Summary
Methodological aspects of quantitative X-ray microanalysis of semi-thick cryosections (2–6 μm) of biological soft tissue were investigated. The preparation of a low background specimen holder is described. Scanning and scanning transmission images of the sections could be obtained, allowing identification and separate analysis of nuclei and cytoplasm. Parallel observations of histochemically stained adjacent sections in the light microscope allowed correlation of the microanalytical data with tissue morphology and histochemistry. Quantitative analysis could be carried out with the help of a standard: a gelatin/glycerol matrix containing mineral salts in known quantities, frozen and sectioned in the same way as the specimen. Mass loss under the electron beam was found to be comparable in specimen and standard. Comparison of various theoretical models for quantitative analysis showed that the ‘P/B-method’ (determination of the background intensity under the characteristic peak) is the most suitable for semi-thick sections. Factors determining the choice of accelerating voltage were analyzed. The usefulness of this specimen type is illustrated in some biological applications (human oral mucosa, rat salivary gland).
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References
Anniko M, Wroblewski R (1983) X-ray microanalysis of developing and mature inner ear. Scan Electron Microsc, in press
Boekestein A, Stols ALH, Stadhouders AM (1980) Quantitation in X-ray microanalysis of biological bulk specimens. Scan Electron Microsc 2:309–320
Bogart BI, Picarelli J (1978) Agonist induced secretions and potassium release from rat submandibular gland slices. Am J Physiol 235:C256–268
Cameron IL, Sheridan PJ, Smith NR (1978) Differences in concentration of elements in brain cells due to: opiates, cell type and subcellular location. An X-ray analysis study. J Neurosci Res 3:397–410
Cameron IL, Smith NKR (1980) Energy dispersive X-ray microanalysis of the concentration of elements in relation to cell reproduction in normal and in cancer cells. Scan Electron Microsc 2:463–474
Chandler JA (1976) A method for preparing absolute standards for quantitative calibration and measurement of section thickness with X-ray microanalysis of biological ultrathin specimens in EMMA. J Microsc 106:291–303
Echlin P, Lai C, Hayes TL (1981) The distribution and relative concentration of potassium in the root-tips of Lemna minor L. analyzed by low-temperature X-ray microanalysis. Scan Electron Microsc 2:489–498
Edström L, Wroblewski R (1982) Genuine myotubular myopathy. Muscle Nerve 5:1–11
Fiori CE, Newbury DE (1980) Artifacts in energy-dispersive X-ray spectrometry in the scanning electron microscope. Scan Electron Microsc 2:251–258
Hall TA (1971) The microprobe assay of chemical elements. In: Oster G (ed) Physical techniques in biochemical research, vol 1A. Academic Press, New York, pp 157–275
Hall TA (1975) Methods of quantitative analysis. J Microsc Biol Cell 22:271–282
Hall TA, Clarke Anderson H, Appleton T (1973) The use of thin specimens for X-ray microanalysis in biology. J Microsc 99:177–182
Hall TA, Gupta BL (1974) Measurement of mass loss in biological specimens under an electron microbeam. In: Hall T, Echlin P, Kaufmann R (eds) Microprobe analysis as applied to cells and tissues. Academic Press, London, pp 147–158
Heinrich KFJ (1966) X-ray absorption uncertainty. In: Mc Kinley TD, Withry DB (eds) The electron microprobe. John Wiley & Sons, New York, pp 296–324
Hobbs LW (1979) Radiation effects in analysis of inorganic specimens by TEM. In: Hren JJ, Goldstein JI, Joy DC (eds) Introduction to analytical electron microscopy. Plenum Press, New York, pp 437–480
Kyser DF (1979) Monte Carlo simulation in analytical electron microscopy. In: Hren JJ, Goldstein JI, Joy DC (eds) Introduction to analytical electron microscopy. Plenum Press, New York, pp 199–222
Marshall AT, Forrest OG (1977) X-ray microanalysis in the transmission microscope at high accelerating voltage. Micron 8:135–138
Martin PM, Poole DM (1971) Electron probe microanalysis: the relation between intensity ratio and concentration. Metall Rev 150:1946
Reed SJB (1975) Electron microprobe analysis. Cambridge Univeristy Press, Cambridge
Reed SJB (1982) The single scattering model and spatial resolution in X-ray microanalysis of thin foils. Ultramicroscopy 7:405–409
Reggio HA, Palade GE (1978) Sulfated compounds in the zymogen granules of the guinea pig pancreas. J Cell Biol 77:288–314
