Before preparation for conventional thin-section TEM and QFDE, the globes were inspected internally with epi- and retroillumination, and the maculas were determined to lack grossly visible drusen or pigmentary change. However, it was found on histopathologic evaluation that the two older eyes had small patches of basal laminar deposit located between the RPE and its basal lamina, a typical finding for eyes of this age.
10 The eye of the 76-year-old donor also had numerous small protuberances on the inner surface of Bruch’s membrane that were too small to be considered drusen.
Oblique sections of Bruch’s membrane, as viewed by TEM, are shown for a 27-year-old eye
(Fig. 2A) , a 41-year-old eye
(Fig. 3A) , a 76-year-old eye
(Fig. 3C) , and a 78-year-old eye
(Fig. 2C) . In the younger eyes, the basal infoldings of the RPE, the RPE basal lamina and the inner collagenous layer (ICL) are apparent
(Figs. 2A 3A) . Collagen fibrils and an unidentified banded material were apparent within the ICL. The ends of collagen fibrils could be seen closely apposed to, perhaps embedded in, the RPE basal lamina
(Fig. 3A) . Consistent with previous TEM descriptions, the most prominent differences between the older
(Figs. 2C 3C) and younger
(Figs. 2A 3A) eyes were disorganization or disappearance of the RPE basal infoldings, increased electron density of the ICL, a paucity of collagen fibrils immediately adjacent to the RPE basal lamina, and the presence of numerous round electron-lucent profiles. These profiles were distributed along fibrils in the ICL and in a band between the ICL and the RPE basal lamina in the older donors
(Figs. 2C 3C) . This band, two to four particles thick in cross-sections of many older eyes,
2 looks wide in
Figures 2C and 3C , because of the oblique plane of section. These profiles were previously identified as cholesterol-containing particles, because they are solid when viewed with lipid-preserving ultrastructural techniques, they are extractable with lipid solvents, and they become more numerous with age along with histochemically detectable and directly assayed esterified and unesterified cholesterol.
2
Oblique fractures of Bruch’s membrane viewed in QFDE preparations of a 27-year-old eye
(Fig. 2B) , a 41-year-old eye
(Fig. 3B) , a 76-year-old eye
(Fig. 3D) , and a 78-year-old eye
(Fig. 2D) revealed the same ultrastructural features, but in much greater detail. The RPE basal lamina appeared as a dense meshwork, and the ICL contained a loose network of 45-nm diameter fibrils. Of note were spherical and elliptical particles 80.8 ± 20.7 nm (mean ± SD) in diameter that were minimally etched by the QFDE process and found in abundance only in the older eyes
(Figs. 2D 3D) . In all the micrographs of the older eyes, low density (+) of these particles was found in the basal lamina itself and high density (+++) was found in the region between the basal lamina and the ICL. Because these particles were solid, of similar dimensions, and present in the same locations and in the same age group as those seen in thin-section TEM, we identified them as EC-containing particles.
More lipid particles appear embedded in the basal lamina in the thin-section TEM images
(Figs. 2C 3C) than in the QFDE images
(Figs. 2D 3D) . The apparent discrepancy between the two imaging modalities can be reconciled by considering
Figure 2A , in which RPE basal infoldings also appear to be embedded in the basal lamina. This underscores that sections imaged by thin-section TEM are sufficiently thick to permit the superimposition of two very thin sublayers. Therefore it is likely that
Figures 2C and 3C represent superimposition of the RPE basal lamina and the adjacent layer of Bruch’s membrane rather than numerous particles embedded in basal lamina.
The main difference between the two older eyes examined was the number of these lipid particles. At the interface between the basal lamina and the ICL of the 76-year-old donor eye was a wall of tightly packed lipid particles appreciable by both thin-section TEM
(Fig. 3C) and QFDE
(Fig. 3D) . This wall was not apparent in the 78-year-old eye
(Figs. 2C 2D) .
Figure 4 demonstrates the high density of these embedded lipid rich particles showing a region where the fracture plane jumped from the basal lamina down to the ICL (the bottom of the groove) and then back up to the basal lamina.
In addition to extending results from a previous study using sections viewed by conventional thin-section and lipid-preserving TEM,
2 QFDE also revealed new details about the morphology of both the lipid-rich particles and the extracellular matrix containing them. An exterior shell was apparent on many lipid-rich particles
(Fig. 5) . Regarding the extracellular matrix, QFDE revealed a dense weave (interfibril spacing <15 nm) in the RPE basal lamina of the younger eyes and a perhaps somewhat less dense weave in the older eyes
(Figs. 6 7) . A further age-related difference involved the surface of collagen fibrils. These fibrils displayed an extensive fine-scale decoration
31 in the younger eyes
(Fig. 8A) that was reduced or absent in the older eyes
(Fig. 8B) . Collagen was identified by its characteristic banding pattern. Elastin-like material identification was based on correspondence with standard thin-section TEM images of the same regions.