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Quantitative comparison of preparation methodologies for x-ray fluorescence microscopy of brain tissue

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Abstract

X-ray fluorescence microscopy (XFM) facilitates high-sensitivity quantitative imaging of trace metals at high spatial resolution over large sample areas and can be applied to a diverse range of biological samples. Accurate determination of elemental content from recorded spectra requires proper calibration of the XFM instrument under the relevant operating conditions. Here, we describe the manufacture, characterization, and utilization of multi-element thin-film reference foils for use in calibration of XFM measurements of biological and other specimens. We have used these internal standards to assess the two-dimensional distribution of trace metals in a thin tissue section of a rat hippocampus. The data used in this study was acquired at the XFM beamline of the Australian Synchrotron using a new 384-element array detector (Maia) and at beamline 2-ID-E at the Advanced Photon Source. Post-processing of samples by different fixation techniques was investigated, with the conclusion that differences in solvent type and sample handling can significantly alter elemental content. The present study highlights the quantitative capability, high statistical power, and versatility of the XFM technique for mapping trace metals in biological samples, e.g., brain tissue samples in order to help understand neurological processes, especially when implemented in conjunction with a high-performance detector such as Maia.

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References

  1. Gray HB, Stiefel EI, Valentine JS, Bertini I (2006) Biological inorganic chemistry: structure and reactivity. University Science Books, Sausalito, CA

    Google Scholar 

  2. Haas KL, Franz KJ (2009) Application of metal coordination chemistry to explore and manipulate cell biology. Chem Rev 109:4921–4960

    Article  CAS  Google Scholar 

  3. Eichert D, Gregoratti L, Kaulich B, Marcello A, Melpignano P, Quaroni L, Kiskinova M (2007) Imaging with spectroscopic micro-analysis using synchrotron radiation. Anal Bioanal Chem 389:1121–1132

    Article  CAS  Google Scholar 

  4. Shen K, Johnson S (2010) Ca2+ influx through NMDA-gated channels activates ATP-sensitive K+ currents through a nitric oxide-cGMP pathway in subthalamic neurons. J Neurosci 30:1882

    Article  CAS  Google Scholar 

  5. Bitanihirwe BKY, Cunningham MG (2009) Zinc: the brain's dark horse. Synapse 63:1029–1049

    Article  CAS  Google Scholar 

  6. Duce JA, Bush AI (2010) Biological metals and Alzheimer's disease: implications for therapeutics and diagnostics. Prog Neurobiol 92(1):1–18

    Article  CAS  Google Scholar 

  7. Gh Popescu BF, George MJ, Bergmann U, Garachtchenko AV, Kelly ME, Mccrea RPE, Lüning K, Devon RM, George GN, Hanson AD, Harder SM, Chapman LD, Pickering IJ, Nichol H (2009) Mapping metals in Parkinson’s and normal brain using rapid-scanning x-ray fluorescence. Phys Med Biol 54:651–663

    Article  Google Scholar 

  8. Lopez-Garcia C, Varea E, Palop JJ, Nacher J, Ramirez C, Ponsoda X, Molowny A (2002) Cytochemical techniques for zinc and heavy metals localization in nerve cells. Microsc Res Tech 56:318–331

    Article  CAS  Google Scholar 

  9. Perls M (1867) Nachweis von eisenoxyd in gewissen pigmenten. Virchows Arch Pathol Anat 39:42–48

    Google Scholar 

  10. Zaleski SS (1887) Das eisen der organe bei morbus maculosus werlhoffi. Arch Experiment Patholol Pharmacol 23:77–90

    Article  Google Scholar 

  11. Ferenci P, Steindl-Munda P, Vogel W, Jessner W, Gschwantler M, Stauber R, Datz C, Hack lF, Wrba F, Bauer P, Lorenz O (2005) Diagnostic value of quantitative hepatic copper determination in patients with Wilson's disease. Clin Gastroenterol Hepatol 3:811

