Image analysis of tendon helical superstructure using interference and polarized light microscopy
Introduction
Waveform or wave-like structures (WLS), also known as crimp, are thought to represent a planar organization of collagen fibers. Polarized light microscopy is generally recommended as the most appropriate method for detecting, describing and interpreting WLS (Baer et al., 1974, Gathercole and Keller, 1991, Gathercole and Keller, 1974). The use of polarized light microscopy can reveal the source of the collagen fiber birefringence that in turn allows for the accurate measurement, interpretation and validation of higher order organization.
The theory and mathematical treatment of collagen birefringence are based on experiments done with flow or streaming birefringence and on data from tissue sections (Vidal, 1964, Vidal, 1986). In principle, intrinsic birefringence is determined by the orientation and oscillator strength of all electronic transitions of the molecule that make up the fibril or filaments. In contrast form or textural birefringence depends on the geometry of the molecule (rod-like in the case of collagen), and varies with the volume fraction of the collagen molecules.
The intrinsic birefringence, which is an average of all electronic transitions in the molecules, provides the same information as linear dichroism (Cassim and Taylor, 1965, Oriel and Schellman, 1966, Casssim and Tobias, 1968, Nordén, 1978). Collagen shows linear dichroism only in the ultra-violet region, at a wavelength of 190/220 nm. The extent to which collagen fibers are packed into bundles plays an important role in the total birefringence (Vidal, 1986). Even when the main axis of the tendon is oriented at 45° relative to the polarizers, accurate measurements of optical path differences reveal the influence of WLS in the final results. This influence is reflected in the standard deviation of the optical path measurements. Morphological observations and measurements have suggested that WLS may reflect a helical organization of collagen fibers/bundles (Vidal, 1986).
Axial sections and whole mounts from acetic acid-treated bovine tendons examined under polarized light using Senarmont's and red first order compensators support the helical bundle supra-organization of collagen in these tendons; a similar organization has been detected in rat tendons (Vidal, 1995a). Reconstituted fibers obtained from collagen bundles self-assemble into a fibrous gel with precise viscoelastic properties that produce a helical supra-organization (Vidal, 1995b, Figs. 2–5).
A final helical supra-organization of collagen fibers, as predicted by the helical model of nucleation and propagation (Silver et al., 1992), agrees with the physico-chemical principles that regulate the self-assembly mechanisms, and confirms that “The chirality of macromolecules results in the formation of helical super-structures” and that “chirality may be used as a tool to assemble molecules into dissymmetric layered structures with (e.g. helical) shapes” (Cornelissen et al., 1998).
Considering that (i) the evidence for a helical arrangement of collagen fibers and bundles in tendons and in extruded fibrous gels of type I collagen (Vidal, 1995a, Vidal, 1995b), (ii) the reports that in many structures there is a helical arrangement of collagen fibrils (Reale et al., 1981) and (iii) experiments using sonicated collagen solutions to establish a correlation between crimp and helical arrangements, have proven inconclusive with regard to tendon supra-organization (Giraud-Guille and Besseau, 1998), we have reexamined this question both qualitatively and a quantitatively.
In this study, various methodological approaches were used to detect and quantify WLS, including polarized light microscopy and compensators, and video image analysis. The latter was used to characterize WLS by mathematical descriptors.
The main thesis of this work is that collagen fibers and/or bundles of fibers in tendons are not arranged in planar structures and that WLS are biological constructs which reflect the supramolecular helical arrangement of such fibers.
Section snippets
Material and methods
Fixed flexor bovine tendons and peeled rat-tails were used throughout this work. All preparative procedures were done at 10 °C. The fixative used was 2% paraformaldehyde in 0.1 M phosphate buffer, pH 7.4.
Bovine tendons were cleaned and freed of fat, muscle and sheaths. Fragments 10 mm long and 6 mm thick were fixed for 24 h in a refrigerator, temperature 8.5–9.0 °C, with the first 2 h of fixation being done under vacuum.
After fixation, the tendons were washed in distilled water and embedded in
Results
WLS or crimp showing various aspects of helical arrangements were observed in the tendons examined. For thick (20–50 μm) sections cut longitudinally to the main axis of bovine tendon, rotating the microscope stage starting at 45° relative to the polarizers revealed interference colors which were produced by the fiber thickness and variations of fiber orientation within the bundles. The orientation of the fibers and bundles was also responsible for the variations in the sinusoidal appearance of
Discussion
Collagen fiber orientation is best detected and described by physical properties such as birefringence and its dispersion. Collagen fiber birefringence is a well-established, accurate method for studying these fibers in tendon. Laser second harmonic transformations (Roth and Freund, 1982) have confirmed the usefulness of this approach as initially proposed by Vidal (1964). In this context, it is necessary to consider that birefringence as studied in this work is a sum of intrinsic plus textural
Acknowledgements
This work was supported by grants from the Brazilian agencies FAPESP, FAEP and CNPq. Animated movie of frames obtained during rotation of the microscope stage are available from the author.
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2017, Journal of BiomechanicsCitation Excerpt :These units, in turn, self-assemble into larger hierarchical structures (Duenwald et al., 2009; Franchi et al., 2010; Orgel et al., 2006; Ramachandran and Kartha, 1955; Silver et al., 2003). Fiber and fascicle level helical windings have been visualized as well (Jozsa et al., 1991; Kalson et al., 2012; Kannus, 2000; Khodabakhshi et al., 2013; Thorpe et al., 2013; Vidal, 2003). Additionally, computer modeling supports the theory of higher level helices (Reese et al., 2010; Zhao et al., 2016).