Instant polarized light microscopy pi (IPOLπ) for quantitative imaging of collagen architecture and dynamics in ocular tissues

Abstract

Collagen architecture determines the biomechanical environment in the eye, and thus characterizing collagen fiber organization and biomechanics is essential to fully understand eye physiology and pathology. We recently introduced instant polarized light microscopy (IPOL) that encodes optically information about fiber orientation and retardance through a color snapshot. Although IPOL allows imaging collagen at the full acquisition speed of the camera, with excellent spatial and angular resolutions, a limitation is that the orientation-encoding color is cyclic every 90 degrees ({pi}/2 radians). In consequence, two orthogonal fibers have the same color and therefore the same orientation when quantified by color-angle mapping. In this study, we demonstrate IPOL{pi}, a new variation of IPOL, in which the orientation-encoding color is cyclic every 180 degrees ({pi} radians). Herein we present the fundamentals of IPOL{pi}, including a framework based on a Mueller-matrix formalism to characterize how fiber orientation and retardance determine the color. The improved quantitative capability of IPOL{pi} enables further study of essential biomechanical properties of collagen in ocular tissues, such as fiber anisotropy and crimp. We present a series of experimental calibrations and quantitative procedures to visualize and quantify ocular collagen orientation and microstructure in the optic nerve head, a region in the back of the eye. There are four important strengths of IPOL{pi} compared to IPOL. First, IPOL{pi} can distinguish the orientations of orthogonal collagen fibers via colors, whereas IPOL cannot. Second, IPOL{pi} requires a lower exposure time than IPOL, thus allowing faster imaging speed. Third, IPOL{pi} allows visualizing non-birefringent tissues and backgrounds from tissue absorption, whereas both appear dark in IPOL images. Fourth, IPOL{pi} is cheaper and less sensitive to imperfectly collimated light than IPOL. Altogether, the high spatial, angular, and temporal resolutions of IPOL{pi} enable a deeper insight into ocular biomechanics and eye physiology and pathology. HighlightsO_LIWe introduce IPOL{pi}, addressing IPOL limitations for characterizing eye collagen. C_LIO_LIIPOL{pi} orientation-encoded color cycle is 180{degrees} ({pi} radians) instead of 90{degrees} in IPOL. C_LIO_LIIPOL{pi} requires a lower exposure time than IPOL, allowing faster imaging speed. C_LIO_LIIPOL{pi} visualizes non-birefringent tissues and backgrounds from brightness. C_LIO_LIIPOL{pi} is cheaper and less sensitive to imperfectly collimated light than IPOL. C_LI