    Article  CAS  Google Scholar 

  12. Henwood A (2003) Current applications of orcein in histochemistry. A brief review with some new observations concerning influence of dye batch variation and aging of dye solutions on staining. Biotech Histochem 78:303–308

    Article  CAS  Google Scholar 

  13. Pilloni L, Lecca S, Van Eyken P, Flore C, Demelia L, Pilleri G, Nurchi AM, Farci AMG, Ambu R, Callea F, Faa G (1998) Value of histochemical stains for copper in the diagnosis of Wilson's disease. Histopathology 33:28–33

    Article  CAS  Google Scholar 

  14. Danscher G, Howell G, Perez-Clausell J, Hertel N (1985) The dithizone, Timm's sulphide silver and the selenium methods demonstrate a chelatable pool of zinc in CNS. A proton activation (PIXE) analysis of carbon tetrachloride extracts from rat brains and spinal cords intravitally treated with dithizone. Histochemistry 83:419–422

    Article  CAS  Google Scholar 

  15. Beckhoff B (2008) Reference-free x-ray spectrometry based on metrology using synchrotron radiation. J Anal At Spectrom 23:845

    Article  CAS  Google Scholar 

  16. Yang L, McRae R, Henary M, Patel R, Lai B, Vogt S, Fahrni C (2005) Imaging of the intracellular topography of copper with a fluorescent sensor and by synchrotron x-ray fluorescence microscopy. Proc Natl Acad Sci 102:11179–11184

    Article  CAS  Google Scholar 

  17. Finney L, Mandava S, Ursos L, Zhang W, Rodi D, Vogt S, Legnini D, Maser J, Ikpatt F, Olopade O (2007) X-ray fluorescence microscopy reveals large-scale relocalization and extracellular translocation of cellular copper during angiogenesis. Proc Natl Acad Sci 104:2247

    Article  CAS  Google Scholar 

  18. National Institute of Standards and Technology (2000) Certificate of Analysis—Standard Reference Material 1833, National Institute of Standards and Technology, Gaithersburg, 20899

  19. National Institute of Standards and Technology (2000) Certificate of Analysis—Standard Reference Material 1832, National Institute of Standards and Technology, Gaithersburg, 20899

  20. Linkous DH, Flinn JM, Koh JY, Lanzirotti A, Bertsch PM, Jones BF, Giblin LJ, Frederickson CJ (2008) Evidence that the ZNT3 protein controls the total amount of elemental zinc in synaptic vesicles. J Histochem Cytochem 56:3–6

    Article  CAS  Google Scholar 

  21. Paterson DJ, Boldeman JW, Cohen DD, Ryan CG (2007) Microspectroscopy beamline at the Australian synchrotron. AIP Conf Proc 879:864–867

    Article  CAS  Google Scholar 

  22. Paterson DJ, de Jonge MD, Howard DL, Lewis W, McKinlay J, Starritt A, Kusel M, Ryan CG, Kirkham R, Moorhead G, Siddons DP (2011) The x-ray fluorescence microscopy beamline at the Australian Synchrotron. AIP Conference Proceedings, Melbourne, Australia (in press)

  23. Kirkham R, Dunn PA, Kucziewski A, Siddons DP, Dodanwela R, Moorhead G, Ryan CG, De Geronimo G, Beuttenmuller R, Pinelli D, Pfeffer M, Davey P, Jensen M, Paterson DJ, de Jonge MD, Kusel M, McKinlay J (2010) The Maia spectroscopy detector system: engineering for integrated pulse capture. Low-latency scanning and real-time processing. AIP Conference Series, Melbourne, p 240

    Google Scholar 

  24. Ryan CG, Siddons DP, Kirkham R, Dunn PA, Kuczewski A, Moorhead G, De Geronimo G, Paterson DJ, de Jonge MD, Hough RM, Lintern MJ, Howard DL, Kappen P, Cleverley JS (2010) The new Maia detector system: methods for high definition trace element imaging of natural material. AIP Conference Series, Melbourne, pp 9–17

