Population-based eye pathology is made possible by a 25-year collaboration with the Alabama Eye Bank, which has high volume for transplantable tissues and the fastest tissue recovery time among US eye banks. Between 1996 and 2010, our laboratory accessioned >900 pairs of nondiabetic donor eyes within 4 hours (before 2001) or 6 hours (after 2001) of death. In 2010, federal and private funding enabled creation of the Project MACULA Web site as a resource for clinical imaging, using 142 maculas (53 advanced AMD, 13 GA eyes from 12 donors and 40 neovascular AMD eyes from 40 donors; 29 early AMD; 60 age-matched control eyes). Prior to histology, all eyes were subjected to ex vivo imaging including color fundus photography using a dissecting microscope with epi- and oblique transillumination, and SDOCT using a custom tissue holder for a Spectralis HRA+OCT (Heidelberg Engineering, Heidelberg, Germany).12 (link) This holder holds a donor eye lacking an anterior segment, so that the fundus looks horizontally into the instrument through a 60-diopter lens, thus emulating clinical imaging. Tracking from scans obtained during a donor's lifetime can be used to align macular specimens after death. Thus, SDOCT has become essential for the pathology laboratory.
Because contemporary SDOCT provides exquisite structural detail, clinical interpretation is best served by morphologic descriptions that are comprehensive, quantitative, pegged to precise retinal locations, and digitally available. Thus, we sought views that were high magnification, high resolution, color, and panoramic, with the original histology accessible online. Transmission electron microscopy, a comprehensive visualization technique, reveals all organelles including spindle-shaped melanosomes unique to RPE. Submicrometer-thick sections of epoxy resin blocks can emulate the benefits of low-magnification color electron microscopy over wide tissue samples. Postfixation with osmium tannic acid paraphenylenediamine (OTAP) preserves extracellular lipids59 (link)–62 (link, link, link) and imparts polychromaticity to toluidine blue–stained sections. We surveyed standard locations in superior and central macula (through rod ring and foveola, respectively).24 (link) At 25 locations in central sections and 13 locations in superior sections (1050 locations per eye; >150,000 total for all eyes), 21 chorioretinal layers were measured and RPE morphology was annotated with a custom ImageJ (https://fiji.sc/; in the public domain) drop-down menu. Systematic sampling enabled unbiased estimates of phenotype frequency.