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Scanning Electron Microscopy and X-Ray Microanalysis of Frozen-Hydrated Bulk Samples

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Cryotechniques in Biological Electron Microscopy

Abstract

Living organisms normally contain a high concentration of water, thus when frozen-hydrated biological samples are examined this is the closest representation of the living state that is currently possible to obtain in the electron microscope. An exception is the “environmental microscope” (Danilatos and Postle 1982), but this has considerably more limitations in its use than the “low temperature microscope”. The methods and techniques of low temperature microscopy of bulk samples evolved for both morphological purposes (Echlin et al. 1970; Echlin 1971; Nei et al. 1972; Echlin and Moreton 1973; Nei et al. 1973; Tokunaga and Tokunaga 1973; Turner and Smith 1974; Robinson 1975; Echlin and Moreton 1976; Echlin and Burgess 1977; Echlin 1978; Echlin et al. 1979) and for X-ray microanalysis (Gehring et al. 1973; Marshall and Wright 1973; Gullasch and Kaufmann 1974; Echlin and Moreton 1974; Fuchs and Lindeman 1975; Brom-bach 1975; Lechene et al. 1975; Marshall 1975a,b; Forrest and Marshall 1976; Zierold 1976; Marshall 1977; Yeo et al. 1977; Fuchs et al. 1978a,b; Kramer and Preston 1978; Zierold and Schäfer 1978; Echlin et al 1980; Fuchs and Fuchs 1980; Marshall 1980a,b; Echlin et al. 1981; Marshall 1981; Marshall 1982; Marshall 1984 a; Marshall and Condron 1985a,b; Marshall et al. 1985 b; Echlin and Taylor 1986).

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References

  1. Bald WB (1985) The relative merits of various cooling methods. J Microsc (Oxford) 140:17–40.

    Article  Google Scholar 

  2. Beckett A, Porter R (1982) Uromyces viciae-fabae on Vicia faba: Scanning electron microscopy of frozen-hydrated material. Protoplasma 111:28–37.

    Article  Google Scholar 

  3. Birks LS (1963) Electron probe microanalysis. John Wiley & Sons, New York.

    Google Scholar 

  4. Boekestein A, Stols ALH, Stadhouders AM (1980) Quantitation in X-ray microanalysis of biological bulk specimens. Scanning Electron Microsc 1980/II:321–334.

    Google Scholar 

  5. Boekestein A, Stadhouders AM, Stols ALH, Roomans GM (1983) Quantitative biological X-ray microanalysis of bulk specimens: an analysis of inaccuracies involved in ZAF-correction. Scanning Electron Microsc 1983/II:725–736.

    Google Scholar 

  6. Boyde A, Franc F (1981) Freeze-drying shrinkage of glutaraldehyde fixed liver. J Microsc (Oxford) 122:75–86.

    Article  CAS  Google Scholar 

  7. Brombach JD (1975) Electron beam X-ray microanalysis of frozen biological bulk specimens below 130 K. 2. The electrical charging of the sample in quantitative analysis. J Microsc Biol Cell 22:233–238.

    Google Scholar 

  8. Brown JD, Robinson WH (1979) Quantitative analysis by ø (ϱZ) curves. Microbeam Anal 1979:238–240.

    Google Scholar 

  9. Danilatos GD, Postle R (1982) The environmental scanning electron microscope and its applications. Scanning Electron Microsc 1982/I:1–16.

    Google Scholar 

  10. Durand M, Deleplanque M, Kahane A (1967) Bulk conductivity of ice between −25° and −100°C with ion exchange membranes. Sol Statn Commun 5:759–760.

    Article  CAS  Google Scholar 

  11. Echlin P (1971) The examination of biological material at low temperature. Scanning Electron Microsc 1971:225–232.

    Google Scholar 

  12. Echlin P (1978) Low temperature scanning electron microscopy: a review. J Microsc (Oxford) 112:47–61.

    Article  CAS  Google Scholar 

  13. Echlin P, Burgess A (1977) Cryofracturing and low temperature scanning electron microscopy of plant material. Scanning Electron Microsc 1977:491–500.

    Google Scholar 

  14. Echlin P, Moreton R (1973) The preparation coating and examination of frozen biological materials in the SEM. Scanning Electron Microsc 1973:325–331.

