Skip to main content
SpringerLink
Log in

Polarized light microscopy in the study of the molecular structure of collagen and reticulin

  • Published:
Histochemistry Aims and scope Submit manuscript

Summary

Although collagen structure has been studied by polarized light microscopy since the early 19th century and continued since, modern studies and reviews failed to correlate the conclusions based on data obtained by the techniques with those of polarized light microscopy. Collagen I is intensely positively birefringent in respect to length of the fibres; the positive intrinsic birefringence indicates a quasi-crystalline alignment parallel to the fibre and molecule axis of the amino acid residues of the polypeptide chains. This would not have been compatible with a helical structure but has been achieved by similar tilt angles and opposite directions of the coiling and supercoiling.

Birefringence characteristics of collagen are also affected by chemical treatments, extractions and staining procedures. Attachment of chemical groups to the anionic charges present on the surface of collagen molecules results in increased positive birefringence in the case of bipolar molecules attached to two or more anionic residues. Unipolar attachment to the same groups, or to the cationic groups of the associated proteoglycans, as well as sulfation or acetylation of hydroxyls of the protein and/or the carbohydrate, reduced or reversed the sign of birefringence. Increased birefringence caused by stretching cannot be due to intramolecular events and is caused by intermolecular changes. The same applies to changes in collagen during aging.

Reticulin is a group of different substances which mostly contain collagen III. The pliability and deformability of this collagen is related to its weakly negative birefringence due to large side chains and presence of different and greater amounts of interstitial proteoglycans and other molecules. The so-called reticulin of healing wounds differs in its constitution from other reticulins but is also rich in intermolecular carbohydrate components.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

References

  • Bailey AJ, Bazin S, Sims TJ, Le Lous M, Nicoletis C, Delaunay A (1975) Characterization of the collagen of human hypertrophic and normal scars. Biochim Biophys Acta 405:412–421

    Google Scholar 

  • Banga I, Balo J, Szabo D (1956) Submicroscopic structure of collagen fibres: their contraction and relaxation. Acta Morphol Acad Sci Hung 6:391–402

    Google Scholar 

  • Berrens L, van Driel LMY (1962) Vergleichende Untersuchungen über menschliches Kollagen und Reticulin. Naturwissenschaften 49:608

    Google Scholar 

  • Bornstein P, Sage H (1980) Structurally distinct collagen types. Annu Rev Biochem 49:957–1003

    Google Scholar 

  • Bouteille M, Pease DC (1971) The tridimensional structure of native collagenous fibrils, their proteinaceous filaments. J Ultrastruct Res 35:314–338

    Google Scholar 

  • Brodsky B, Eikenberry EF (1982) Characterization of fibrous forms of collagen. Methods Enzymol 82:127–174

    Google Scholar 

  • Brewer DB (1957) Differences in the fine structure of collagen and reticulin as revealed by the polarizing microscope. J Pathol Bacteriol 74:371–385

    Google Scholar 

  • Castellani PP, Morocutti M, Franchi M, Ruggeri A, Bigi A, Roveri N (1983) Arrangement of microfibrils in collagen fibrils of tendons in the rat tail. Ultrastructural and x-ray diffraction investigation. Cell Tissue Res 234:735–743

    Google Scholar 

  • Eyre DR (1980) Collagen: molecular diversity in the body's protein scaffold. Science 207:1315–1322

    Google Scholar 

  • Fleischmajer R, Gay S, Perlish JS, Cesarinni JP (1980) Immunoelectron microscopy of type III collagen in normal and scleroderma skin. J Invest Dermatol 75:189–191

    Google Scholar 

  • Folkhard W, Knorzer E, Mosler E, Nemetschek T (1984) Packing of collagen molecules modified with 2-propanol. J Mol Biol 177:841–844

    Google Scholar 

  • Fraser RD, MacRae TP, Miller A, Suzuki E (1983) Molecular conformation and packing in collagen fibrils. J Mol Biol 167:497–521

    Google Scholar 

  • Gallop PM (1964) Concerning some special structural features of the collagen molecule. In: New York Heart Association (ed) Connective tissue: intercellular macromolecules. Little Brown & Co, Boston, pp 77–92

    Google Scholar 

  • Gay S, Miller EJ (1978) Collagen in the physiology and pathology of connective tissue. Gustav Fischer, Stuttgart

    Google Scholar 

  • Gay S, Vijanto J, Raekallio J, Penttinen R (1978) Collagen types in early phases of wound healing in children. Acta Chir Scand 144:205–211

