Effects of sample orientation in Raman microspectroscopy of collagen fibers and their impact on the interpretation of the amide III band

In most tissues, collagen is present in the form of fibrils, large supramolecular assemblies of fundamental structural units of polypeptide chains. In this contribution we report a clear correlation between the intensity profile of the Raman spectrum of collagen and the orientation of a collagen fiber, depending on the laser polarization. More specifically, fiber orientation is shown to affect the relative intensity ratio between the bands at 1245 and 1268 cm−1 (amide III doublet). In view of this finding based on the analysis of a collagen fiber, the customary use of this parameter as an exclusive marker for collagen secondary structure in tissues has to be reconsidered, taking into account fibrils orientation. Moreover, in Raman microspectroscopy of tissues this effect could be exploited to yield information, which could be relevant for diagnostic purposes or fundamental research, about the orientation of collagen fibrils.