under the fixed designation E1881; the number immediately following the designation indicates the year oforiginal adoption or, in the case of revision, the year of last revision. A number in parentheses in
2、dicates the year of last reapproval. Asuperscript epsilon () indicates an editorial change since the last revision or reapproval.1. Scope1.1 This guide provides the Secondary Ion Mass Spectrom-etry (SIMS) analyst with a cryogenic method for analyzingindividual tissue culture cells growing in vitro.
3、This guide issuitable for frozen-hydrated and frozen-freeze-dried sampletypes. Included are procedures for correlating optical, laserscanning confocal and secondary electron microscopies tocomplement SIMS analysis.1.2 This guide is not suitable for cell cultures that do notattach to the substrate.1.
4、3 This guide is not suitable for any plastic embedded cellculture specimens.1.4 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 safety and health practices and determi
5、ne the applica-bility of regulatory limitations prior to use.2. Referenced Documents2.1 ASTM Standards:2E673 Terminology Relating to Surface Analysis (Withdrawn2012)33. Terminology3.1 Definitions:3.1.1 See Terminology E673 for definitions of terms used inSIMS.4. Summary of Guide4.1 This guide descri
6、bes a cryogenic freeze-fracture methodof sample preparation for cell culture specimens for SIMSanalysis. In brief, cell cultures are grown on a conductingsubstrate, such as silicon. When cells reach about 80 %confluency, they are fast frozen and fractured by using asandwich method (1).4This allows f
7、reeze-fixation of cellularcontents and removal of the EF-leaflet of the apical plasmamembrane. Since this kind of fracture occurs in groups of cellsgrowing together, fractured cells are easily recognized foroptical, SEM and SIMS imaging.4.2 By correlative laser scanning confocal microscopy andSIMS,
8、the same frozen freeze-dried cell can be analyzed fororganelle localization in relation to elemental content (2).5. Significance and Use5.1 The presence of cell growth medium complicates adirect analysis of cells with SIMS. Attempts to wash out thenutrient medium results in the exposure of cells to
9、unphysi-ological reagents that may also alter their chemical composi-tion. This obstacle is overcome by using a sandwich freeze-fracture method (1). This cryogenic method has provided aunique way of sampling individual cells in their native state forSIMS analysis.5.2 The procedure described here has
10、 been successfullyused for imaging Na+and K+ion transport (3), calciumalterations in stimulated cells (4,5), and localization of thera-peutic drugs and isotopically labeled molecules in single cells(6). The frozen freeze-dried cells prepared according to thismethod have been checked for SIMS matrix
11、effects (7). Ionimage quantification has also been achieved in this sample type(8).5.3 The procedure described here is amenable to a widevariety of cell cultures and provides a way for studying theresponse of individual cells for chemical alterations in the stateof health and disease and localizatio
12、n of isotopically-labeledmolecules and theraputic drugs in cell culture models.6. Apparatus6.1 This guide can be used for the analysis of cell cultureswith virtually any SIMS instrument.6.2 A cold stage in the SIMS instrument is needed toanalyze frozen-hydrated specimens (9).1This guide is under the
13、 jurisdiction of ASTM Committee E42 on SurfaceAnalysis and is the direct responsibility of Subcommittee E42.06 on SIMS.Current edition approved Nov. 1, 2012. Published December 2012. Originallyapproved in 1997. Last previous edition approved in 2006 as E1881 06. DOI:10.1520/E1881-12.2For referenced
14、ASTM standards, visit the ASTM website, www.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.3The last approved version of this historical standard is referenced onwww.as
15、tm.org.4The boldface numbers in parentheses refer to a list of references at the end ofthis guide.Copyright ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States17. Procedure7.1 Cells are grown on silicon wafer pieces (approximately1cm2area) of any s
16、hape.Alternatively, high purity germaniumwafer pieces are used for cell growth for studies involving theuse of44Ca stable isotope. These substrates are nontoxic tocells and have been used for growing various cell lines (1,2,8).Sterilize the silicon or germanium pieces prior to cell seeding.After the
