1、Designation: E1880 12Standard Practice forTissue Cryosection Analysis with SIMS1This standard is issued under the fixed designation E1880; 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 paren
2、theses indicates the year of last reapproval. Asuperscript epsilon () indicates an editorial change since the last revision or reapproval.1. Scope1.1 This practice provides the Secondary Ion Mass Spec-trometry (SIMS) analyst with a method for analyzing tissuecryosections in the imaging mode of the i
3、nstrument. Thispractice is suitable for frozen-freeze-dried and frozen-hydratedcryosection analysis.1.2 This practice does not describe methods for optimalfreezing of the specimen for immobilizing diffusible chemicalspecies in their native intracellular sites.1.3 This practice does not describe meth
4、ods for obtainingcryosections from a frozen specimen.1.4 This practice is not suitable for any plastic embeddedtissues.1.5 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-pri
5、ate safety and health practices and determine 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 inSI
6、MS.4. Summary of Practice4.1 This practice describes a method for the analysis oftissue cryosections with SIMS. Tissue cryosections for SIMSanalysis need to be mounted flat on an electrically conductingsubstrate. Cryosections should remain flat and adhere well tothe substrate for SIMS analysis. This
7、 is achieved by pressingfrozen cryosections into an indium substrate. Indium, being amalleable metal (Moh hardness = 1.2, Youngs modulus = 10.6GPa), provides a “cushion” for pressing and holding the frozencryosections flat for SIMS analysis. Indium substrates areprepared by pressing sheet indium ont
8、o a polished siliconwafer.An approximately 1 m thick layer of indium (99.999 %purity) is then vapor deposited on this surface. This top layerprovides “fluffy” indium that helps in holding cryosections flatfor SIMS analysis.5. Significance and Use5.1 Pressing cryosections flat onto a conducting subst
9、ratehas been one of the most challenging problems in SIMSanalysis of cryogenically prepared tissue specimens. Frozencryosections often curl or peel off, or both, from the substrateduring freeze-drying. The curling of cryosections results in anuneven sample surface for SIMS analysis. Furthermore, iff
10、reeze-dried cryosections are not attached tightly to thesubstrate, the impact of the primary ion beam may result infurther curling and even dislodging of the cryosection from thesubstrate. These problems render SIMS analysis difficult,frustrating and time consuming. The use of indium as asubstrate f
11、or pressing cryosections flat has provided a practicalapproach for analyzing cryogenically prepared tissue speci-mens (1).45.2 The procedure described herein has been successfullyused for SIMS imaging of calcium and magnesium transportand localization of anticancer drugs in animal models (2, 3, 4,5)
12、.5.3 The procedure described here is amenable to soft tissuesof both animal and plant origin.6. Apparatus6.1 The procedure described here can be used for tissuecryosection analysis with virtually any SIMS instrument.6.2 A cold stage in the SIMS instrument is needed toanalyze frozen-hydrated specimen
13、s (6).1This practice is under the 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 E1880 06. DOI:
14、10.1520/E1880-12.2For referenced 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 historica
15、l standard is referenced onwww.astm.org.4The boldface numbers in parentheses refer to a list of references at the end ofthis standard.Copyright ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States17. Procedure7.1 Prepare the indium substrate by pres
16、sing sheet indiumonto polished silicon wafer pieces of approximately 15 to 25mm2surface area, which can be irregularly shaped. A simplemechanical Pellet Press5can be used effectively for pressingfor pressing sheet indium onto the surface of polished siliconwafer. Next, vapor deposit an approximately
17、 1 m thick layerof high purity (99.999 %) indium onto the pressed indiumsheet. The high purity of indium is emphasized only due to thefact that it should not impart any significant contamination tothe sample. The vapor deposition can be achieved by vacuum-based processes such as evaporation from a h