Roinel N (1982) Radiation damage to microdroplets at low temperature. J Microsc 126:253–258
Roomans GM (1979a) Standards for X-ray microanalysis of biological specimens. Scan Electron Microsc 2:649–657
Roomans GM (1979b) Quantitative X-ray microanalysis of halogen elements in biological specimens. Histochemistry 65:49–58
Roomans GM (1980) Quantitative X-ray microanalysis of thin sections. In: Hayat MA (ed) X-ray microanalysis in biology. University Park Press, Baltimore, pp 401–453
Roomans GM (1981) Quantitative electron probe X-ray microanalysis of biological bulk specimens. Scan Electron Microsc 2:345–356
Roomans GM, Kuypers GAJ (1980) Background determination in quantitative X-ray microanalysis of biological thin sections. Ultramicroscopy 5:81–83
Roomans GM, Sevéus LA (1977) Preparation of thin cryosectioned standards for quantitative microprobe analysis. J Submicrosc Cytol 9:31–35
Roomans GM, Wei X, Ceder O, Kollberg H (1982) The reserpinized rat in the study of cystic fibrosis: X-ray microanalysis of submandibular gland and pancreas. Ultrastruct Pathol 3:285–293
Russ JC (1974) X-ray microanalysis in the biological sciences. J Submicrosc Cytol 6:55–79
Seale TW, Flux M, Rennert OM, Quissel DO, Barzen KA, Lafferty JL (1980) Cystic fibrosis factors: effect of serum on the secretory response of dispersed rat submandibular cells. Pediatr Res 14:1398–1402
Shuman H, Somlyo AV, Somlyo AP (1976) Quantitative electron probe microanalysis of biological thin sections: methods and validity. Ultramicroscopy 1:317–339
Small JA, Heinrich KFJ, Fiori CE, Myklebust RL, Newbury DE, Dilmore MF (1978) The production and characterization of glass fibers and spheres for microanalysis. Scan Electron Microsc 1:445–454
Smith NKR, Cameron IL (1981) Observations on electron probe X-ray microanalysis compared to other methods for measuring intracellular elemental concentration. Scan Electron Microsc 2:395–408
Spurr AR (1975) Choice and preparation of standards for X-ray microanalysis with special reference to microcyclic polyether complexes. J Microsc Biol Cell 22:287–302
Statham PJ, Pawley JB (1978) A new method for particle X-ray microanalysis based on peak to background measurements. Scan Electron Microsc 1:469–478
Sumner AT (1978) Quantitation in biological X-ray microanalysis with particular reference to histochemistry. J Microsc 114:19–30
Theisen R (1965) Quantitative electron microprobe analysis. Springer, Berlin Göttingen Heidelberg
Trump BJ, Berezesky IK, Pendergrass R (1978) X-ray microanalysis of diffusible elements in scanning electron microscopy of biological thin sections. Studies of pathologically altered cells. Scan Electron Microsc 2:1027–1041
Von Euler AM, Müller RM, Ceder O, Roomans GM (1983) Effects of cystic fibrosis serum and fibroblast culture medium on ion distribution in rat submandibular gland. Ultrastruct Pathol (in press)
Warner RR, Coleman JR (1975) A biological thin specimen microprobe quantitation procedure that calculates composition and ρx. Micron 6:79–84
Wroblewski J, Anniko M, Wroblewski R (1983) Healthy and diseased epithelium of the oral and maxillary sinus mucosa studied by X-ray microanalysis. J Submicr Cytol (in press)
Wroblewski R (1982) Healthy and diseased striated muscle studied by analytical scanning electron microscopy with special reference to fibre type. Scan Electron Microsc 3:1173–1189
Wroblewski R, Edström L (1983) Changes in elemental composition following tenotomy of rat soleus muscle. Submitted for publication
Wroblewski R, Roomans GM, Jansson E, Edström L (1978) Electron probe X-ray microanalysis of human muscle biopsies. Histochemistry 55:281–292
Young JA, Van Lennep EW (1978) The morphology of salivary glands. Academic Press, London
Young JA, Van Lennep EW (1979) Transport in salivary and salt glands. In: Giebisch G, Tosteson DC, Ussing HH (eds) Membrane transport in biology, vol 4B. Springer, Berlin Heidelberg New York, pp 563–692
Zs Nagy I, Pieri C, Giuli C, Bertoni-Freddari C, Zs Nagy V (1977) Energy-dispersive X-ray microanalysis of the electrolytes in biological bulk specimens. J Ultrastruct Res 58:22–33
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Wroblewski, J., Müller, R.M., Wroblewski, R. et al. Quantitative X-ray microanalysis of semi-thick cryosections. Histochemistry 77, 447–463 (1983). https://doi.org/10.1007/BF00495800
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DOI: https://doi.org/10.1007/BF00495800