    Google Scholar 

  15. Echlin P, Moreton R (1974) The preparation of biological materials of X-ray microanalysis. In: Hall T, Echlin P, Kaufmann R (eds) Microprobe analysis as applied to cells and tissues. Academic Press, London New York, pp 159–174.

    Google Scholar 

  16. Echlin P, Moreton R (1976) Low temperature techniques for scanning electron microscopy. Scanning Electron Microsc 1976:753–762.

    Google Scholar 

  17. Echlin P, Taylor SE (1986) The preparation and X-ray microanalysis of bulk frozen hydrated vacuolate plant tissue. J Microsc (Oxford) 141:329–348.

    Article  Google Scholar 

  18. Echlin P, Lai C, Hayes TL (1981) The distribution and relative concentration of potassium in the root-tips of Lemna minor analysed using low temperature X-ray microanalysis. Scanning Electron Microsc 1981/II:489–498.

    Google Scholar 

  19. Echlin P, Paden R, Dronzek B, Wayte R (1970) Scanning electron microscopy of labile biological tissues maintained under controlled conditions. Scanning Electron Microsc 1970:51–56.

    Google Scholar 

  20. Echlin P, Pawley JB, Hayes TL (1979) Freeze-fracture scanning electron microscopy of Lemna minor. Scanning Electron Microsc 1979/III:69–76.

    Google Scholar 

  21. Echlin P, Lai CE, Hayes TL, Hook G (1980) Elemental analysis of frozen-hydrated differentiating phloem parenchyma in roots of Lemna minor L. Scanning Electron Microsc 1980/II:383–394.

    Google Scholar 

  22. Echlin P, Lai CE, Hayes TL (1982) Low temperature X-ray microanalysis of the differentiating vascular tissue in root tips of Lemna minor L. J Microsc (Oxford) 126:285–306.

    Article  CAS  Google Scholar 

  23. Forrest QG, Marshall AT (1976) Comparative X-ray microanalysis of frozen-hydrated and freeze-substituted specimens. In: Ben Shaul Y (ed) Electron microscopy 1976, vol 2. Proc 6th Eur Congr Electron Microsc, Israel Soc Electron Microsc, Jerusalem, pp 218–220.

    Google Scholar 

  24. Fuchs W, Fuchs H (1980) The use of frozen-hydrated bulk specimens for X-ray microanalysis. Scanning Electron Microsc 1980/II:371–382.

    Google Scholar 

  25. Fuchs W, Lindemann B (1975) Electron beam X-ray microanalysis of frozen biological bulk specimens below 130 K. 1. Instrumentation and specimen preparation. J Microsc Biol Cell 22:227–232.

    Google Scholar 

  26. Fuchs W, Lindemann B, Brombach JD, Trosch W (1978a) Instrumentation and specimen preparation for electron beam X-ray microanalysis of frozen hydrated bulk specimens. J Microsc (Oxford) 112:75–87.

    Article  CAS  Google Scholar 

  27. Fuchs W, Brombach JD, Trosch W (1978 b) Charging effect in electron-irradiated ice. J Microsc (Oxford) 112:63–74.

    Article  Google Scholar 

  28. Gehring K, Dörge A, Wunderlich P, Thurau K (1973) On the application of the electron microscope for the analysis of freeze-dried cryosections and deep frozen water containing tissue of the rat kidney. Beitr Elektronenmikrosk Direktabb 5:937–950.

    Google Scholar 

  29. Green M (1963) The efficiency of production of characteristic X-ray radiation. In: Pattee HH, Cosslett VE, Engstrom A (eds) X-ray optics and X-ray microanalysis. Academic Press, London New York, pp 185–189.

    Google Scholar 

  30. Green M, Cosslett VE (1961) The efficiency of production of characteristic X-radiation in thick targets of a pure element. Proc Phys Soc 78:1206–1214.

    Article  CAS  Google Scholar 

  31. Gullasch J, Kaufmann R (1974) Energy-dispersive X-ray microanalysis in soft biological tissues: relevance and reproducibility of the results as depending on specimen preparation (air drying, cryofixation, cool-stage techniques). In: Hall T, Echlin P, Kaufmann R (eds) Microprobe analysis as applied to cells and tissues. Academic Press, London New York, pp 175–190.