    Google Scholar 

  • Glegg RE, Edinger D, Leblond CP (1953) Some carbohydrate components of reticular fibers. Science 118:614–616

    Google Scholar 

  • Gotoh T, Sugi Y, Hirakow R (1983) Ultrastructural construction of collagen fibrils as revealed by the freeze-fracture technique. J Electron Microsc (Tokyo) 32:213–215

    Google Scholar 

  • Grant ME, Freeman IL, Schofield JD, Jackson DS (1969) Variations in the carbohydrate content of human and bovine polymeric collagens from various tissues. Biochim Biophys Acta 177:682–685

    Google Scholar 

  • Hofmann H, Fietzek PP, Kühn K (1978) The role of polar and hydrophobic interactions for the molecular packing of type I collagen: a three-dimensional evaluation of the amino acid sequence. J Mol Biol 125:137–165

    Google Scholar 

  • Hofmann H, Voss T, Kühn K, Engel J (1984) Localization of flexible sites in thread-like molecules from electron micrographs. Comparison of interstitial, basement membrane and intima collagens. J Mol Biol 172:325–343

    Google Scholar 

  • Hosemann R (1973) Paracrystals in biopolymer and synthetic polymers. Endeavour 32:99–105

    Google Scholar 

  • Hukins DWL (1978) Bone stiffness explained by the liquid crystal model for the collagen fibril. J Theor Biol 71:661–667

    Google Scholar 

  • Jackson DS, Williams G (1956) Nature of reticulin. Nature 178:915–916

    Google Scholar 

  • Jozsa L, Reffy A, Balint JB (1984) Polarization and electron microscopic studies on the collagen of intact and ruptured human tendons. Acta Histochem 74:209–215

    Google Scholar 

  • Junqueira LCU, Bignolas G, Brentani RR (1979) Picrosirius staining plus polarization microscopy, a specific method for collagen detection in tissue sections. Histochem J 11:447–455

    Google Scholar 

  • Junqueira LCU, Cossermelli W, Brentani RR (1978) Differential staining of collagens type I, II and III by Sirus red and polarization microscopy. Arch Histol Jpn 41:267–274

    Google Scholar 

  • Junqueira LCU, Montes GS, Sanchez EM (1982) The influence of tissue section thickness on the study of collagen by the Picrosirius-polarization method. Histochemistry 74:153–156

    Google Scholar 

  • Kurkinen M, Vaheri A, Roberts PJ, Stenman S (1980) Sequential appearance of fibronectin and collagen in experimental granulation tissue. Lab Invest 43:47–51

    Google Scholar 

  • Laufer M, Ashkenazi C, Katz D, Wolman M (1974) Orientation of collagen in wound healing. Br J Exp Pathol 55:233–236

    Google Scholar 

  • Lillie RD (1977) HJ Conn's biological stains, 9th edn., Williams and Wilkins, Baltimore, p 54

    Google Scholar 

  • Lindner J, Graf HA, Schweinitz V, Freytag G (1960) Reactionsformen von Kollagenfasern. Acta Histochem 9:231–246

    Google Scholar 

  • Loewe G (1953) Changes in the ground substance of ageing cartilage. J Pathol Bacteriol 65:381–388

    Google Scholar 

  • Massari F, Marsico G (1952) Precisazione sulla minuta struttura delle fibre reticolari. Boll Soc Ital Biol Sper 28:1141–1143

    Google Scholar 

  • Miller EJ (1976a) Biochemical characteristics and biological significance of the genetically-distinct collagens. Mol Cell Biochem 13:165–192

    Google Scholar 

  • Miller EJ (1976b) Molecular packing in collagen fibrils. In: Ramachandran GN, Reddi AH (eds) Biochemistry of collagen. Plenum Press, New York, pp 85–136

    Google Scholar 

  • Miller EJ, Gay S (1982) Collagen: an overview. Methods Enzymol 82:3–32

    Google Scholar 

  • Missmahl HP (1957) Doppelbrechung der retikulären Faser und sich hieraus ergebender Nachweis von gerichtet eingelagerten Lipoiden in die retikuläre Faser. Z Zellforsch 45:612–619

    Google Scholar 

  • Montes GS, Krisztán RM, Shigihara KM, Tokoro R, Mourao PAS, Junqueira LCU (1980) Histochemical and morphological characterization of reticular fibers. Histochemistry 65:131–141

    Google Scholar 

  • Ordman LJ, Gillman T (1966) Studies in the healing of cutaneous wounds. I. The healing of incisions through the skin of pigs. Arch Surg 93:857–882

