Abstract
In this study, polarized Raman spectroscopy (PRS) was used to characterize the anisotropic response of the amide I band of collagen as a basis for evaluating three-dimensional collagen fibril orientation in tissues. Firstly, the response was investigated theoretically by applying classical Raman theory to collagen-like peptide crystal structures. The theoretical methodology was then tested experimentally, by measuring amide I intensity anisotropy in rat tail as a function of the orientation of the incident laser polarization. For the theoretical study, several collagen-like triple-helical peptide crystal structures obtained from the Protein Data Bank were rotated “in plane” and “out of plane” to evaluate the role of molecular orientation on the intensity of the amide I band. Collagen-like peptides exhibit a sinusoidal anisotropic response when rotated “in plane” with respect to the polarized incident laser. Maximal intensity was obtained when the polarization of the incident light is perpendicular to the molecule and minimal when parallel. In the case of “out of plane” rotation of the molecular structure a decreased anisotropic response was observed, becoming completely isotropic when the structure was perpendicular to the plane of observation. The theoretical Raman response of collagen was compared to that of alpha helical protein fragments. In contrast to collagen, alpha helices have a maximal signal when incident light is parallel to the molecule and minimal when perpendicular. For out-of-plane molecular orientations alpha-helix structures display a decreased average intensity. Results obtained from experiments on rat tail tendon are in excellent agreement with the theoretical predictions, thus demonstrating the high potential of PRS for experimental evaluation of the three-dimensional orientation of collagen fibers in biological tissues.
Highlights
Collagen is an important structural component in many biological tissues including bone, teeth and skin [1]
Raman spectroscopy is based on the analysis of the inelastic scattering of light interacting with molecules in which the frequency shift between the incident and the scattered light is associated with a particular vibration mode of a chemical bond
The alpha helix structure exhibits the opposite behaviour: the minimal amide I intensity response is observed when the polarization direction of the incident light is perpendicular to the structure and maximal when parallel
Summary
Collagen is an important structural component in many biological tissues including bone, teeth and skin [1] It imparts toughness [2] and deformability to these tissues and by controlling its fibrillar arrangement at multiple hierarchical levels [3] organisms can produce tissues both with highly directional (anisotropic) material properties as well as more isotropic properties [4]. Raman spectroscopy is based on the analysis of the inelastic scattering of light interacting with molecules in which the frequency shift between the incident and the scattered light is associated with a particular vibration mode of a chemical bond Considering their hierarchical structure from atomic up to macroscopic scale, several biological materials have already been the object of studies by PRS. Bone has been extensively studied using PRS since several material property parameters such as mineral to organic matrix ratio can be extracted from the Raman spectrum [17,18,19]
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