Methods for digital section preparation

  • The foveal center was recognized by GCL and INL gaps, Henle fibers in longitudinal rather than cross-sectional profile, a central ONL lacking rod nuclei, and markers for central bouquet cones (distinctive chromatin, thin inner segments, and long inner fibers).
  • Foveal sections were digitally scanned in their entirety with a 40X numerical aperture 0.95 objective, a robotic microscope stage, and image-stitching software (CellSens, Olympus).
  • Retinal sections are represented as a Cartesian coordinate system with the origin at foveal center.
    • Sections from all eyes are shown as left eyes
    • The negative x-axis, to the left, is the peripapillary terminus of Bruch’s membrane, nasal to the fovea.
    • The positive x-axis, to the right, is temporal to the fovea.
    • Annotation locations are assigned to the mid-point of each layer’s thickness