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

Abstract

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.

Paracoccidioidomycosis: a sequential histopathologic study of lesions in experimentally-infected rats

“…The reticular fibers in net-like pattern around the granulomas seen at the onset of infection, and not always detected by the Picrosirius-polarization method, preceding the presence of collagen types I and III, could corresponding to other types of proteins and glycoproteins of the matrix 39 .…”

Techniques to assess bone ultrastructure organization: orientation and arrangement of mineralized collagen fibrils

“…the mineral-to-matrix ratio), which is not uniform throughout a bone section, and thus complicates quantification of the 3D orientation of the mineralized collagen fibres from PLM images [64,68]. In general, there is a lack of standardization in the analysis of polarized light images, mainly due to the challenging technical demands and the complex theory of polarized light and birefringence, which can lead to incorrect interpretations of PLM outcomes [62,64].…”

“…Polarized light microscopy (PLM) has been used since the early nineteenth century to study collagen structure in different biological tissues, exploiting the positive intrinsic and form birefringence of collagen [5,38,62]. Especially for bone, use of PLM has been mostly driven by the early observation of the alternating bright and dark appearance of the lamellae in the osteons (figure 2).…”

A review of some recent developments in polarization-sensitive optical imaging techniques for the study of articular cartilage

“…Relating the quantitative value of birefringence to underlying physical properties is clearly more complicated in this case. Studies on Type-I collagen in tendon over the course of several decades has led to the consensus that this collagen type displays intense intrinsic positive birefringence due to the quasi-crystalline arrangement of the amino-acid residues that comprise the polypeptide chains of the collagen molecule alpha-chains 22 . This intrinsic birefringence can be further enhanced by binding acidic dyes such as picrosirius red to the collagen molecule.…”