Figure 1.
Polarized Raman spectroscopy of RTT.
Spectra taken at the same spot of the sample were collected with two different laser polarization orientations [parallel (laser X, blue line) and perpendicular (laser Z, red line) to the tendon axis]. A large anisotropy of the amide I band in the two different laser to fiber configurations is due to the preferential orientation of vibrational units along the main axis of the tendon.
Figure 2.
Global coordinate system and the Euler angles that describe the position of the molecular structures of collagen-like peptide and alpha helix.
The directions of propagation of the incident and scattered beam (Ei and Es) are represented by the red arrows parallel to the X axis while yellow bar represents the position of molecular structures. The “in plane” rotations are performed in the plane YZ and the “out of plane” rotations are performed in the ZX plane.
Figure 3.
Theoretical prediction of the anisotropic response of amide I band for collagen-like and alpha helix molecules.
Normalized anisotropic response of the amide I band of a collagen-like peptide molecule (ID:1CAG) and alpha helix (ID:1XQ8) located at A) (φ = 90°, θ = 0°,ω = 0°) on the plane ZY, B) (φ = 90°, θ = 90°, ω = 0°) on the plane ZY and C) (φ = 0°, θ = 0°,ω = 0°) on the plane ZX. For the collagen-like peptide structure located “in plane” (A and B) the maximum response of the amide I band is obtained when the polarization of the light is parallel to the molecule position, the opposite response is observed for the alpha helix. In the “out of plane” (C) response both structures give rise to a much more isotropic response of the amide I band.
Figure 4.
Averaged theoretical amide I response of collagen-like peptide for rotation around the main axis of the molecule.
A) Amide I band response of a collagen-like peptide (ID: 1BKV) oriented (φ = 0°, θ = 0°,) plane (i.e. perpendicular to the ZY plane) rotated at different ω angles around the c-axis and its average response. B) Amide I band response of a collagen-like peptide (ID:1BKV) lying in the ZY plane (φ = 90°, θ = 0°) that has been rotated at different ω angles around the c-axis of the molecule and its average response. All graphs are plotted as functions of the polarization angle β of the incident laser beam (according to eq.3).
Figure 5.
Averaged theoretical amide I response of collagen-like peptide molecules for “out of plane” rotation.
Normalized amide I response for four different collagen-like peptide structures (ID: 1BKV, 1CGD, 1QSU) that are rotated in the plane XZ [from (φ = 90°, θ = 90°) to (φ = 0°, θ = 90°)] vs the polarization angle of the incident light. The responses have been averaged at angles ω = 0°, ω = 90°, ω = 180°, ω = 270°. All the molecules exhibit a similar trend independent from which collagen-like peptide crystal structure. The average responses for all the selected structures are marked in bold.
Figure 6.
In situ PRS mapping of the collagen orientation in −45 and 45 degrees tilted dried RTT.
(A) and (C) show maps obtained by fitting thirteen Raman images collected with different polarization angles of the incident laser light. The direction of arrows indicates the orientation of collagen molecules, their length represents the amplitude of the fitting curve, and the color code is the average intensity of the amide I band. (B) and (D) are example of experimental points extracted from the area marked in (1) and (2), respectively.
Figure 7.
Experimental PRS results for RTT fibers in both “in plane” and “out of plane” configuration.
Sinusoidal anisotropic response was found for fiber placed at φ = 90°, θ = 90° (collagen parallel to Z-axis, “in plane” configuration) with maximal intensity when the polarization of the light is perpendicular to the fiber and minimal when parallel. In the case of φ = 0°, θ = 0° configuration (collagen parallel to laser beam, “out of plane” configuration) isotropic response was observed.