Reflected cross-polarized light microscopy as a method for measuring collagen fiber crimp in musculoskeletal tissues

Many musculoskeletal tissues are composed primarily of type I collagen, which takes on a periodic crimp morphology that allows large tensile strains in the tissue. The spatial period of collagen fiber crimp may be used to infer internal strains in a tissue and is typically measured using transmitted cross-polarized light imaging of thin slices. However, slicing may induce specimen distortion and precludes mechanical loading of the specimen during imaging. We hypothesized that reflected cross-polarized light imaging of thick tissue explants would yield crimp period measurements comparable to those obtained from transmitted light imaging of thin slices. We further hypothesized that these measurements would be sensitive to applied uniaxial strain in the fiber direction. These hypotheses were tested by imaging both intervertebral disc outer annulus fibrosus and medial collateral ligament tissue specimens. We found that both transmitted and reflected light yielded similar crimp period measurements for intervertebral disc tissue, with an overall average of 43.5 ± 11.5 μm. Reflected light yielded a significantly higher crimp period with lower variance than transmission through thin specimens (54.1 ± 10.6 μm versus 50.4 ± 16.0 μm) in the ligament. Upon application of axial tension, crimp periods in both fibers increased at a rate of approximately three times the applied strain (with 3.17% applied strain yielding a 9.64 ± 4.4% increase in crimp period in the disc and an 11.7 ± 3.7% increase in the ligament), indicating significant fibril sliding. In support of our hypotheses, these findings suggest that reflected cross-polarized light is a suitable method for measuring collagen fiber crimp in musculoskeletal tissues, both statically and under tension.