17、 cells reach about 80 % confluency, replace thenutrient growth medium with new medium containing 11 mpolystyrene beads (approximately 50 000 beads per 100 mmplastic dish, see Ref (1) for details on size of the beads). Thesebeads act as spacers during the sandwich-fracture technique. Ittakes approxim
18、ately 30 min for the beads to settle down on thesubstrate. After beads settle down on the substrate the cells canbe subjected to desired treatments and cryogenic sampling.7.1.1 After the desired treatments fast freeze and freeze-fracture the cells by a sandwich technique which involves thefollowing
19、steps: (1) remove the silicon piece containing thecells from the nutrient medium, (2) remove excess nutrientmedium from the cells by touching one edge of the siliconpiece with filter paper, (3) place a new and clean silicon waferpiece on top, sandwiching the cells between two polishedsurfaces, (4) f
20、ast freeze the sandwich in cryogenic fluids(supercooled isopentane, propane, liquid nitrogen, and soforth), (5) transfer the sandwich quickly to liquid nitrogen, and(6) fracture the sandwich by prying apart the two halves underliquid nitrogen. At this stage the silicon piece used for growingthe cell
21、s contains a group of cells fractured together at the basalor dorsal cells surfaces, and randomly scattered individualcross fractured cells where the fracture plane has passedthrough the cytoplasm and/or nucleus (10). In a group of cellsfractured at the dorsal cell surface the apical plasma membrane
22、fracture removes the extracellular nutrient medium and theEF-leaflet of the plasma membrane on the top silicon piece (1,10). The fractured cells on the silicon substrate can be analyzedfrozen-hydrated or after freeze-drying with SIMS imagingtechniques.7.1.2 Depending on the need of a particular SIMS
23、 analysis,the freeze-dried cells may be analyzed directly or gold coatedto enhance electrical conductivity.7.1.3 For correlative optical, SEM and SIMS, fracturedfreeze-dried cells can be imaged with a reflected light micro-scope or SEM prior to SIMS analysis (11).7.1.4 For organelle localization in
24、relation to SIMS isotopeimages, a correlative laser scanning confocal microscopy andSIMS approach has been developed (2). This approach relieson labeling the organelles with specific fluorescent markers inlive cells and then mapping the organelle localization in 3-Dwith a laser scanning confocal mic
25、roscope in a fracturedfreeze-dried cell prior to SIMS analysis of the same cell(2,4,5).7.1.5 This sandwich freeze-fracture method has been suc-cessfully used for dynamic SIMS studies of quantitativesubcellular localization of anticancer agents in human cancercell lines (12, 13), and 3-D quantitative
26、 imaging of subcellularcalcium stores in cells undergoing cell division (14).7.1.6 This sandwich freeze-fracture method has found us-ages in static Time-of-flight SIMS and Laser-SNMS techniquesfor molecular and atomic localization studies in mammaliancells and single cell organisms (15-17).8. Keywor
27、ds8.1 SIMSREFERENCES(1) Chandra, S., Morrison, G. H., and Wolcott C. C., “Imaging Intracel-lular Elemental Distribution and Ion Fluxes in Cultured Cells UsingIon Microscopy: Freeze-fracture Methodology,” Journal of Micros-copy (Oxford), Vol 144, 1986, p. 15.(2) Chandra, S., Kable, E. P. W., Morrison
28、, G. H., and Webb W. W.,“Calcium Sequestration in the Golgi Apparatus of Cultured Mamma-lian Cells Revealed by Laser Scanning Confocal Microscopy and IonMicroscopy,” Journal of Cell Science , Vol 100, 1991, p. 747.(3) Chandra, S., and Morrison, G. H., “Imaging Elemental Distributionand Ion Transport
29、 in Cultured Cells with Ion Microscopy,” Science,Vol 228, 1985, p. 1543.(4) Chandra, S., Fewtrell, C., Millard, P. J., Sandison, D. R., Webb, W. W.,and Morrison, G. H., “Imaging of Total Intracellular Calcium andCalcium Influx and Efflux in Individual Resting and StimulatedTumor Mast Cells Using Ion
30、 Microscopy,” Journal of BiologicalChemistry, Vol 269, 1994, p. 15186.(5) Zha, X., Chandra, S., Ridsdale, A., and Morrison, G. H.,“ GolgiApparatus is Involved in Intracellular Ca2+Regulation in RenalEpithelial LLC-PK1Cells,” American Journal of Physiology (CellPhysiology 38), Vol 269, 1995, p. C1133
