Fiber alignment imaging during mechanical testing of soft tissues

A method to image fiber alignment during mechanical testing of soft tissues was developed based on quantitative polarized light microscopy. Images were acquired after passing light through a rotating polarizer, a tissue sample, and an effective circular analyzer at multiple polarizer positions during uniaxial mechanical testing. The image set was analyzed offline using harmonic analysis to generate an alignment image, which contains the direction and strength of alignment at each image pixel. Alignment images of the entire tissue sample were generated every 3-5 s during the mechanical test allowing stress-strain behavior to be correlated with fiber alignment. Loading of fresh tissue-equivalent samples in the direction normal to the initial direction of fiber alignment revealed a spatially inhomogeneous realignment into the loading direction, with most realignment occurring near the free edges undergoing maximum lateral contraction and prior to significant load developing. Glutaraldehyde-fixed samples, in contrast, showed little realignment until yielding occurred.