    Google Scholar 

  32. Hall TA (1971) The microprobe assay of chemical elements. In: Oster E (ed) Physical techniques in biological research, vol 1A, 2nd edn. Academic Press, London New York, pp 157–275.

    Google Scholar 

  33. Hall TA (1986) Properties of frozen sections relevant to quantitative microanalysis. J Microsc (Oxford) 141:319–328.

    Article  CAS  Google Scholar 

  34. Hall TA, Gupta BL (1982) Quantification for the X-ray microanalysis of cryosections. J Microsc (Oxford) 126:333–345.

    Article  CAS  Google Scholar 

  35. Hayes TL, Koch G (1975) Some problems associated with low temperature micromanipulation in the SEM. Scanning Electron Microsc 1975/II:35–42.

    Google Scholar 

  36. Henke BL, Lee P, Tanaka TJ, Shimabukuro RL, Fujikawa BK (1982) Low energy X-ray interaction coefficients: Photoabsorption, scattering and reflection. Atom Data Nucl Data Tab 27:1–44.

    Article  CAS  Google Scholar 

  37. Hook G, Lai C, Bastacky J, Hayes T (1980) Conductive coatings studied on inflated lung in the frozen hydrated and freeze-dried states. Scanning Electron Microsc 1980/IV:27–32.

    Google Scholar 

  38. Jones D, McHardy WJ, Tait JM (1984) Low temperature scanning electron microscopy of biological specimens. Trans Br Mycol Soc 82:164–170.

    Article  Google Scholar 

  39. Koehler JK (1968) The technique and application of freeze-etching in ultrastructure research. Adv Biol Med Phys 12:1–15.

    PubMed  CAS  Google Scholar 

  40. Kramer D, Preston J (1978) A modified method of X-ray microanalysis of bulk frozen plant tissue and its application to the problem of salt exclusion in mangroove roots. Microsc Acta Suppl 2:193–200.

    CAS  Google Scholar 

  41. Lechene C, Strunk T, Warner R (1975) Perspectives in electron probe microanalysis of biological samples kept frozen. Microbeam Anal 1975:49A–49E.

    Google Scholar 

  42. Lustyik G, Zs.-Nagy I (1985) Alterations of the intracellular water and ion concentrations in brain and liver cells during aging as revealed by energy dispersive X-ray microanalysis of bulk specimens. Scanning Electron Microsc 1985/I:323–337.

    Google Scholar 

  43. Marshall AT (1975a) X-ray microanalysis of frozen hydrated biological specimens: the effect of charging. Micron 5:272–280.

    Google Scholar 

  44. Marshall AT (1975b) Electron probe microanalysis. In: Hayat MA (ed) Principles and techniques of scanning electron microscopy, vol. 4. Van Nostrand Reinhold, New York, pp 103–173.

    Google Scholar 

  45. Marshall AT (1977) Electron probe X-ray microanalysis of frozen hydrated biological specimens. Microsc Acta 79:254–266.

    PubMed  CAS  Google Scholar 

  46. Marshall AT (1980a) Quanitative X-ray microanalysis of frozen-hydrated bulk biological specimens. Scanning Electron Microsc 1980/II:335–348.

    Google Scholar 

  47. Marshall (1980b) Frozen-hydrated bulk specimens. In: Hayat MA (ed) X-ray microanalysis in biology. Univ Park Press, Baltimore, pp 167–205.

    Google Scholar 

  48. Marshall (1980c) Principles and instrumentation. In: Hayat MA (ed) X-ray microanalysis in biology. Univ Park Press,Baltimore, pp 1–64.

    Google Scholar 

  49. Marshall AT (1981) Simultaneous use of EDS, windowless EDS, BE and SE detectors and digital real-time line scanning for the X-ray microanalysis of frozen-hydrated biological specimens. Scanning Electron Microsc 1981/II:327–343.

    Google Scholar 

  50. Marshall AT (1982) Application of ø(ϱz) curves and a windowless detector to the quantitative X-ray microanalysis of frozen-hydrated bulk biological specimens. Scanning Electron Microsc 1982/I:243–260.

    Google Scholar 

  51. Marshall AT (1983) X-ray microanalysis of the filter chamber of the cicada, Cyclochila australasiae Don. A water shunting epithelial complex. Cell Tissue Res 231:215–277.

