Polarization controlled second harmonic generation imaging of stretched collagen fibrils reveals collagen deformation pathway in situ
The tensile properties of single collagen fibrils, the building block of load-bearing tissues, have been studied extensively by nanomechanical techniques and molecular dynamics simulation. However, the deformation pathway of collagen molecules within fibrils has not yet been observed experimentally. In addition, the role played by divalent and trivalent enzymatic crosslinks in modulating this deformation pathway is poorly understood. Here we used polarization controlled second harmonic generation (SHG) imaging combined with atomic force microscopy (AFM) to characterize the molecular state of collagen triple helices within stretched single collagen fibrils. The fibrils were extracted from a pair of bovine leg tendons from the same animal in order to compare fibrils with a high amount of immature divalent crosslinks to fibrils with a high amount of mature trivalent crosslinks. By selecting fibrils with a large SHG intensity gradient along their length and then imaging the same fibrils by AFM we were able to link the observed intensity gradient with a gradient in D-band strain and a gradient in molecular strain as estimated from the SHG anisotropy parameter rho. In contrast to previous studies at the tendon scale, we observed that the SHG molecular strain is always larger than the D-band strain for all fibrils with this difference being largest for fibrils rich in divalent crosslinks. By analyzing the behavior of the relative density of SHG emitters as a function of molecular strain, we observe a two-state transition from an SHG producing to a non SHG producing state with a free energy barrier between 6 and 10 kBT that we propose corresponds to the local untwisting of the collagen triple helix superhelical twist which likely preceded bond rupture and loss of the SHG signal. We also show that trivalent crosslinks tend to delay the transition onset compared to divalent crosslinks.