1、Designation: E 1881 06Standard Guide forCell Culture Analysis with SIMS1This standard is issued under the fixed designation E 1881; 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
2、indicates the year of last reapproval. Asuperscript epsilon (e) 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 vitr
3、o. 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
4、.1.3 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 dete
5、rmine the applica-bility of regulatory limitations prior to use.2. Referenced Documents2.1 ASTM Standards:2E 673 Terminology Relating to Surface Analysis3. Terminology3.1 Definitions:3.1.1 See Terminology E 673 for definitions of terms used inSIMS.4. Summary of Guide4.1 This guide describes a cryoge
6、nic 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).3This allows freeze-fixati
7、on 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, the same fro
8、zen 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 unphysi-olog
9、ical 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 been succes
10、sfullyused 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 effects (7).
11、 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 localization of isotopi
12、cally-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).7. Procedure7.1 Cells are grown on s
13、ilicon wafer pieces (approximately1cm2area) of any shape.Alternatively, high purity germaniumwafer pieces are used for cell growth for studies involving theuse of44Ca stable isotope. These substrates are nontoxic to cellsand have been used for growing various cell lines (1,2,8).Sterilize the silicon
14、 or germanium pieces prior to cell seeding.After the cells reach about 80 % confluency, replace thenutrient growth medium with new medium containing 11 m1This guide is under the jurisdiction of ASTM Committee E42 on SurfaceAnalysis and is the direct responsibility of Subcommittee E42.06 on SIMS.Curr
15、ent edition approved Nov. 1, 2006. Published November 2006. Originallyapproved in 1997. Last previous edition approved in 2002 as E 1881 97 (2002).2For referenced ASTM standards, visit the ASTM website, www.astm.org, orcontact ASTM Customer Service at serviceastm.org. For Annual Book of ASTMStandard
16、s volume information, refer to the standards Document Summary page onthe ASTM website.3The boldface numbers in parentheses refer to a list of references at the end ofthis guide.1Copyright ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959, United States.polystyr
17、ene 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 approximately 30 min for the beads to settle down on thesubstrate. After beads settle down on the substrate the cells
18、 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 steps: (1) remove the silicon piece containing thecells from the nutrient medium, (2) remove excess nutrientm
19、edium 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) fast freeze the sandwich in cryogenic fluids(supercooled isopentane, propane, liquid nitrogen, and soforth), (
20、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 cells contains a group of cells fractured together at the basalor dorsal cells surfaces, and randomly scattered i
21、ndividualcross 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 membranefracture removes the extracellular nutrient medium and theEF-leaflet of the plasma membrane on the top silico
22、n 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 analysis,the freeze-dried cells may be analyzed directly or gold coatedto enhance electrical conductivity.7.
23、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 relation to SIMS isotopeimages, a correlative laser scanning confocal microscopy andSIMS approach has been de
24、veloped (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 microscope in a fracturedfreeze-dried cell prior to SIMS analysis of the same cell(2,4,5).7.1.5 This sandwich fr
25、eeze-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 imaging of subcellularcalcium stores in cells undergoing cell division (14).7.1.6 This sandwich freeze-fract
26、ure 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. Keywords8.1 SIMSREFERENCES(1) Chandra, S., Morrison, G. H., and Wolcott C. C., “Imaging Intracel-lular Elemental Di
27、stribution 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, G. H., and Webb W. W.,“Calcium Sequestration in the Golgi Apparatus of Cultured Mamma-lian Cells Revealed b
28、y 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 in Cultured Cells with Ion Microscopy,” Science,Vol 228, 1985, p. 1543.(4) Chandra, S., Fewtrell, C., Millard
29、, 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 Stimulated TumorMast Cells Using Ion Microscopy,” Journal of Biological Chemistry,Vol 269, 1994, p. 15186.(5) Zha, X., Chandra, S., Ridsdale, A.,
30、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.(6) Chandra, S., and Morrison, G. H., “Imaging Ion and MolecularTransport at Subcellular Resolution by Second
31、ary Ion Mass Spectrom-etry,” International Journal of Mass Spectrometry and Ion Processes,Vol 143, 1995, p. 161.(7) Chandra, S., Ausserer, W. A., and Morrison, G. H., “Evaluation ofMatrix Effects in Ion Microscopic Analysis of Freeze-fractured,Freeze-dried Cultured Cells,” Journal of Microscopy (Oxf
32、ord),Vol148, 1987, p. 223.(8) Ausserer, W. A., Ling, Y. C., Chandra, S., and Morrison, G. H.,“Quantitative Imaging of Boron, Calcium, Magnesium, Potassium andSodium Distributions in Cultured Cells with Ion Microscopy,” Ana-lytical Chemistry, Vol 61, 1989, p. 2690.(9) Chandra, S., Bernius, M. T., and
33、 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 Morrison, G. H., “Evaluation of fracture planes andcell morphology in complimentary fractures of cultured cel
34、ls in thefrozen-hydrated state by field-emission secondary electron micros-copy: feasibility for ion localization and fluorescence imaging stud-ies,” Journal of Microscopy (Oxford), Vol 186, 1997, p. 232.(11) Chandra, S., and Morrison, G. H., “Sample Preparation of AnimalTissues and Cell Cultures fo
35、r 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 boron-10 and boron-11 iso-topes in the same cell delivered by two combined BNCT drugs: Invitro studies o
36、n human glioblastoma T98G cells,” Radiation Re-search, Vol 157, 2002, p. 700.(13) Chandra, S., Kabalka, G. W., Lorey, II, D. R., Smith, D. R., andCoderre, J. A., “Imaging of fluorine and boron from fluorinated-boronophenylalanine in the same cell at organelle resolution bycorrelative SIMS ion micros
37、copy and confocal laser scanning mi-croscopy,” Clinical Cancer Research, Vol 8, 2002, p.2675.(14) Chandra, S., “Quantitative imaging of subcellular calcium stores inmammalian LLC-PK1 epithelial cells undergoing mitosis by SIMSion microscopy,” European Journal of Cell Biology, Vol 84, 2005, p.783.(15
38、) Roddy, T. P., Cannon, D. M., Ostrowski, S. G., Winograd, N., andEwing, A. G., “Identification of cellular sections with imaging massE1881062spectrometry following freeze-fracture,” Analytical Chemistry, Vol74, 2002, p. 4020.(16) Fartmann, M., Kriegeskotte, C., Dambach, S., Wittig, A., Sauerwein,W.
39、, and Arlinghouse, H. F., “Quantitative imaging of atomic andmolecular species in cancer cell cultures with TOF-SIMS andLaser-SNMS,” Applied Surface Science, Vol 231-232, 2004, p. 428.(17) Gazi, E., Lockyer, N. P., Vickerman, J. C., Gardner, P., Dwyer, J.,Hart, C. A., Brown, M. D., Clarke, N. W., an
40、d Miyan, J., “ImagingToF and synchrotron-based FT-IR microspectroscopic studies ofprostate cancer cell lines,” Applied Surface Science, Vol 231-231,2004, p. 452.ASTM International takes no position respecting the validity of any patent rights asserted in connection with any item mentionedin this sta
41、ndard. 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 standard is subject to revision at any time by the responsible technical committee and must be revi
42、ewed 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 International Headquarters. Your comments will receive careful consideration at a meeting of theresponsib
43、le 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 below.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).E1881063