18、eated filament orsputtering from an indium target. The indium substrates arenow ready for use.7.1.1 Chill an individual indium substrate by immersing itinto liquid nitrogen prior to its use for pressing cryosections.Quickly transfer the indium substrate to the cryomicrotome andkeep at the desired te
19、mperature of cryosectioning. Place afrozen tissue cryosection on the indium substrate and gentlypress by using a new chilled silicon piece. Make sure that thepolished surface of the top silicon piece is used to press thecryosection onto indium substrate in order to avoid introducingthe irregular top
20、ography of the rough silicon surface. Removethe top silicon piece by sliding it off using chilled tweezers.The pressed frozen cryosection on the indium substrate is nowready for frozen-hydrated analysis with a cold stage in theSIMS instrument. Alternatively, the pressed cryosection on theindium subs
21、trate can be freeze-dried by transferring the indiumsubstrate to a freeze-drier.7.1.2 Upon completion of freeze-drying, the freeze-driershould be opened by introducing dry gasses (N2, Ar, etc.) inorder to avoid rehydration of tissue sections. The indiumsubstrates containing freeze-dried tissue secti
22、ons should bequickly transferred to a desiccator for storage. The freeze-driedcryosections are now ready for SIMS analysis.7.1.3 Depending on the need of a particular SIMS analysis,the freeze-dried cryosections may be analyzed directly or goldcoated to enhance electrical conductivity.7.1.4 A quick v
23、isual inspection of the cryosection surfaceshould be made prior to its insertion into the sample chamberof the SIMS instrument. A reflected light microscope can beused to observe any folds, ripples or loosely attached regions inthe section. At this stage, it is always desirable to “repress” thefreez
24、e-dried section gently into the indium with a polishedsilicon piece. It is also desirable to remove the loosely attachedpieces of tissue section from the substrate by using tweezers.7.1.5 Correlative morphological information to complementthe SIMS analysis can be made by using adjacent cryosectionsf
25、or optical microscopy and SIMS analysis (2).8. Keywords8.1 SIMSREFERENCES(1) Sod, E. W., Crooker, A. R., and Morrison, G. H., “BiologicalCryosection Preparation and Practical Ion Yield Evaluation for IonMicroscopic Analysis,” Journal of Microscopy (Oxford), Vol 160,1990, p. 55.(2) Chandra, S., Fullm
26、er, C. S., Smith, C. A., Wasserman, R. H., andMorrison, G. H. “Ion Microscopic Imaging of Calcium Transport inthe Intestinal Tissue of Vitamin D-deficient and Vitamin D-repleteChickens: A44Ca Stable Isotope Study,” Proceedings of the NationalAcademy of Sciences (USA), Vol 87, 1990, p. 5715.(3) Chand
27、ra, 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.(4) Chandra, S., Bernius, M. T., and Morrison, G. H., “IntracellularLocalization of Diffusible Elements in Frozen-hydrat
28、ed BiologicalSpecimens with Ion Microscopy,” Analytical Chemistry, Vol 58, 1986,p. 493.(5) Smith, D. R., Chandra, S., Barth, R.F., Yang, W., Joel, D.D., andCoderre, J.A., “Quantitative Imagining and Microlocalization ofBoron-10 in Brain Tumors and Infiltrating Tumor Cells by SIMS IonMicroscopy: Rele
29、vance to Neutron Capture Therapy,” CancerResearch, Vol 61, 2001, p. 8179.(6) Chandra, S., Bernius, M.T., and Morrison, G.H. “Intracellular Local-ization of Diffusible Elements in Frozen-Hydrated Biological Speci-mens with Ion Microscopy,” Analytical Chemistry, Vol 58, 1986, p.493.ASTM International
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31、 responsibility.This standard 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 add
32、ressed to ASTM International 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
33、 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
34、(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/).5The sole source of supply of the Pellet Press apparatus known to the committeeat this time is Parr Instrument Co., Moline, IL. If you are aware of alternativesuppliers, please provide this information to ASTM International Headquarters.Your comments will receive careful consideration at a meeting of the responsibletechnical committee,1which you may attend.E1880 122
