Imaging the mechanical properties of a developing embryonic tendon using Brillouin microscopy.

Tendon mechanical properties are critical for proper musculoskeletal movement. Yet, current understanding of how tendons develop their mechanical properties remains incomplete. Atomic force microscopy and tensile testing are used to characterize early-stage and late-stage embryonic tendon mechanical properties respectively, but both require contact with the tissue, which can alter or destroy tissue structure or integrity and render the tissue unsuitable for subsequent assays. Brillouin confocal microscopy is an emerging tool used to measure mechanical properties of tissues in an all-optical, contact-free, label-free manner. Here, Brillouin confocal microscopy was successfully implemented to measure chick embryo Achilles tendon mechanical properties, which were then validated by comparisons to previously published tensile modulus data. Significant changes in mechanical properties that occur with development and due to experimentally induced changes in extracellular matrix crosslinking were quantifiable. Brillouin microscopy also detected increasing mechanical heterogeneity of developing tendons. The potential to map the spatial distribution of the mechanical properties of developing extracellular matrix using Brillouin microscopy was demonstrated. Measurement of mechanical properties of the tendon within the limb, and sensitivity to changes in tendon strain imposed by changing ankle flexion demonstrated the potential for Brillouin microscopy to quantify tendon properties in vivo. Collectively, these results provide an exciting first study using Brillouin microscopy for quantitative and spatial mapping of embryonic tendon mechanical properties in an all-optical, label-free manner, which could lead to mechanistic discoveries not possible with conventional mechanical testing methodologies. STATEMENT OF SIGNIFICANCE: Tendon injuries and disorders are problematic, as tendons lack the ability to regenerate, and current clinical solutions are insufficient. Knowledge gained from elucidating how embryonic tendons develop their mechanical properties could be used to inform the development of therapeutics to treat injured or abnormal tendons. However, conventional mechanical testing methods require challenging manipulations of small tissues and render tissues unusable for additional assays. Brillouin microscopy is a powerful technique that can image mechanical properties of tissues in a non-contact and label-free manner, which enables in vivo analysis of mechanical properties. In this study, we show that Brillouin microscopy can image mechanical properties of tendon in developing embryos, thus opening new avenues for investigation into the development of tendon mechanical properties.