Reflection Contrast Microscopy

The Bridge Between Light and Electron Microscopy
  • F. A. Prins
  • I. Cornelese-ten Velde
  • E. de Heer
Part of the Methods in Molecular Biology™ book series (MIMB, volume 319)


Reflection contrast microscopy (RCM) is a light microscopic method to image cells at high definition and enhanced sensitivity compared to conventional bright-field microscopy. RCM images have very high contrast, which makes them easily applicable for digital image analysis. Because ultrathin sections are mostly used in this method, RCM also functions by bridging light with electron microscopy: the combination of ultrastructural with histochemical studies. RCM can also replace electron microscopy for rapid and simple screening of large quantities of samples for immunocytochemical staining. Special attention is paid to small biological objects, which have to be processed for RCM. If you encounter the limits of brightfield microscopy, in resolution, sensitivity or handling of the specimen, RCM will be a feasible option.

Reflection contrast microscopy methods use only slightly adjusted electron microscopy methods for specimen preparation. Therefore, many familiar techniques for ultrathin specimen preparation can be applied. It is essential that only refractive index differences exist in those areas that are of interest and that the further specimen is as optically homogenic as possible, with a refractive index as close to that of glass as possible. Therefore, plastic embedding is recommended.

Key Words

Ultrathin section immunocytochemistry high definition specimen preparation reflection mode of CLSM reflection contrast microscopy enhanced detection sensitivity image analysis bright-field microscopy transmission electron microscopy high-resolution high-contrast microscopy combining light and electron microscopy 