31、.(6) Chandra, S., and Morrison, G. H., “Imaging Ion and MolecularTransport at Subcellular Resolution by Secondary Ion MassSpectrometry,” International Journal of Mass Spectrometry and IonProcesses, Vol 143, 1995, p. 161.(7) Chandra, S., Ausserer, W. A., and Morrison, G. H., “Evaluation ofMatrix Effe
32、cts in Ion Microscopic Analysis of Freeze-fractured,Freeze-dried Cultured Cells,” Journal of Microscopy (Oxford),Vol148, 1987, p. 223.(8) Ausserer, W. A., Ling, Y. C., Chandra, S., and Morrison, G. H.,“Quantitative Imaging of Boron, Calcium, Magnesium, Potassiumand Sodium Distributions in Cultured C
33、ells with Ion Microscopy,”Analytical Chemistry, Vol 61, 1989, p. 2690.(9) Chandra, S., Bernius, M. T., and Morrison, G. H. “IntracellularLocalization of Diffusible Elements in Frozen-hydrated BiologicalSpecimens with Ion Microscopy,” Analytical Chemistry, Vol 58, 1986,p. 493.(10) Chandra, S. and Mor
34、rison, G. H., “Evaluation of fracture planes andcell morphology in complimentary fractures of cultured cells in thefrozen-hydrated state by field-emission secondary electron micros-copy: feasibility for ion localization and fluorescence imagingstudies,” Journal of Microscopy (Oxford), Vol 186, 1997,
35、 p. 232.(11) Chandra, S., and Morrison, G. H., “Sample Preparation of AnimalTissues and Cell Cultures for Secondary Ion Mass Spectrometry(SIMS) Microscopy,” Biology of the Cell, Vol 74, 1992, p. 31.(12) Chandra, S., Lorey II, D. R., and Smith, D. R., “Quantitativesubcellular dynamic SIMS imaging of
36、boron-10 and boron-11isotopes in the same cell delivered by two combined BNCT drugs: Invitro studies on human glioblastoma T98G cells,” RadiationResearch, Vol 157, 2002 , p. 700.E1881 122(13) Chandra, S., Kabalka, G. W., Lorey, II, D. R., Smith, D. R., andCoderre, J. A., “Imaging of fluorine and bor
37、on from fluorinated-boronophenylalanine in the same cell at organelle resolution bycorrelative SIMS ion microscopy and confocal laser scanningmicroscopy,” Clinical Cancer Research, Vol 8, 2002, p. 2675.(14) Chandra, S., “Quantitative imaging of subcellular calcium stores inmammalian LLC-PK1 epitheli
38、al cells undergoing mitosis by SIMSion microscopy,” European Journal of Cell Biology, Vol 84, 2005, p.783.(15) Roddy, T. P., Cannon, D. M., Ostrowski, S. G., Winograd, N., andEwing, A. G., “Identification of cellular sections with imaging massspectrometry following freeze-fracture,” Analytical Chemi
39、stry, Vol74, 2002, p. 4020.(16) Fartmann, M., Kriegeskotte, C., Dambach, S., Wittig,A., Sauerwein,W., and Arlinghouse, H. F., “Quantitative imaging of atomic andmolecular species in cancer cell cultures with TOF-SIMS andLaser-SNMS,”Applied Surface Science, Vol 231232 , 2004, p. 428.(17) Gazi, E., Lo
40、ckyer, N. P., Vickerman, J. C., Gardner, P., Dwyer, J.,Hart, C. A., Brown, M. D., Clarke, N. W., and Miyan, J., “ImagingToF and synchrotron-based FT-IR microspectroscopic studies ofprostate cancer cell lines,” Applied Surface Science, Vol 231232,2004, p. 452.ASTM International takes no position resp
41、ecting the validity of any patent rights asserted in connection with any item mentionedin this standard. Users of this standard are expressly advised that determination of the validity of any such patent rights, and the riskof infringement of such rights, are entirely their own responsibility.This s
42、tandard is subject to revision at any time by the responsible technical committee and must be reviewed every five years andif not revised, either reapproved or withdrawn. Your comments are invited either for revision of this standard or for additional standardsand should be addressed to ASTM Interna
43、tional Headquarters. Your comments will receive careful consideration at a meeting of theresponsible technical committee, which you may attend. If you feel that your comments have not received a fair hearing you shouldmake your views known to the ASTM Committee on Standards, at the address shown bel
44、ow.This standard is copyrighted by ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959,United States. Individual reprints (single or multiple copies) of this standard may be obtained by contacting ASTM at the aboveaddress or at 610-832-9585 (phone), 610-832-9555 (fax), or serviceastm.org (e-mail); or through the ASTM website(www.astm.org). Permission rights to photocopy the standard may also be secured from the ASTM website (www.astm.org/COPYRIGHT/).E1881 123
