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

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, diffusion-driven SWS increase from 1.7 {+/-} 0.4 m/s to 2.5 {+/-} 0.5 m/s, 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. Key Results- Point-excitation OMTHE at microscopic resolution maps dynamic stiffness changes in collagen gels during polymerization and crosslinking at high frame rates. - Polymerization and crosslinking of collagen show distinct time courses with polymerization being in the order of minutes ahead of crosslinking. - Collagen stiffening due to polymerization precedes changes in optical density as seen by light microscopy.