    Google Scholar 

  • Parry DAD, Barnes GRG, Craig AS (1978) A comparison of the size distribution of collagen fibrils in connective tissues as a function of age and a possible relation between fibril size distribution and mechanical properties. Proc R Soc London (Biol) 203:305–321

    Google Scholar 

  • Pease DC, Bouteille M (1971) The tridimensional ultrastructure of native collagenous fibrils, cytochemical evidence for a carbohydrate matrix. J Ultrastruct Res 35:339–358

    Google Scholar 

  • Pras M, Glynn LE (1973) Isolation of a non-collagenous reticulin component and its primary characterization. Br J Exp Pathol 54:449–456

    Google Scholar 

  • Pras M, Johnson GD, Holborrow EJ, Glynn LE (1974) Antigenic properties of a non-collogenous-reticulin component of normal connective tissue. Immunology 27:469–478

    Google Scholar 

  • Prockop DJ, Berg RA, Kivirikko KI, Uitto J (1976) Intracellular steps in the biosynthesis of collagen. In: Ramachandran GN, Reddi AH (eds) Biochemistry of collahen. Plenum Press, New York, pp 163–273

    Google Scholar 

  • Puchtler H, Waldrop FS (1978) Silver impregnation methods for reticulum fibers and reticulin: a re-investigation of their origins and specificity. Histochemistry 57:177–188

    Google Scholar 

  • Ramachandran GN (1963) Molecular structure of collagen. In: Hall DA (ed) International review of connective tissue research, vol 1. Academic Press, New York, pp 127–182

    Google Scholar 

  • Ramachandran GN, Ramakrishnan C (1976) Molecular structure of collagen. In: Ramachandran GN, Reddi AH (eds) Biochemistry of collagen. Plenum Press, New York, pp 45–84

    Google Scholar 

  • Ramachandran GN, Reddi AH (1976) Biochemistry of collagen. Plenum Press, New York

    Google Scholar 

  • Romhanyi G, Bukovinszky A, Deak G (1972) On the ultrastructure and proteolytic susceptibility of acetylcollagen as studied by topo-optical reactions. Acta Morphol Acad Sci Hung 20:49–69

    Google Scholar 

  • Romhyani G, Bukovinszky A, Deak G (1973) Sulfation as a collagen-specific reaction. The ultrastructure of sulfate collagen, basement membranes and reticulin fibers as shown by topo-optical staining reactions. Histochemie 36:125–138

    Google Scholar 

  • Romhanyi G, Deak G (1967) The submicroscopic structure of collagen fibers as revealed by topo-optical staining reactions with cationic dyes. Acta Morphol Acad Sci Hung 15:147–161

    Google Scholar 

  • Romhanyi G, Deak G, Bukovinszky A (1970) Collagen-specific topo-optical staining reaction with Congo red and its ultrastructural interpretation. Acta Morphol Acad Sci Hung 18:261–282

    Google Scholar 

  • Romhanyi G, Deak G, Fischer J (1975) Aldehyde bisulfite-toluidine blue (ABT) staining as a topo-optical reaction for demonstration of linear order of vicinal OH groups in biological structures. Histochemistry 43:333–348

    Google Scholar 

  • Ruggeri A, Benazzo F, Reale E (1979) Collagen fibrils with straight and helicoidal microfibrils: a freeze-fracture and thin-section study. J Ultrastruct Res 68:101–108

    Google Scholar 

  • Schmidt WJ (1934) Polarisationsoptische Analyse des submikroskopischen Baues von Zellen und Geweben. In: Abderhalden E (ed) Handbuch der biologischen Arbeitsmethoden, Abt V, Teil 10. Urban & Schwarzenberg, Berlin, pp 435–665

    Google Scholar 

  • Schmidt WJ (1957) Einiges über die Polarisationsoptik von Proteinstrukturen. Acta Histochem 4:175–192

    Google Scholar 

  • Schnabel R (1966) Zur mikroskopischen Untersuchung optisch anisotroperstructuren des Nervensystems mit circular polarisiertem Licht. Acta Neuropathol 7:180–184

    Google Scholar 

  • Scott JE (1980) Collagen-proteoglycan interactions. Localization of proteoglycans in tendon by electron microscopy. Biochem J 887–891

  • Silbert JE (1973) Biosynthesis of mucopolysaccharides and protein-polysaccharides. In: Perez-Tamayo R, Roikind M (eds) Molecular pathology of connective tissues. Marcel Dekker, New York, pp 323–353

    Google Scholar 

  • Squire JM, Freundlich A (1980) Direct observation of a transverse periodicity in collagen fibrils. Nature 288:410–413