  1. 1.
    Yamaguchi, H., Maat-Schieman, M. L. C., van Duinen, S. G., et al. (2000) Amyloid β protein (Aβ) starts to deposit as plasma membrane-bound form in diffuse plaques of brains from hereditary cerebral hemorrhage with amyloidosisdutch type, alzheimer disease, and nondemented age subjects. J. Neuropath. Exp. Neurol. 59, 723–732.PubMedGoogle Scholar
  2. 2.
    Natté, R., Yamaguchi, H., Maat-Schieman, M. L. C., et al. (1999) Ultrastructural evidence of early non-fibrillar Aβ42 in capillary basement membrane of patients with hereditary cerebral hemorrhage with amyloidosis, dutch type. Acta Neuropathol. 98, 577–582.PubMedCrossRefGoogle Scholar
  3. 3.
    Ploem, J. S., Cornelese-ten Velde, I., Prins, F. A., Bonnet, J., and de Heer, E. (1997) Reflection contrast microscopy. Sci. Tech. Inf. XI(4), 98–113.Google Scholar
  4. 4.
    Nasser Hajibagheri, M. A. (1999) Electron Microscopy: Methods and Protocols, Humana, Totowa, NJ.CrossRefGoogle Scholar
  5. 5.
    Filler, T. J., Rickert, C. H., Fassnacht, U. K., and Pera, F. (1994) Reflection contrast microscopy within chrome-alum hematoxylin stained thick tissue-sections. Histochemistry 101, 375–378.PubMedCrossRefGoogle Scholar
  6. 6.
    Filler, T. J. and Peuker, E. T. (2000) Reflection contrast microscopy (RCM): a forgotten technique? J. Pathol. 190, 635–638.PubMedCrossRefGoogle Scholar
  7. 7.
    Ploem, J. S. (1975) Reflection-contrast microscopy as a tool for investigation of the attachment of living cells to a glass surface, in Mononuclear Phagocytes in Immunity (van Furth, R., ed.), Blackwell, Oxford, pp. 405–421.Google Scholar
  8. 8.
    Verscheuren, H. (1985) Interference reflection microscopy in cell biology: methodology and applications. J. Cell Sci. 75, 279–301.Google Scholar
  9. 9.
    Piller, H. (1959) Enhancement of contrast in reflected-light microscopy. Zeiss-Werkschrift 34, 87–90.Google Scholar
  10. 10.
    Stach, E. (1949) Verkommungen de Kohlen-Auflichtmikroskopie. Glückauf 85, 117–122.Google Scholar
  11. 11.
    Cornelese-ten Velde, I., Bonnet, J., Tanke, H. J., and Ploem, J. S. (1988) Reflection contrast microscopy. Visualization of (peroxidase-generated) diaminobenzidine polymer products and its underlying optical phenomena. Histochemistry 89, 141–150.PubMedCrossRefGoogle Scholar
  12. 12.
    Carlemalm, E., Villiger, W., Hobot, J. A., Acetarin, J. D., and Kellenberger, E. (1985) Low temperature embedding with Lowicryl resins: two new formulations and some applications. J. Microsc. 140, 55–72.PubMedGoogle Scholar
  13. 13.
    Danscher, G. (1981) Localization of gold in biological tissue. A photochemical method for light and electronmicroscopy. Histochemistry 71, 81–88.PubMedCrossRefGoogle Scholar
  14. 14.
    Pluta, M. (1989) Advanced Light Microscopy. Specialized Methods. Elsevier PWN-Polish Scientific Publishers, Warszawa, pp. 197–210.Google Scholar
  15. 15.
    Hoetelmans, R. W., Prins, F. A., Cornelese-ten Velde, I., van der Meer, I., van de Velde, C. J., and van Dierendonck, J. H. (2001) Effects of acetone, methanol, or paraformaldehyde on cellular structure, visualized by reflection contrast microscopy and transmission and scanning electron microscopy. Appl. Immunohistochem. Mol. Morphol. 9, 346–351.PubMedCrossRefGoogle Scholar
  16. 16.
    Speel, E. J. M., Kramps, M., Bonnet, J., Ramaekers, F. C. S., and Hopman, A. H. (1993) Multicolour preparations for in situ hybridization using precipitating enzyme cytochemistry in combination with reflection contrast microscopy. Histochemistry 100, 357–366.PubMedCrossRefGoogle Scholar
  17. 17.
    Prins, F. A., Bruijn, J. A., and de Heer, E. (1996) Applications in renal immunopathology of reflection contrast microscopy, a novel superior light microscopical technique. Kidney Int. 49, 261–266.PubMedCrossRefGoogle Scholar
  18. 18.
    Kootstra, C. J., Bergijk, E. C., Veninga, A., et al. (1995) Qualitative alterations in laminin expression in experimental lupus nephritis. Am. J. Pathol. 147, 476–488.PubMedGoogle Scholar
  19. 19.
    Bergijk, E. C., Munaut, C., Baelde, J. J., et al. (1992) A histologic study of the extracellular matrix during the development of glomerulosclerosis in murine chronic graft-versus host disease. Am. J. Pathol. 140, 1147–1156.PubMedGoogle Scholar
  20. 20.
    Leer van, E. H. G., Ronco, P., Verroust, P., van der Wal, A. M., Hoedemaeker, P. J., and de Heer, E. (1993) Epitope specificity of anti-gp330 autoantibodies determines the development of proteinuria in active Heymann nephritis. Am. J. Pathol. 141, 821–829.Google Scholar
  21. 21.
    Cornelese-ten Velde, I. and Prins, F.A. (1990) New sensitive light microscopical detection of colloidal gold on ultrathin sections by RCM. Combination of reflection contrast and electron microscopy in post-embedding Immunogold. Histochemistry 94, 61–71.PubMedCrossRefGoogle Scholar
  22. 22.
