Principles, Applications and Protocols of Microwave Technology for Morphological Analysis

  • Anthony S-Y. Leong
  • F. Joel Leong


Since the introduction of microwave irradiation for morphologic diagnosis and research just over 20 years ago, the applications of microwaves have expanded considerably beyond their initial use as a safe, clean and rapid method of tissue fixation. Microwaves can now be employed for the fixation of large specimens, including brain, to accelerate the action of cross-linking fixatives, as well as to greatly reduce the various stages of tissue processing so that a paraffin-embedded block can be produced in about 30 min. Almost every histochemical stain can be greatly accelerated by microwaves, and immunohistochemical staining procedures can be completed in 20 min by employing microwaves. Cellular antigens are distinctly better preserved in tissues fixed by microwaves than by conventional cross-linking fixatives, and the cytomorphology of cryostat sections irradiated in Wolman’s solutions is vastly improved. Microwaves can similarly be employed in transmission and electron microscopy both for fixation and staining of ultrathin sections, and they have the potential to greatly accelerate the polymerization of resins. They also have applications in newer molecular techniques; they not only increase the speed of the procedures but also improve sensitivity. This wide range of applications makes microwave technology invaluable in the pathology laboratory.


Microwave Irradiation Microwave Oven Tissue Block Cryostat Section Tissue Fixation 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. 1.
    Bernard, J.R. 1974. Microwave irradiation as a generator of heat for histological fixation. Stain Tech- nol. 49:215–224PubMedGoogle Scholar
  2. 2.
    Boon, M.E., L.P. Kok and E. Ouwerkerk-Noordan. 1986. Microwave-stimulated diffusion for fast processing of tissue: reduced dehydrating, clearing and impregnating times. Histopathology 10:303- 309PubMedCrossRefGoogle Scholar
  3. 3.
    Boon, M.E., L.P. Kok, H.E. Moorlag, P.O. Gerrits and A.J.H. Suurmeijer. 1987. Microwave-stimulated staining of plastic-embedded bone marrow sections with the Romanowsky-Giemsa stain: improved staining patterns. Stain Technol. 62:257–264PubMedGoogle Scholar
  4. 4.
    Boon, M.E. and L.P. Kok. 1988. Microwave Cookbook of Pathology. The Art of Microscopic Visualisation, 2nd ed., Coulomb Press, Leyden.Google Scholar
  5. 5.
    Boon, M.E., E. Marani, P.J.M. Adriolo, J.W. Steffelaar, G.T. Bots and L.P. Kok. 1988. Mirowave irradiation of human brain tissue: production of microscopic slides within one day. J. Clin. Pathol. 47:590–593CrossRefGoogle Scholar
  6. 6.
    Boon, M.E., L.P. Kok, H.E. Moorlag and A.J.H. Suurmeijer. 1989. Accelerated immunugold-silver staining of paraffin sections using microwave irradiation: factors influencing results. Am. J. Clin. Pathol. 97:137–143Google Scholar
  7. 7.
    Brinn, N.T. 1983. Rapid metallic histological staining using the microwave oven. J. Histotechnol. 6:125–129Google Scholar
  8. 8.
    Chiu, K.Y. and K.W. Chin. 1987. Rapid immunofluorescence staining of human renal biopsy specimens using microwave irradiation. J. Clin. Pathol. 40:689–692PubMedCrossRefGoogle Scholar
  9. 9.
    Churukian, C.J. and E.A. Schenk. 1988. A Warthin-Starry method for spirochetes and bacteria using a microwave oven. J. Histotechnol. 77:149–151CrossRefGoogle Scholar
  10. 10.
    Clearly, S.F. 1978. Survey of microwave and radio frequency: biological effects and mechanisms. D.H.E.W. Publication (FDA) 78–8055:1–33Google Scholar
  11. 11.
    Coates, P.J., P.A. Hall, M.G. Butler and A.J. D’Ardenne. 1987. Rapid technique of DNA-DNA in situ hybridisation on formalin fixed tissue sections using microwave irradiation. J. Clin. Pathol. 40:865- 869PubMedCrossRefGoogle Scholar
  12. 12.
    Cuevas, E.C., A.C. Bateman, B.S. Wilkins, P.A. Johnson, J.H. Williams, A.H.F. Lee, D.B. Jones and D.H. Wright. 1994. Microwave antigen retrieval in immunocytochemistry: a study of 80 antibodies. J. Clin. Pathol. 1:448–452CrossRefGoogle Scholar
  13. 13.
