Instant polarized light microscopy for imaging collagen microarchitecture and dynamics

Yang

Hua

et al. 2021

Preprint

Self Cite

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.

Section: Ipol Imaging Systemmentioning

confidence: 99%

“…Shown on the right are five example IPOL images of the same chicken tendon section at known orientations. Details of the calibration are described elsewhere (Yang et al, 2021).…”

mentioning

confidence: 99%

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

Yang

Hua

et al. 2021

Preprint

Self Cite

“…Instant polarized light microscopy (IPOL) is a recently introduced technique that optically encodes information about fiber orientation and retardance through a color snapshot. (Lee et al, 2022; Yang et al, 2021) IPOL allows quantitative imaging of collagen at the full acquisition speed of the camera, with excellent spatial and angular resolutions. IPOL, however, has the limitation that the orientation-encoded colors are cyclic every 90 degrees (π/2 radians).…”

Section: Introductionmentioning

confidence: 99%

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

Schilpp

Naylor

et al. 2023

Preprint

Self Cite

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.

“…The collagen fibers of the eye [13][14][15][16][17], like those of other soft tissues, have natural undulations or waviness called crimp [18][19][20]. Crimp is central to the nonlinear biomechanics of soft tissue through a process known as fiber recruitment [21][22][23].…”

Section: Introductionmentioning

confidence: 99%

Stretch-Induced Uncrimping of Equatorial Sclera Collagen Bundles

Jan

Wallace

et al. 2022

Preprint

Self Cite

Stretch-induced collagen uncrimping underlies the nonlinear mechanical behavior of the sclera according to what is often called the process of recruitment. We recently reported experimental measurements of sclera collagen crimp and pressure-induced uncrimping. Our studies, however, were cross-sectional, providing statistical descriptions of crimp with no information on the effects of stretch on specific collagen bundles. Data on bundle-specific uncrimping is necessary to better understand the effects of macroscale input on the collagen microscale and tissue failure. Our goal in this project was to measure bundle-specific stretch-induced collagen uncrimping of sclera. Three goat eyes were cryosectioned sagittally (30 μm). Samples of equatorial sclera were isolated, mounted to a custom uniaxial stretcher and imaged with polarized light microscopy at various levels of clamp-to-clamp stretch until failure. At each stretch level, local strain was measured using image tracking techniques. The level of collagen crimping was determined from the bundle waviness, defined as the circular standard deviation of fiber orientation along a bundle. Eye-specific recruitment curves were then computed using eye-specific waviness at maximum stretch before sample failure to define fibers as recruited. Nonlinear mixed effect models were used to determine the associations of waviness to local strain and recruitment to clamp-to-clamp stretch. Waviness decreased exponentially with local strain (p<0.001), whereas bundle recruitment followed a sigmoidal curve with clamp-to-clamp stretch (p<0.001). Individual bundle responses to stretch varied substantially, but recruitment curves were similar across sections and eyes. In conclusion, uniaxial stretch caused measurable bundle-specific uncrimping, with the sigmoidal recruitment pattern characteristic of fiber-reinforced soft tissues.