The Histochemical Journal

, Volume 14, Issue 6, pp 857–878 | Cite as

Polarization microscopy and microspectrophotometry of Sirius Red, Picrosirius and Chlorantine Fast Red aggregates and of their complexes with collagen

  • Benedicto De Campos Vidal
  • Maria Luiza S. Mello
  • Édson R. Pimentel


A detailed quantitative analysis of the anisotropic properties of Sirius Red F3B, Picrosirius, and Chlorantine Fast Red crystals, and of their complexes with a macromolecularly oriented protein either in a pure form or as part of a tissue structure was carried out. Collagen I was used as the protein model. Linear dichroism and dispersion of birefringence were investigated in dye aggregates, in stained filaments of collagen I and in collagen bundles in sections of tendon. A positive linear dichroism, the characteristics of which varied as a function of the dye type used, was demonstrated for the dye aggregates and stained substrates. However, even thin regions of the stained tendon collagen bundles showed very high absorbances, differing from the pattern reported previously, for collagen stained with another sulphonated azo dye, Xylidine Ponceau. Consequently, not all these dyes enable protein concentration and orientation to be determined in collagen-containing structures. From the linear dichroism patterns it is assumed that the long axis of the molecules of these azo dye is mostly parallel to that of filaments of pure collagen I and statistically parallel to the long axis of collagen bundles of tendon sections. The dye aggregates and, stained pure collagen I and tendon collagen bundles exhibited birefringent images with interference colours that varied as a function of thickness and packing state of the preparations, which is in agreement with reports in the literature. The optical retardations of the collagen bundles increased by a factor of 5–6 times after staining with Picrosirius. From data on form dichroism it is concluded that when studying the macromolecular orientation of collagen preparations stained with azo dyes, the choice of the mounting medium deserves consideration.


Sulphonated Polarization Microscopy Ponceau Collagen Bundle Pure Collagen 
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. Conn, H. J. (1969)Biological stains. A handbook of the nature and uses of the dyes employed in the biological laboratory. Baltimore: Williams & Wilkins.Google Scholar
  2. Deitch, A. D. (1966) Cytophotometry of proteins. InIntroduction to quantitative Cytochemistry (edited byWied, G. L.), pp. 451–68. New York, London: Academic Press.Google Scholar
  3. Ewing, G. W. (1969)Instrumental Methods of Chemical Analysis. New York: McGraw-Hill.Google Scholar
  4. Frey-Wissling, A. (1948)Submicroscopy morphology of protoplasm and its derivatives. Amsterdam: Elsevier.Google Scholar
  5. Goldstein, D. J. (1969) Detection of dichroism with the microscope.J. Microsc. 89, 19–36.Google Scholar
  6. Joiner, D. W., Puchtler, H. &Sweat, F. (1968) Staining of immature collagen by resorcinfuchsin in infant kidneys.J. R. microsc. Soc. 88, 461–71.Google Scholar
  7. Junqueira, L. C. U., Bignolas, G. &Brentani, R. R. (1979) Picrosirius staining plus polarization microscopy, a specific method for collagen detection in tissue sections.Histochem. J. 11, 447–55.Google Scholar
  8. Lison, L. (1960)Histochimie et Cytochimie Animales. Paris: Gauthier-Villars.Google Scholar
  9. Missmahl, H. P. (1966) Birefringence and dichroism of dyes and their significance in the detection of oriented structures. InIntroduction to Quantitative Cytochemistry (edited byWied, G. L.), pp. 539–47. New York, London: Academic Press.Google Scholar
  10. Nordén, B. (1980) Applications of linear dichroism spectroscopy.Appl. Spectr. Rev. 14, 157–248.Google Scholar
  11. Pérez-Tamayo, R. &Montfort, J. (1980) The susceptibility of hepatic collagen to homologous collagenase in human and experimental cirrhosis of the liver.Am. J. Pathol. 100, 427–42.Google Scholar
  12. Pimentel, E. R. & Vidal, B. C. (1980) Propriedades anisotrópicas de feixes de colágeno e glicoproteínas ligadas ao sirius red.II Congr. Bras. Biol Celular, Rio de Janeiro, Abstracts, pp. 156–7.Google Scholar
  13. Puchtler, H., Sweat, F., Jackson, J. G. &Joiner, D. W. (1966) Collagen-like staining, polarization and fluorescence microscopic properties of ‘elastic fibers’ in hyperplastic arteriosclerosis. InBiochemistry and Physiology of Connective Tissue (edited byCompte, P.). Lyon: Société Ormeco et Imprimerie du Sud-Est.Google Scholar
  14. Schmidt, W. J. (1937)Doppelbrechung von Karyoplasma, Zytoplasma und Metaplasma. Berlin: Borntraeger.Google Scholar
  15. Schmidt, W. J. &Keil, A. (1958)Die gesunden und die erkrankten Zahngewebe des Menschen und der Wirbeltiere im Polarizations mikroskop. München: Carl Hanser Verlag.Google Scholar
  16. Sweat, F., Puchtler, H. &Rosenthal, S. I. (1964) Sirius red F3BA as a stain for connective tissueArch. Path. 78, 69–72.Google Scholar
  17. Venkataraman, K. (1952)The Chemistry of Synthetic Dyes. New York: Academic Press.Google Scholar
  18. Vidal, B. C. (1970) Dichroism in collagen bundles stained with xylidine ponceau 2R.Ann. Histochim. 15, 289–96.Google Scholar
  19. Vidal, B. C. (1972) Toluidine blue: Cotton effect-like phenomena of crystal aggregates obtained by drying on microscopical slides.Ann. Histochim. 17, 151–7.Google Scholar
  20. Vidal, B. C. (1980) The part played by proteoglycans and structural glycoproteins in the molecular orientation of collagen bundles.Cell. molec. Biol. 26, 415–21.Google Scholar
  21. Wood, G. C. (1964) The precipitation, of collagen fibres from solution.Int. Rev. Connect. Tiss. Res. 2, 1–28.Google Scholar
  22. Zeiss Information (1977) Continuous filter monochromator b with motor drive.Mikro Bull. 18, 1–2.Google Scholar

Copyright information

© Chapman and Hall Ltd 1982

Authors and Affiliations

  • Benedicto De Campos Vidal
    • 1
  • Maria Luiza S. Mello
    • 1
  • Édson R. Pimentel
    • 1
  1. 1.Department of Cell Biology, Institute of BiologyUNICAMPCampinasBrazil

Personalised recommendations