Instant polarized light microscopy for real-time wide-field visualization of collagen architecture

Collagen fiber architecture plays an important role in the mechanical properties of soft tissues. Conventional polarized light microscopy done using linear polarizers and, sometimes, quarter-wave plates is a label-free imaging technique for quantifying collagen fiber architecture, specifically distribution and orientation. However, this technique has several limitations. First, it requires acquiring multiple images with different polarization states, which precludes many time-sensitive applications. Second, post-processing, especially image registration, reduces the level of detail discernible. Third, the added optical elements may cause glare under coaxial illumination, thus complicating the use of reflected light microscopy. We have recently demonstrated instant polarized light microscopy (IPOL), that requires only one image and therefore no registration. IPOL utilizes wavelength-dependent polarization to modify the spectrum of the illumination, generating visible colors that depend on fiber orientation and density. Herein we present two further advances on IPOL: we extend it to work with coaxial illumination allowing transmitted and reflected light microscopy, and we integrate it in a dissecting microscope. This permits real-time imaging, limited only by the camera frame rate, making it possible to track dynamic events, such as fast-acting responses to external forces or moving objects. We demonstrate IPOL with a field of view of 11 mm and a long working distance of 65 mm, which simplifies testing of large samples. IPOL provides both fiber distribution and orientation information in a single true-color snapshot, and therefore, it is suitable for time-sensitive applications.