High-Speed Full-Color Polarized Light Imaging of Collagen Using a Polarization Camera

Polarized light imaging (PLI) has been effective in visualizing and quantifying collagen content. Collagen-specific data are often overlaid over the tissue image for visualization. However, such contextual tissue images are typically in grayscale and lack important color information, limiting the usefulness of PLI in imaging the stained histology slides and for surgical guidance. The objective of this study was to develop a robust and easy-to-implement PLI technique to capture both true color and birefringent collagen data, and we call it ColorPOL. ColorPOL uses only one polarization-sensitive camera to capture information at 75 frames per second. The true color images were synthesized from individual RGB images, and collagen-specific information (fiber orientation and retardance) was derived from the green channel image. We implemented ColorPOL in transmission mode on an upright microscope and in reflection mode for wide-field thick tissue imaging. The color images in both implementations provided valuable color tissue context that facilitated the identification and localization of collagen content. Additionally, we demonstrated that in reflection mode, the high imaging speed enabled us to record and visualize continuous deformations of the collagenous tissues (tendons, sciatic nerves, and blood vessels) overlaid on the processed collagen-specific information. Robust performance and flexible configuration will make ColorPOL a valuable tool in basic research and translational applications.