    Daymon, M.E. and A.S-Y. Leong. 1984. Microwave fixation for surgical and autopsy tissues. Pathology 16:418Google Scholar
  14. 14.
    Estrada, J.C., N.T. Brinn and E.H. Bossen. 1985. A rapid method of staining ultra-thin sections for surgical pathology TEM with the use of the microwave oven. Am. J. Clin. Pathol. 83:639–641PubMedGoogle Scholar
  15. 15.
    Giammara, B.L., J. Pierce, A. Rustioni, M. Borowitz, D. Chandler, P. Yates and J. Hanker. 1985. Microwave acceleration of the metallization and intensification of cytochemical and immunocyto- chemical reaction products with silver methenamine, p. 704–705. Proceedings of the 43rd Annual Meeting of the Electron Microscopy Society of America.Google Scholar
  16. 16.
    Giammara, B.L. 1992. Microwave embedding methods. Scanning (Suppl. I) 74:60–61Google Scholar
  17. 17.
    Gown, A.M., N. de Wever and H. Battifora. 1993. Microwave-based antigenic unmasking. A revolutionary new technique for routine immunohistochemistry. Appl. Immunohistochem. 7:256–266Google Scholar
  18. 18.
    Hafiz, S., R.C. Spencer, M. Lee, H. Gooch and B.I. Duerden. 1985. Use of microwaves for acid and alcohol fast staining. J. Clin. Pathol. 38:1073–1076PubMedCrossRefGoogle Scholar
  19. 19.
    Hanker, J.S. and B.L. Giammara. 1992. Microwave-accelerated cytochemical stains for the electron microscopic examination and the image analysis of light microscopy diagnostic slides. Scanning (Suppl.II) 74:61–64Google Scholar
  20. 20.
    Haruna, N., T. Monden and H. Morimoto. 1990. Use of a rapid microwave fixation technique for im- munocytochemical demonstration of tumor necrosis factor, interleukin-la, and interleukin-lb in activated human peripheral mononuclear cells. Acta Histochem. Cytochem. 23:563–572CrossRefGoogle Scholar
  21. 21.
    Hjerpe, A., M.E. Boon and L.P. Kok. 1988. Microwave stimulation of an immunological reaction (CEA/anti-CEA) and its use in immunohistochemistry. Histochem. J. 20:388–396PubMedCrossRefGoogle Scholar
  22. 22.
    Hopwood, D., G. Coghill, J. Ramsay, G. Milne and M. Kerr. 1984. Microwave fixation. Its potential for routine techniques: Histochemistry, immunocytochemistry and electron microscopy. Histochem. J. 76:1171–1191PubMedCrossRefGoogle Scholar
  23. 23.
    Hopwood, D., G. Yeaman and G. Milne. 1988. Differentiating the effects of microwave and heat on tissue proteins and their cross linking by formaldehyde. Histochem. J 20:341–346PubMedCrossRefGoogle Scholar
  24. 24.
    Horobin, R.W. and M.E. Boon. 1988. Understanding microwave-stimulated Romanowsky-Giemsa staining of plastic embedded bone marrow. Histochem. J. 20:329–334PubMedCrossRefGoogle Scholar
  25. 25.
    Jackson, P., E.N. Lalani and J. Boutsen. 1988. Microwave stimulated immunogold-silver staining. Histochem. J. 20:353–358PubMedCrossRefGoogle Scholar
  26. 26.
    Kawasaki, Y., T. Monden, H. Morimoto, M. Murotani, Y. Miyoshi, T. Kobayashi, T. Shimano and T. Mori. 1992. Immunohistochemical study of p53 expression in microwave fixed, paraffin-embedded sections of colorectal carcinoma and adenoma. Am. J. Clin. Pathol. 97:244–249PubMedGoogle Scholar
  27. 27.
    Kok, L.P., M.E. Boon and A.J.H. Suurmeijer. 1987. Major improvement in microscope-image quality of cryostat sections combining freezing and microwave-stimulated fixation. Am. J. Clin. Pathol. 88:620–623PubMedGoogle Scholar
  28. 28.
    Kok, L.P. and M.E. Boon. 1990. Microwaves for microscopy. J. Microsc. 158:191–322CrossRefGoogle Scholar
  29. 29.
    Kok, L.P. and M.E. Boon. 1995. Ultrarapid vacuum-microwave histoprocessing. Histochem. J. 27:411–419PubMedGoogle Scholar
  30. 30.
