Spatiotemporal mapping of microscale stiffness during collagen polymerization and crosslinking by optical multifrequency time-harmonic elastography

Abstract

Optical multifrequency time-harmonic elastography (OMTHE) was used for rapid mechanical characterization of extra-cellular matrix-derived collagen networks at micrometer resolution. OMTHE was optimized for point-wise shear wave excitation in small sample volumes and compared to tabletop magnetic resonance elastography (ttMRE) and optical intensity changes. Dynamic stiffening due to the fluid–gel transition during collagen polymerization and chemical crosslinking using glutaraldehyde was tracked by shear waves speed (SWS) at vibration frequencies between 3 and 10 kHz and frame rates up to 4 kHz. During collagen polymerization, after an initial lag phase, SWS increased on average 6 ± 3 min earlier than optical density, suggesting that a load-bearing percolating fiber network was established before fibril thickening enhanced light scattering. In contrast, chemical crosslinking showed a lag-free, SWS increase driven by the diffusion and subsequent action of the crosslinker from 1.7 ± 0.4 m s⁻¹ to 2.5 ± 0.5 m s⁻¹, matching the relative SWS change from ground-truth ttMRE. In conclusion, OMTHE provides a unique research tool that quantifies biomechanical property changes in small biological samples with spatiotemporal resolutions of micrometers and seconds.