Structured polarized light microscopy for collagen fiber structure and orientation quantification in thick ocular tissues

Abstract

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.

Quantification of the regional bioarchitecture in the human aorta

“…Due to anatomical variance between cadavers, including length, curvature and tortuosity, histological results are presented as a function of eight anatomical sites rather than as a function of physical distance from the aortic root. Following dissection, specimens are dehydrated through a graded series of ethanol (50-100%) before being embedded in paraffin wax and sectioned at a thickness of 5 lm in order to maximise collagen to tissue contrast and limit tissue scattering (Yang et al 2018). For area fraction (AF) analyses, sectioning is performed in the r-c (radial-circumferential) plane to enable investigation through the thickness of the aortic wall, while for fibre orientation (FO) analyses sectioning is performed in the a-c (axial-circumferential) plane on a separate sample obtained from the same site ( Fig.…”

Lamina Cribrosa Capillaries Straighten as Intraocular Pressure Increases

“…The labeled vasculature and LC collagen beams were imaged using a recently developed structured polarized light microscopy (SPLM) imaging system. 46 Briefly, the images were acquired using an Olympus MVX10 upright microscope (6.3× magnification setting) with a scientific-grade camera (Hamamatsu, Flash 4.0 LT) and a 1× objective (numerical aperture [NA], 0.25) at an effective magnification of 6.3×. Structured light illumination was achieved with a digital light projector (LightCrafter evaluation module; Texas Instruments).…”

“…Imaging was performed as described elsewhere. 46 Briefly, for each IOP step, three 2D-sinusoidal patterns with a phase shift of 0 degrees, 120 degrees, and 240 degrees were projected onto the sample sequentially and corresponding reflectance or fluorescence images were acquired. The spatial frequency was 3 mm −1 .…”

“…The spatial frequency was 3 mm −1 . 46 Both pattern projection and image acquisition were controlled by a custom LabVIEW program. For image acquisition, first, the collagen beams were brought into focus and imaged, then, we switched to the fluorescence filter and acquired the capillary image.…”

Self-assembly of progenitor cells under the aegis of platelet factors facilitates human skin organoid formation and vascularized wound healing

“…6ab). Polarized light microscopy 42 highlighted maturing collagen fibers produced by hiPSC-FBs (Fig. 6c).…”