    Lai, F.M., K.N. Lai, E.C. Chew and J.C.K. Lee. 1987. Microwave fixation in diagnostic renal pathology. Pathology 79:17–21PubMedCrossRefGoogle Scholar
  31. 31.
    Lan, H.Y., W. Mu, D.J. Nikolic-Paterson and R. Atkins. 1995. A novel, simple, reliable and sensitive method for multiple immunoenzyme staining; use of microwave oven heating to block antibody cross- reactivity and retrieve antigens. J. Histotech Cytochem 43:97–102CrossRefGoogle Scholar
  32. 32.
    Leong, A.S-Y. 1988. Applications of microwave irradiation in histopathology. Pathol. Annu. 2 213- 234Google Scholar
  33. 33.
    Leong, A.S-Y. 1991. Microwave fixation and rapid processing in a large throughput histopathology laboratory. Pathology 23:271–273PubMedCrossRefGoogle Scholar
  34. 34.
    Leong, A.S-Y. 1991. Microwave irradiation-applications in tissue fixation: processing and staining for light microscopy and electron microscopy, p. 47–60. In World Health Organisation Bi Regional Training Course on Electron Microscopy in Biomedical Research and Diagnosis of Human Diseases. University of Adelaide Press, Adelaide.Google Scholar
  35. 35.
    Leong, A.S-Y., M.E. Daymon and J. Miiios. 1985. Microwave irradiation as a form of fixation for light and electron microscopy. J. Pathol. 746:313–321PubMedCrossRefGoogle Scholar
  36. 36.
    Leong, A.S-Y. and J. Miiios. 1986. Rapid immunoperoxidase staining of lymphocyte antigens using microwave irradiation. J. Pathol. 748:183–187CrossRefGoogle Scholar
  37. 37.
    Leong, A.S-Y. and C.G. Duncis. 1986. A method of rapid fixation of large biopsy specimens using microwave irradiation. Pathology 78:222–225PubMedCrossRefGoogle Scholar
  38. 38.
    Leong, A.S-Y., J. Miiios and C.G. Duncis. 1988. Antigen preservation in microwave-irradiated tissues: a comparison with formaldehyde fixation. J. Pathol. 756:275–282PubMedCrossRefGoogle Scholar
  39. 39.
    Leong, A.S-Y. and P.N. Gilham. 1989. The effects of progressive formaldehyde fixation on the preservation of tissue antigens. Pathology 27:266–271CrossRefGoogle Scholar
  40. 40.
    Leong, A.S-Y. and P. Gilham. 1989. A new, rapid microwave-stimulated method of staining melanocytic lesions. Stain Technol. 64:81–87PubMedGoogle Scholar
  41. 41.
    Leong, A.S-Y. and S. Pulbrook. 1989. Microwave-stimulated staining of reticulin fibres in plastic sections by an ammoniacal silver nitrate method. J. Histotechnol. 72:289–292Google Scholar
  42. 42.
    Leong, A.S-Y. and D.W. Gove. 1990. Microwave techniques for tissue fixation, processing and staining. EMSA Bull. 20:61–65Google Scholar
  43. 43.
    Leong, A.S-Y. and D.W. Gove. 1990. Applications of microwave irradiation in electron microscopy, cf Ref. 62. Proc. Int. Congr. Electron. Microsc. 72:140–141Google Scholar
  44. 44.
    Leong, A.S-Y. and J. Miiios. 1990. Accelerated immunohistochemical staining by microwaves. J. Pathol. 767:327–334CrossRefGoogle Scholar
  45. 45.
    Leong, A.S-Y. and J. Miiios. 1993. An assessment of the efficacy of the microwave antigen-retrieval procedure on a range of tissue antigens. Appl. Immunohistochem. 7:267–274Google Scholar
  46. 46.
    Leong, A.J.-Y. and J. Miiios. 1993. A comparison of two antibodies to entrogen and progesterone from commercial sources and the effects of microwave antigen retrieval. Appl. Immunohistochem. 7:282–288Google Scholar
  47. 47.
    Leong, S.S.-Y. 1994. Microwave technology for morphological analysis. Cell Vision 1:278–288Google Scholar
  48. 48.
    Login, G.R. 1978. Microwave fixation versus formalin fixation of surgical and autopsy tissue. Am. J. Med. Technol. 44:435–437PubMedGoogle Scholar
  49. 49.
