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

Lee, Po-Yi; Yang, Bin; Hua, Yi; Brazile, Bryn; Ji, Fengting; Zhu, Ziyi; Fryc, Gosia A.; Sigal, Ian A.

doi:10.1117/12.2542878

Cited by 2 publications

(2 citation statements)

References 15 publications

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“…Implementing IPOL with a dissecting microscope with a broader depth of field and a deeper focal depth can achieve a large field of view of a single image and thus circumvents the need for mosaicking, potentially at the cost of resolving power. (Lee et al, 2020) In conclusion, we have demonstrated that IPOL allows visualization and quantification of changes in ONH collagen morphology and architecture under dynamic loading. This study represents an important step towards using a novel imaging modality to study ONH collagen microstructure and biomechanics, which could help understand the role of collagen microstructure in eye physiology, aging, and in biomechanics-related diseases, such as glaucoma and myopia.…”

Section: Discussionmentioning

confidence: 71%

Real-time imaging of optic nerve head collagen microstructure and biomechanics using instant polarized light microscopy

Lee

Yang

Hua

et al. 2021

Preprint

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Current tools lack the temporal or spatial resolution necessary to image many important aspects of the architecture and dynamics of the optic nerve head (ONH). We evaluated the potential of instant polarized light microscopy (IPOL) to overcome these limitations by leveraging the ability to capture collagen fiber orientation and density in a single image. Coronal sections through the ONH of fresh normal sheep eyes were imaged using IPOL while they were stretched using custom uniaxial or biaxial micro-stretch devices. IPOL allows identifying ONH collagen architectural details, such as fiber interweaving and crimp, and has high temporal resolution, limited only by the frame rate of the camera. Local collagen fiber orientations and deformations were quantified using color analysis and image tracking techniques. We quantified stretch-induced collagen uncrimping of lamina cribrosa (LC) and peripapillary sclera (PPS), and changes in LC pore size (area) and shape (convexity and aspect ratio). The simultaneous high spatial and temporal resolutions of IPOL revealed complex ONH biomechanics: i) stretch-induced local deformation of the PPS was nonlinear and nonaffine. ii) under load the crimped collagen fibers in the PPS and LC straightened, without torsion and with only small rotations. iii) stretch-induced LC pore deformation was anisotropic and heterogeneous among pores. Overall, with stretch the pores were became larger, more convex, and more circular. We have demonstrated that IPOL reveals details of collagen morphology and mechanics under dynamic loading previously out of reach. IPOL can detect stretch-induced collagen uncrimping and other elements of the tissue nonlinear mechanical behavior. IPOL showed changes in pore morphology and collagen architecture that will help improve understanding of how LC tissue responds to load.

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Section: Discussionmentioning

confidence: 71%

Real-time imaging of optic nerve head collagen microstructure and biomechanics using instant polarized light microscopy

Lee

Yang

Hua

et al. 2021

Preprint

Self Cite

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“…In IPOL, the imaging system (e.g., dissecting microscope) requires an extra light source collimator to make sure that a collimated light passes through z-cut quartz in the illumination path. (Lee et al, 2020) This requirement increases the system’s complexity and cost. In IPOLπ, the z-cut quartz can be placed directly into the imaging path.…”

Section: Discussionmentioning

confidence: 99%

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

Lee

Schilpp

Naylor

et al. 2023

Preprint

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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 (π/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 IPOLπ, a new variation of IPOL, in which the orientation-encoding color is cyclic every 180 degrees (π radians). Herein we present the fundamentals of IPOLπ, including a framework based on a Mueller-matrix formalism to characterize how fiber orientation and retardance determine the color. The improved quantitative capability of IPOLπ 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 IPOLπ compared to IPOL. First, IPOLπ can distinguish the orientations of orthogonal collagen fibers via colors, whereas IPOL cannot. Second, IPOLπ requires a lower exposure time than IPOL, thus allowing faster imaging speed. Third, IPOLπ allows visualizing non-birefringent tissues and backgrounds from tissue absorption, whereas both appear dark in IPOL images. Fourth, IPOLπ is cheaper and less sensitive to imperfectly collimated light than IPOL. Altogether, the high spatial, angular, and temporal resolutions of IPOLπ enable a deeper insight into ocular biomechanics and eye physiology and pathology.

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

Cited by 2 publications

References 15 publications

Real-time imaging of optic nerve head collagen microstructure and biomechanics using instant polarized light microscopy

Real-time imaging of optic nerve head collagen microstructure and biomechanics using instant polarized light microscopy

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

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