    Google Scholar 

  25. Ryan CG, Kirkham R, Hough RM, Moorhead G, Siddons DP, De Jonge MD, Patersone DJ, De Geronimod G, Howard DL, Cleverley JS (2010) Elemental x-ray imaging using the Maia detector array: the benefits and challenges of large solid-angle. Nucl Instrum Methods Phys Res Sect A 619:37–43

    Article  CAS  Google Scholar 

  26. Ryan CG (2000) Quantitative trace element imaging using PIXE and the nuclear microprobe. Int J Imaging Syst Technol 11:219–230

    Article  Google Scholar 

  27. Ryan C, Etschmann B, Vogt S, Maser J, Harland C, Van Achterbergh E, Legnini D (2005) Nuclear microprobe-synchrotron synergy: towards integrated quantitative real-time elemental imaging using PIXE and SXRF. Nucl Instrum Methods Phys Res Sect B 231:183–188

    Article  CAS  Google Scholar 

  28. Lombi E, Smith E, Hansen TH, Paterson D, De Jonge MD, Howard DL, Persson DP, Husted S, Ryan C, Schjoerring JK (2011) Megapixel imaging of (micro)nutrients in mature barley grains. J Exp Bot 62:273–282

    Article  CAS  Google Scholar 

  29. van Genderen IL, van Meer G, Slot JW, Geuze HJ, Voorhout WF (1991) Subcellular localization of Forssman glycolipid in epithelial MDCK cells by immuno-electronmicroscopy after freeze-substitution. J Cell Biol 115:1009–1019

    Article  Google Scholar 

  30. Leskovjan AC, Kretlow A, Lanzirotti A, Barrea R, Vogt S, Miller LM (2011) Increased brain iron coincides with early plaque formation in a mouse model of Alzheimer's disease. Neuroimage 55:32–38

    Article  CAS  Google Scholar 

  31. Carmona A, Cloetens P, Devès G, Bohic S, Ortega R (2008) Nano-imaging of trace metals by synchrotron X-ray fluorescence into dopaminergic single cells and neurite-like processes. J Anal At Spectrom 23:1083

    Article  CAS  Google Scholar 

  32. Corezzi S, Urbanelli L, Cloetens P, Emiliani C, Helfen L, Bohic S, Elisei F, Fioretto D (2009) Synchrotron-based x-ray fluorescence imaging of human cells labeled with CdSe quantum dots. Anal Biochem 388:33–39

    Article  CAS  Google Scholar 

  33. Matsuyama S, Shimura M, Fujii M, Maeshima K, Yumoto H, Mimura H, Sano Y, Yabashi M, Nishino Y, Tamasaku K, Ishizaka Y, Ishikawa T, Yamauchi K (2010) Elemental mapping of frozen-hydrated cells with cryo-scanning X-ray fluorescence microscopy. X-ray Spectrom 39:260–266

    Article  CAS  Google Scholar 

  34. Vogt S (2003) MAPS: a set of software tools for analysis and visualization of 3D X-ray fluorescence data sets. J Phys IV France 104:635–638

    Article  CAS  Google Scholar 

  35. Twining BS, Baines SB, Fisher NS, Maser J, Vogt S, Jacobsen C, Tovar-Sanchez A, Sañudo-Wilhelmy SA (2003) Quantifying trace elements in individual aquatic protist cells with a synchrotron x-ray fluorescence microprobe. Anal Chem 75:3806–3816

    Article  CAS  Google Scholar 

  36. Jones NC, Cardamone L, Williams JP, Salzberg MR, Myers DE, O’Brien TJ (2008) Experimental traumatic brain injury induces a pervasive hyperanxious phenotype in rats. J Neurotrauma 25:1367–1374

    Article  Google Scholar 

  37. Bouilleret V, Cardamone L, Liu YR, Fang K, Myers DE, O’Brien TJ (2009) Progressive brain changes on serial manganese-enhanced MRI following traumatic brain injury in the rat. J Neurotrauma 26:1999–2013

