Application of Array Tomography for High Resolution 3d Reconstruction of the Optic Nerve Head

Abstract

Purpose: : Our goal was to 3-dimensionally reconstruct the entire collagen and elastin network of the ONH at high lateral and axial resolution.

Methods: : Eyes were fixed in 4% paraformaldehyde in PBS overnight at 4°C. The ONH was then removed, embedded in LR White resin and serially sectioned at 2 um using a Leica Ultracut Ultramicrotome with a diamond knife. Sections were collected in continuous ribbons and floated onto gelatin coated glass slides to maintain orientation. Nonlinear optical images of two photon excited fluorescence (elastin) and second harmonic generated signals (collagen) were acquired using a Zeiss 510 Meta LSM and Chameleon femtosecond laser. For each section a total of 49 overlapping single-plane images (512 x 512 pixels, 0.9 um lateral resolution) arranged in a quadratic grid (7 X7) were collected. Images from each section were then concatenated into a single mosaic of 3584x3584 pixels as a 8-bit multichannel image file. Mosaics were then processed using a median filter (2x2 pixel) to reduce noise, normalized to match gray levels, assembled sequentially into to a Z-stack and each plane rotated and aligned manually using Amira software program (Visage Imaging, Carlsbad, CA). The aligned Z-stacks were then visualized using the 3D volume rendering subroutine of Amira and/or exported for further image analysis.

Results: : As presented, high resolution reconstructions of the ONH were obtained. These examples represent the reconstruction of the ONH of a 53 yr. old Hispanic male and a 73 yr. old Caucasian female with no history of ocular disease.

Conclusions: : Array tomography allows one to create high resolution, detailed reconstructions of structural components of entire ONH. Importantly, these reconstructions can be objectively and quantitatively analyzed to determine the microscopic density of collagen and elastin within the ONH.

Keywords: optic nerve • anatomy • extracellular matrix