    Article  PubMed  CAS  Google Scholar 

  52. Marshall AT (1984a) The windowless energy dispersive X-ray detector: prospects for a role in biological X-ray microanalysis. Scanning Electron Microsc 1984/II:493–504.

    Google Scholar 

  53. Marshall AT (1984b) Residual gas analysis in a SEM. J Microsc (Oxford) 133:119–120.

    Article  CAS  Google Scholar 

  54. Marshall AT, Carde D (1983) Beryllium coating for biological X-ray microanalysis. J Microsc (Oxford) 134:113–116.

    Article  Google Scholar 

  55. Marshall AT, Condron RJ (1985a) X-ray microanalytical resolution in frozen-hydrated biological bulk samples. J Microsc (Oxford) 140:109–118.

    Article  Google Scholar 

  56. Marshall AT, Condron RJ (1985b) Normalisation of light element X-ray intensities for surface topography effects in frozen-hydrated biological bulk samples. J Microsc (Oxford) 140:99–108.

    Article  Google Scholar 

  57. Marshall AT, Condron RJ (1987) A simple method of using ø(ϱz) curves for the X-ray microanalysis of frozen-hydrated bulk biological samples. Micron Microsc Acta 18:23–26.

    Article  CAS  Google Scholar 

  58. Marshall AT, Wright A (1973) Detection of ions in insect osmoregulatory systems by electron probe X-ray microanalysis using scanning electron microscopy and a cryoscopic technique. Micron 4:31–45.

    Google Scholar 

  59. Marshall AT, Carde D, Kent MJ (1982) Low temperature stages for the JSM35 SEM and the JEE4B coating unit. Micron 13:313–314.

    Google Scholar 

  60. Marshall AT, Carde D, Kent MJ (1985a) Improved vacuum evaporation unit for beryllium coating for biological X-ray microanalysis. J Microsc (Oxford) 139:335–337.

    Article  CAS  Google Scholar 

  61. Marshall AT, Hyatt AD, Phillips JG, Condron RJ (1985b) Isosmotic secretion in the avian nasal salt gland: X-ray microanalysis of luminal and intracellular ion distribution. J Comp Physiol B 156:213–227.

    Article  Google Scholar 

  62. Nei T, Yotsumoto H, Hasegawa Y, Nagasawa Y (1972) Electron microscopic observation of biological specimen in their native state by employing cryogenic techniques. In: Arceneaux CJ (ed) Proc 30th Annu Electron Microsc Soc Am, Los Angeles, pp 410–411.

    Google Scholar 

  63. Nei T, Yotsumoto H, Hasegawa Y, Nagasawa Y (1973) Direct observation of frozen specimens with a scanning electron microscope. J Electron Microsc 22:185–190.

    CAS  Google Scholar 

  64. Oates K, Potts WTW (1985) Electron beam penetration and X-ray excitation depth in ice. Micron Microsc Acta 16:1–4.

    Article  CAS  Google Scholar 

  65. Parobek L, Brown JD (1978) The atomic number and absorption corrections in electron microprobe analysis at low electron energies. X-ray Spectrometry 7:26–30.

    Article  CAS  Google Scholar 

  66. Pawley JB, Norton JT (1978) A chamber attached to the SEM for fracturing and coating frozen biological samples. J Microsc (Oxford) 112:169–182.

    Article  CAS  Google Scholar 

  67. Plattner H, Bachmann L (1982) Cryofixation: A tool in biological ultrastructural research. Int Rev Cytol 79:237–304.

    Article  PubMed  CAS  Google Scholar 

  68. Potts WTW, Oates K (1983) The ionic concentrations in the mitochondria rich or chloride cell of Fundulus heteroclitus. J Exp Zool 227:349–359.

    Article  CAS  Google Scholar 

  69. Reed SJB (1975) Electron microprobe analysis. Univ Press, Cambridge, pp 1–400.

    Google Scholar 

  70. Robards AW, Crosby P (1979) A comprehensive freezing, fracturing and coating system for low temperature scanning electron microscopy. Scanning Electron Microsc 1979/II:325–344.

    Google Scholar 

  71. Robards AW, Sleytr UB (1985) Low temperature methods in biological electron microscopy. In: Glauert AM (ed) Practical methods in electron microscopy, vol 10. Elsevier, Amsterdam.