    Login, G.R. and A.M. Dvorak. 1985. Microwave energy fixation for electron microscopy. Am. J. Pathol. 720:230–243PubMedGoogle Scholar
  50. 50.
    Login, G.R., W.B. Stavinoha and A.M. Dvorak. 1986. Ultrafast microwave energy fixation for electron microscopy. J. Histochem. Cytochem. 34:381–387PubMedCrossRefGoogle Scholar
  51. 51.
    Login, G.R., S J. Galli, E. Morgan, N. Arizono, L.B. Schwartz and A.M. Dvorak. 1987. Microwave fixation of rat mast cells. 1. Localization of granule chymase with an ultrastructural post-embedding immunogold technique. Lab. Invest. 57:592–599PubMedGoogle Scholar
  52. 52.
    Login, G.R., B.K. Dwyer and A.M. Dvorak. 1990. Rapid primary microwave-osmium fixation. I.Preservation of structure for electron microscopy in seconds. J. Histochem. Cytochem. 38:755–762PubMedCrossRefGoogle Scholar
  53. 53.
    Login, G.R. and A.M. Dvorak. 1994. Methods of Microwave Fixation for Microscopy. A Review of Research and Clinical Applications: 1970–1992. Gustav Fischer Verlag, StuttgartGoogle Scholar
  54. 54.
    Loughman, N.T. 1989. Pneumocystis carinii: rapid diagnosis with a microwave oven. Acta Cytol. 53:416–417PubMedGoogle Scholar
  55. 55.
    Marani, E., J.M. Guldemond, P.J.M. Adriolo, M.E. Boon and L.P. Kok. 1987. The microwave Rio- Hortega technique: A 24 hour method. Histochem. J. 79:658–664PubMedCrossRefGoogle Scholar
  56. 56.
    Marani, E., M.E. Boon, P.J.M. Adriolo, W.J. Rietveld and L.P. Kok. 1987. Microwave-cryostat technique for neuro-anatomical studies. J. Neurosci. Methods 22:97–101PubMedCrossRefGoogle Scholar
  57. 57.
    Maruyama, Y. 1981. Rapid tissue fixation by microwave irradiation Trends Pharmacol. Sci. 2:239- 241CrossRefGoogle Scholar
  58. 58.
    Matthews, K. and J.K. Kelly. 1989. A microwave oven method for the combined alcian blue-periodic acid-Schiff stain. J. Histotechnol. 12:295–303Google Scholar
  59. 59.
    Mayers, C.P. 1970. Histological fixation by microwave heating. J. Clin. Pathol. 23:273–275PubMedCrossRefGoogle Scholar
  60. 60.
    McLay, A.L.C., J.D. Anderson and W. McMeekin. 1987. Microwave polymerisation of epoxy resin. Rapid processing technique in untrastructural pathology. J. Clin. Pathol. 40:350–352PubMedCrossRefGoogle Scholar
  61. 61.
    Merz, H., O. Richers, S. Schrimel, K. Orscheschek and A.C. Feller. 1993. Constant detection of surface and cytoplasmic immunoglobulin heavy and light chain expression in formalin-fixed and paraffin- embedded material. J. Pathol. 770:257–264PubMedCrossRefGoogle Scholar
  62. 62.
    Mizuhira, V. and H. Hasegawa. 1990. Opening images of synaptic vesicles and calcium localisation by means of microwave fixation method. Proc. Int. Congr. Electron Microsc. 72:182–183Google Scholar
  63. 63.
    Momose, H., P. Mehta and H. Battifora. 1993. Antigen retrieval by microwave irradiation in lead thiocyanate: A comparison with protease digestion retrieval. Appl. Immunohistochem. 7:77–82Google Scholar
  64. 64.
    Ni, C., T.C. Chang, S.S. Searl, E. Coughlin-Wilkinson and D.M. Albert. 1981. Rapid paraffin fixation for use in histological examination. Ophthalmology (Rochester) 88:1372–1376CrossRefGoogle Scholar
  65. 65.
    Ohtani, H. 1991. Microwave-stimulated fixation for pre-embedding immunoelectron microscopy. Eur. J. Morphol. 29:64–67PubMedGoogle Scholar
  66. 66.
    Patterson, M.K., Jr. and R. Bulard. 1980. Microwave fixation of cells in tissue culture. Stain Technol. 55:71–75PubMedGoogle Scholar
  67. 67.
    Peracchia, C. and B.S. Mittler. 1972. New glutaraldehyde fixation procedures. J. Ultrastruct. Res. 39:57–64PubMedCrossRefGoogle Scholar
  68. 68.