    de Jong, D., Prins, F. A., Mason, D. Y., Reed, J. C., van Ommen, G. B., and Kluin, P. M. (1994) Subcellular localization of bcl-2 protein in malignant and normal lymphoid cells. Cancer Res. 54, 256–260.PubMedGoogle Scholar
  23. 23.
    Balzar, M. and Prins F. A. (1999) The structural analysis of adhesions mediated by Ep-CAM. Exp. Cell Res. 246, 108–121.PubMedCrossRefGoogle Scholar
  24. 24.
    Curtis, A. S. G. (1964) The mechanism of adhesion of cells to glass. A study by interference reflection microscopy. J. Cell. Biol. 20, 199–215.PubMedCrossRefGoogle Scholar
  25. 25.
    Kruidering, M., Maasdam, D. H., Prins F. A., de Heer, E., Mulder, G. J., and Nagelkerke, J. F. (1994) Evaluation of nephrotoxicity in vitro using a suspension of highly purified porcine proximal tubular cells and characterization of the cells in primary culture. Exp. Nephrol. 2, 334–344.Google Scholar
  26. 26.
    Neelissen, J. A. M., Arth, C., Wolff, M., Schrijvers, A. H. G. J., Jungingen, H. E., and Bode, H. E. (2000) Visualization of percutaneous 3H-estradiol and 3Hnorethindrone acetate transport across human epidermis as a function of time. Acta Derm. Venereol. 208, 36–43.Google Scholar
  27. 27.
    Rickert, C. H., Filler, T. J., and Gullotta, F. (1997) Reflection contrast microscopy on silver-stained amyloid deposits in Alzheimer’s disease. Sci. Tech. Inf. XI(5), 136–142.Google Scholar
  28. 28.
    Diaz, C. L., Melchers, P. J. J., Hooykaas, P. J. J., Lugtenberg, B. J. J., and Kijne, J. W. (1989) Root lectin as a determinant of host-plant specificity in the Rhizobium-legume symbiosis. Nature 338, 579–581.CrossRefGoogle Scholar
  29. 29.
    van der Burg, M. P. M., Guicherit, O. R., Frölich, M., et al. (1994) Assessment of islet isolation efficacy in dogs. Cell Transplant. 3, 91–101.PubMedGoogle Scholar
  30. 30.
    Landegent, J. E., Jansen in de Wal, N., van Ommen, G. B., et al. (1984) Chromosomal localization of a unique gene by non-autoradiographic in situ hybridization. Nature 317, 175–177.CrossRefGoogle Scholar
  31. 31.
    Landegent, J. E., Jansen in de Wal, N., Ploem, J. S., and van der Ploeg, M. (1985) Sensitive detection of hybridocytochemical results by means of reflection-contrast microscopy. J. Histochem. Cytochem. 33, 1241–1246.PubMedGoogle Scholar
  32. 32.
    Macville, M. V. E., van Dorp, A. M., Wiesmeijer, K. C., Dirks, R. W., Fransen, A. M., and Raap, A. (1995) Monitoring morphology and signal during non-radioactive in situ hybridization procedures by reflection-contrast microscopy and transmission electron microscopy. J. Histochem. Cytochem. 43, 665–674.PubMedGoogle Scholar
  33. 33.
    Cornelese-ten Velde, I., Bonnet, J., Tanke, H. J., and Ploem, J. S. (1990) Reflection contrast microscopy performed on epi-illumination microscope stands: comparison of reflection contrast-and epi-polarization microscopy. J. Microsc. 159, 1–13.Google Scholar
  34. 34.
    Cornelese-ten Velde, I. and Prins, F. A. (1990) New sensitive light microscopical detection of colloidal gold on ultrathin sections by RCM. Combination of reflection contrast and electron microscopy in post-embedding immunogold histochemistry. Histochemistry 94, 61–71.PubMedCrossRefGoogle Scholar
  35. 35.
    Cornelese-ten Velde, I., Wiegant, J., Tanke, H. J., and Ploem, J. S. (1989) Improved detection and quantitation of the (immuno) peroxidase product using reflection contrast microscopy. Histochemistry 92, 153–160.PubMedCrossRefGoogle Scholar
  36. 36.
    Ploem, J. S., Cornelese-ten Velde, I., Prins, F. A., and Bonnet, J. (1995) Reflectioncontrast microscopy: an overview. RMS 30/3, 185–192.Google Scholar
  37. 37.
    Ploem, J. S., Prins, F. A., and Cornelese-ten Velde, I. (1998) Reflection-contrast microscopy for high definition images with light microscopy: practical tips using new Leica DM R equipment for RCM. Sci. Tech. Inf. CDR 1, 47–86.Google Scholar
  38. 38.
    Prins, F. A., van Diemen-Steenvoorde, R., Bonnet, J., and Cornelese-ten Velde, I. (1993) Reflection contrast microscopy of ultrathin sections in immunocytochemical localization studies: a versatile technique bridging electron microscopy with light microscopy. Histochemistry 99, 417–425.PubMedGoogle Scholar
  39. 39.
    Ploem, J. S., Prins, F. A., and Cornelese-ten Velde, I. (1999) Reflection-contrast microscopy, in Light Microscopy in Biology: A Practical Approach, 2nd ed., (Lacey, A.J., ed.), Oxford University Press, Oxford, pp. 275–310.Google Scholar
  40. 40.
    Ploem, J. S. (1975) General introduction. Ann. NY Acad. Sci. 254, 40.CrossRefGoogle Scholar

Copyright information

© Humana Press Inc. 2006

Authors and Affiliations

  • F. A. Prins
    • 1
  • I. Cornelese-ten Velde
    • 1
  • E. de Heer
    • 1
  1. 1.Department of PathologyLeiden University HospitalLeidenThe Netherlands

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