    Google Scholar 

  72. Robinson VNE (1975) A simple technique for examining frozen hydrated specimens in the scanning electron microscope. J Microsc (Oxford) 104:287–292.

    Article  CAS  Google Scholar 

  73. Roomans GM (1981) Quantitative electron probe X-ray microanalysis of biological bulk specimens. Scanning Electron Microsc. 1981/II:345–356.

    Google Scholar 

  74. Talmon Y (1984) Radiation damage to organic inclusions in ice. Ultramicroscopy 14:305–316.

    Article  CAS  Google Scholar 

  75. Talmon Y, Thomas EL (1977) Beam heating of a moderately thick cold stage specimen in the SEM/STEM. J Microsc (Oxford) 111:151–164.

    Article  Google Scholar 

  76. Taylor PG, Burgess A (1977) Cold stage for electron probe microanalyser. J Microsc (Oxford) 111:51–64.

    Article  Google Scholar 

  77. Tokunaga J, Tokunaga M (1973) Cryo-scanning microscopy of conidiospores formations in Aspergillus niger. Jeol News 11e(1):3–7.

    Google Scholar 

  78. Turner RH, Smith CB (1974) A simple technique of examining fresh, frozen, biological specimens in the scanning electron microscope. J Microsc (Oxford) 102:209–214.

    Article  CAS  Google Scholar 

  79. Umrath W (1983) Berechnung der Gefriertrocknungszeiten für die elektronenmikroskopische Präparation. Mikroskopie (Wien) 40:9–37.

    CAS  Google Scholar 

  80. Whitecross MI, Price GD, Preston JS (1982) Use of colloidal graphite in frozen-hydrated standard solutions for X-ray microanalysis. J Microsc (Oxford) 128:RP3–RP4.

    Article  Google Scholar 

  81. Yeo AR, Läuchli A, Kramer D, Gullasch J (1977) Ion measurements by X-ray microanalysis in unfixed, frozen, hydrated plant cells of species differing in salt tolerance. Planta 134:35–38.

    Article  CAS  Google Scholar 

  82. Zierold K (1976) X-ray microanalysis of cryo-fractured tissue specimens of the rat. In: Ben Shaul Y (ed) Electron microscopy 1976, vol 2. Proc 6th Eur Congr Electron Microsc, Israel Soc Electron Microsc, Jerusalem, pp 223–225.

    Google Scholar 

  83. Zierold K, Schäfer D (1978) Quantitative X-ray microanalysis of diffusible ions in skeletal muscle bulk specimens. J Microsc (Oxford) 112:89–93.

    Article  CAS  Google Scholar 

  84. Zs.-Nagy I, Pieri C, Giuli C, Bertoni-Freddari C, Zsa.-Nagy V (1977) Energy dispersive X-ray microanalysis of the electrolytes in biological bulk specimens. I. Specimen preparation, beam penetration and quantitative analysis. J Ultrastruct Res 58:22–33.

    Article  PubMed  CAS  Google Scholar 

  85. Zs.-Nagy I, Lustyik G, Bertoni-Freddari C (1982) Intracellular water and dry mass content as measured in bulk specimens by energy-dispersive X-ray microanalysis. Tissue Cell 14:47–60.

    Article  Google Scholar 

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  1. Analytical Electron Microscopy Laboratory, Department of Zoology, La Trobe University, Bundoora (Melbourne), Victoria, 3083, Australia

    Alan T. Marshall

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  1. Max-Planck-Institut für Verhaltensphysiologie, D-8131, Seewiesen, Germany

    Rudolf Alexander Steinbrecht

  2. Max-Planck-Institut für Systemphysiologie, Rheinlanddamm 201, D-4600, Dortmund, Germany

    Karl Zierold

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Marshall, A.T. (1987). Scanning Electron Microscopy and X-Ray Microanalysis of Frozen-Hydrated Bulk Samples. In: Steinbrecht, R.A., Zierold, K. (eds) Cryotechniques in Biological Electron Microscopy. Springer, Berlin, Heidelberg. https://doi.org/10.1007/978-3-642-72815-0_13

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  • DOI: https://doi.org/10.1007/978-3-642-72815-0_13

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