    Petrere, J.A. and J.L. Schardein. 1977. Microwave fixation of foetal specimens. Stain Technol. 52:113–114PubMedGoogle Scholar
  69. 69.
    Riches, D.J. and E.C. Chew. 1984. The use of microwaves for fixation in electron microscopy, p. 257- 260. In M.F. Chung (Ed.), Proceedings of the 3rd Asia-Pacific Conference on Electron Microscopy, Hentexco Trading Co., Hong KongGoogle Scholar
  70. 70.
    Schaffner, R. 1986. The Perls’ iron staining procedure for use in the microwave oven using a temperature probe. J. Histotechnol. 9:107–108CrossRefGoogle Scholar
  71. 71.
    Schneider, D.R., B.T. Felt and H. Goldman. 1982. On the use of microwave irradiation energy for brain tissue fixation. J. Neurochem. 38:749–752PubMedCrossRefGoogle Scholar
  72. 72.
    Shi, S., M.E. Key and K.L. Kaira. 1991. Antigen retrieval in formalin-fixed, paraffin-embedded tissues: an enhancement method for immunohistochemical staining based on microwave oven heating of tissue sections. J. Histochem. Cytochem. 39:741–748PubMedCrossRefGoogle Scholar
  73. 73.
    Stavinoha, W.B., B. Pepelko and P.W. Smith. 1970. Microwave radiation to inactivate cholinesterase in the rat brain prior to analysis for acetylcholine. Pharmacologist 12:257Google Scholar
  74. 74.
    Stavinoha, W.B., J. Frazer and A.T. Modak. 1977. Microwave fixation for the study of acetyl choline metabolism, p. 167–179. In D.J. Jenden (Ed.), Cholinergic Mechanisms and Cycle Pharmacology. Plenum Press, New YorkGoogle Scholar
  75. 75.
    Swischer, B.L. 1987. Modified Steiner procedure for microwave staining of spirochetes and non-filamentous bacteria. J. Histotechnol. 10:241–243CrossRefGoogle Scholar
  76. 76.
    Taylor, C.R., S-R. Shi, B. Chaiwun, L. Young, S.A. Imam and R.J. Cote. 1994. Strategies for improving the immunohistochemical staining of various intranuclear prognostic markers in formalin fixed paraffin sections. Hum. Pathol. 25:263–270PubMedCrossRefGoogle Scholar
  77. 77.
    van den Brink, W.J., H.J.M.A. Zijlmans, L.P. Kok, P. Bolhuis, H.H. Volkers, M.E. Boon and H.J. Houthoff. 1990. Microwave irradiation in labile-detection for diagnostic DNA-m situ hybridisation. Histochem. J. 22:327–334PubMedCrossRefGoogle Scholar
  78. 78.
    van de Kant, H.J.G., M.E. Boon and D.G. de Rooij. 1988. Microwave-added technique to detect BrdU in S-phase cells using immunogold-silver staining and plastic-embedded sections. Histochem. J. 20:335–340PubMedCrossRefGoogle Scholar
  79. 79.
    Volkers, H.H., W.J. van den Brink, R. Rook and F.M. van den Berg. 1992. Microwave label detection technique for DNA in situ hybridisation. Eur. J. Morphol. 29:59–62Google Scholar
  80. 80.
    Wannakrairot, P. and A.S-Y. Leong. 1989. Microwave stimulated immunogold-silver staining. Presented at the National Scientific Meeting of the Australian Institute of Laboratory Scientists, AdelaideGoogle Scholar
  81. 81.
    Yasuda, K., S. Yamashita, M. Shiozawa, S. Aiso and Y. Yasui. 1992. Application of ultrasound for tissue fixation: Combined use with microwave to enhance the effect of chemical fixation. Cytochemistry (Kyoto) 28:237–244Google Scholar
  82. 82.
    Zeikus, J.A. and H.C. Aldridge. 1975. Use of hot formaldehyde fixatives in processing parasitic nematodes for electron microscopy. Stain Technol. 50:219–223.PubMedGoogle Scholar

Copyright information

© Springer Science+Business Media New York 1996

Authors and Affiliations

  • Anthony S-Y. Leong
    • 1
  • F. Joel Leong
    • 1
  1. 1.Division of Tissue PathologyInstitute of Medical & Veterinary ScienceAdelaideAustralia

Personalised recommendations