We characterized the stiffness and anisotropy of the equatorial sclera and its dependence on intraocular pressure (IOP). Porcine eyes were tested using ultrasound elastography under IOP control. Shear waves were induced using a mechanical shaker, and an off-axis 17.8 MHz transducer used to track the wave propagation in the equatorial and anterior-posterior directions. Wave speed was measured and used to estimate directional Young’s moduli. Anisotropy was defined as the ratio of the equatorial moduli to the anterior-posterior moduli. Sclera was stiffer in the equatorial direction, with anisotropy decreasing with IOP, from, 5.1 at 10 mmHg to 3.7 at 30 mmHg.
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 timesensitive 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.
Collagen is a major constituent of the eye and understanding its architecture and biomechanics is critical to preserve and restore vision. We, recently, demonstrated polarized light microscopy (PLM) as a powerful technique for measuring properties of the collagen fibers of the eye, such as spatial distribution and orientation. Our implementation of PLM, however, required sectioning the tissues for imaging using transmitted light. This is problematic because it limits analysis to thin sections. This is not only slow, but precludes study of dynamic events such as pressure-induced deformations, which are central to the role of collagen. We introduce structured polarized light microscopy (SPLM), an imaging technique that combines structured light illumination with PLM to allow imaging and measurement of collagen fiber properties in thick ocular tissues. Using pig and sheep eyes, we show that SPLM rejects diffuse background light effectively in thick tissues, significantly enhancing visualization of optic nerve head (ONH) structures, such as the lamina cribrosa, and improving the accuracy of the collagen fiber orientation measurements. Further, we demonstrate the integration of SPLM with an inflation device to enable direct visualization, deformation tracking, and quantification of collagen fibers in ONHs while under controlled pressure.
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