    Google Scholar 

  • Snodgrass MJ (1977) Ultrastructural distinction between reticular and collagenous fibers with an ammoniacal silver stain. Anat Rec 187:191–205

    Google Scholar 

  • Stenman S, Vaheri A (1978) Distribution of a major connective tissue protein, fibronectin in normal human tissues. J Exp Med 147:1054–1064

    Google Scholar 

  • Svendsen KH, Koch MHJ (1982) X-ray diffraction evidence of collagen molecular packing and cross-linking in fibrils of rat tendon observed by synchrotron radiation. EMBO J 669–674

  • Szendroi M, Vajta G, Kovacs L, Schaff Z, Lapis K (1984) Polarization colours of collagen fibres: a sign of collagen production activity in fibrotic processes. Acta Morphol Hung 32:47–55

    Google Scholar 

  • Tonna EA, Hatzel G (1967) Age changes in the distribution of connective tissue elements in human material studies with polarized light microscopy. J Gerontol 22:281–289

    Google Scholar 

  • Traub W (1978) Molecular assembly in collagen. FEBS Lett 92:114–120

    Google Scholar 

  • Unsworth DJ, Scott DL, Almond TJ, Beard HK, Holborow EJ, Walton KW (1982) Studies on reticulin. I: Serological and immunohistological investigations of the occurrence of collagen type III, fibronectin and the non-collagenous glycoprotein of Pras and Glynn in reticulin. Br J Exp Pathol 63:154–166

    Google Scholar 

  • Valentin G (1861) Die Untersuchung der Pflanzen und der Thiergewebe in polarisiertem Lichte. Wilhelm Engelmann, Leipzig

    Google Scholar 

  • Velican C, Velican D (1968) Studies on the reticulin network of human liver. Virchows Arch (Cell Pathol) 1:297–316

    Google Scholar 

  • Weber L, Kirsch E, Müller P, Krieg T (1984) Collagen type distribution and macromolecular organization of connective tissue in different layers of human skin. J Invest Dermatol 82:156–160

    Google Scholar 

  • Williams JF, McCulagh KG, Silver JA (1984) The distribution of types I and III collagen and fibronectin in the healing equine tendon. Connect Tissue Res 12:211–227

    Google Scholar 

  • Windrum GM, Kent PW, Eastoe JE (1955) The constitution of human renal reticulin. Br J Exp Pathol 36:49–59

    Google Scholar 

  • Wolman M (1970) On the use of polarized light in pathology. Pathol Annu 5:381–416

    Google Scholar 

  • Wolman M (1975) Polarized light microscopy as a tool of diagnostic pathology. J Histochem Cytochem 23:21–50

    Google Scholar 

  • Wolman M, Bubis JJ (1965) The cause of the green polarization color of amyloid stained with Congo red. Histochemie 4:351–356

    Google Scholar 

  • Wolman M, Gillman T (1972) A polarized light study of collagen in dermal wound healing. Br J Exp Pathol 53:85–89

    Google Scholar 

Download references

Author information

Author notes

  1. M. Wolman (Visiting Professor of Anatomy)

    Present address: Louisiana State University Medical Center, New Orleans, USA

Authors and Affiliations

  1. Department of Pathology, Tel Aviv University, Sackler Faculty of Medicine, 69978, Tel-Aviv, Israel

    M. Wolman (Visiting Professor of Anatomy)

  2. Department of Anatomy, Louisiana State University School of Dentistry, 1100 Florida Avenue, 70119, New Orleans, LA, USA

    F. H. Kasten

Authors
  1. M. Wolman
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. F. H. Kasten
    View author publications

    You can also search for this author in PubMed Google Scholar

Additional information

Supported in part by BRSG Grant No. S07-RR05704-13 awarded to the Louisiana State University School of Dentistry by the Biomedical Research Grant Program, Division of Research Resources, National Institutes of Health. The authors are grateful to Mr. Raymond Calvert, Head Illustrator of the Graphic Arts Section, Department of Learning Resources, L.S.U. School of Dentistry for the graphic illustrations and to Ms. Toni M. LaBranch and Ms. Linda Watson for typing the drafts and manuscript

Rights and permissions

Reprints and permissions

About this article

Cite this article

Wolman, M., Kasten, F.H. Polarized light microscopy in the study of the molecular structure of collagen and reticulin. Histochemistry 85, 41–49 (1986). https://doi.org/10.1007/BF00508652

Download citation

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1007/BF00508652

Keywords

Search

Navigation