1、Designation: E 1880 06Standard Practice forTissue Cryosection Analysis with SIMS1This standard is issued under the fixed designation E 1880; 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 par
2、entheses indicates the year of last reapproval. Asuperscript epsilon (e) 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 th
3、e instrument. 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 m
4、ethods 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-
5、priate safety and health practices and determine the applica-bility of regulatory limitations prior to use.2. Referenced Documents2.1 ASTM Standards:E 673 Terminology Related to Surface Analysis23. Terminology3.1 Definitions:3.1.1 See Terminology E 673 for definitions of terms used inSIMS.4. Summary
6、 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 is achieved
7、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 onto a polished
8、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 substratehas been
9、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, iffreeze-dried c
10、ryosections are not attached tightly to the sub-strate, the impact of the primary ion beam may result in furthercurling and even dislodging of the cryosection from thesubstrate. These problems render SIMS analysis difficult,frustrating and time consuming. The use of indium as asubstrate for pressing
11、 cryosections flat has provided a practicalapproach for analyzing cryogenically prepared tissue speci-mens.(1)5.2 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)5.3 The proce
12、dure 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 specimens.(6)7. Proced
13、ure7.1 Prepare the indium substrate by pressing sheet indiumonto polished silicon wafer pieces of approximately 15 to 25mm2surface area, which can be irregularly shaped. Next,vapor deposit an approximately 1 m thick layer of high purity(99.999 %) indium onto the pressed indium sheet. The highpurity
14、of indium is emphasized only due to the fact that itshould not impart any significant contamination to the sample.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, 2006. P
15、ublished November 2006. Originallyapproved in 1997. Last previous edition approved in 2002 as E 1880 97 (2002).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 s
16、tandards Document Summary page onthe ASTM website.1Copyright ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959, United States.The vapor deposition can be achieved by vacuum-basedprocesses such as evaporation from a heated filament orsputtering from an indium ta
17、rget. 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 temperature of cryosectioning. Place afrozen ti
18、ssue 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 topography of the rough silicon surface. Removet
19、he 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 substrate can be freeze-dried by transferring the
20、 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 sections should bequickly transferred to a desicca
21、tor 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 visual inspection of the cryosection surfacesh
22、ould 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” thefreeze-dried section gently into the indium with a
23、 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 cryosectionsfor optical microscopy and SIMS analysis.(2)8.
24、 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., Fullmer, C. S., Smith, C. A., Wasserman, R. H., and
25、Morrison, 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) Chandra, S., and Morrison, G. H., “Sample Preparati
26、on 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-hydrated BiologicalSpecimens with Ion Microscopy,” A
27、nalytical 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: Relevance to Neutron Capture Therapy,” Cancer Re-s
28、earch, 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 takes no position respecting the validity of
29、 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 standard is subject to
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32、pyrighted 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).E1880062