    Article  Google Scholar 

  38. Stedman J, Spyrou N (1998) Hierarchical clustering into groups of human brain regions according to elemental composition. J Radioanal Nucl Chem 236:11–14

    Article  CAS  Google Scholar 

  39. Frederickson CJ, Suh SW, Silva D, Frederickson CJ, Thompson RB (2000) Importance of zinc in the central nervous system: the zinc-containing neuron. J Nutr 130(5S Suppl):1471S–1483S

    CAS  Google Scholar 

  40. De Boer D, Borstrok J, Leenaers A, Van Sprang H, Brouwer P (1993) How accurate is the fundamental parameter approach? XRF analysis of bulk and multilayer samples. X-Ray Spectrom 22:33–38

    Article  Google Scholar 

  41. LeFurgey A, Ingram P (1990) Calcium measurements with electron probe x-ray and electron energy loss analysis. Environ Health Perspect 84:57–73

    Article  CAS  Google Scholar 

  42. Balamurugan K, Schaffner W (2006) Copper homeostasis in eukaryotes: teetering on a tightrope. Biochim Biophys Acta 1763:737–746

    Article  CAS  Google Scholar 

  43. Bancroft JD, Stevens A (1996) Theory and practice of histological techniques. Churchill Livingston, New York

    Google Scholar 

Download references

Acknowledgements

The authors would like to acknowledge Ms. Lisa Cardamone of The Department of Medicine at The Royal Melbourne Hospital for assistance with preparation of samples. This work was supported by funds from the CSIRO OCE Postdoctoral Fellowship Program (SAJ) and the Transport Accident Commission of Victoria through the Victorian Neurotrauma Initiative (DEM). This research was undertaken on the XFM beamline at the Australian Synchrotron and at beamline 2-ID-E at the APS. Use of the APS was supported by the US Department of Energy, Office of Science, Office of Basic Energy Sciences, under Contract No. DE-AC02-06CH11357.

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Authors and Affiliations

  1. Materials Science and Engineering and the Preventative Health Flagship, CSIRO, Gate 5, Normanby Road (Private Bag 33, Clayton South 3169), Clayton, VIC, 3168, Australia

    Simon A. James, Brett A. Sexton, Pamela Hoobin, Sheridan C. Mayo, Gareth F. Moorhead & Stephen W. Wilkins

  2. Department of Surgery/Orthopaedics, St. Vincent s Hospital, Fitzroy, VIC, 3065, Australia

    Damian E. Myers

  3. Department of Surgery, University of Melbourne, Parkville, VIC, 3010, Australia

    Damian E. Myers

  4. Australian Synchrotron, Clayton, VIC, 3168, Australia

    Martin D. de Jonge, David Paterson & Daryl L. Howard

  5. X-ray Science Division, Argonne National Laboratory, Argonne, IL, 60439, USA

    Stefan Vogt

  6. Earth Science and Resources Engineering, CSIRO, Gate 5, Normanby Road, Clayton, VIC, 3168, Australia

    Chris G. Ryan

  7. School of Physics, University of Melbourne, Parkville, VIC, 3010, Australia

    Chris G. Ryan & Gareth F. Moorhead

  8. CODES Centre of Excellence, University of Tasmania, Hobart, TAS, 7001, Australia

    Chris G. Ryan

  9. Melbourne Centre for Nanofabrication, Clayton, VIC, 3168, Australia

    Matteo Altissimo

  10. School of Physics, Monash University, Clayton, VIC, 3800, Australia

    Stephen W. Wilkins

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  1. Simon A. James
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Correspondence to Simon A. James or Damian E. Myers.

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Published in the special issue Imaging Techniques with Synchrotron Radiation with Guest Editor Cyril Petibois.

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James, S.A., Myers, D.E., de Jonge, M.D. et al. Quantitative comparison of preparation methodologies for x-ray fluorescence microscopy of brain tissue. Anal Bioanal Chem 401, 853–864 (2011). https://doi.org/10.1007/s00216-011-4978-3

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