May 2009
Volume 50, Issue 5
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Glaucoma  |   May 2009
Comparison of Clinical and Three-Dimensional Histomorphometric Optic Disc Margin Anatomy
Author Affiliations
  • Nicholas G. Strouthidis
    From the Optic Nerve Head Research Laboratory and the
  • Hongli Yang
    From the Optic Nerve Head Research Laboratory and the
    Department of Biomedical Engineering, Tulane University, New Orleans, Louisiana.
  • J. Crawford Downs
    Ocular Biomechanics Laboratory, Discoveries in Sight Research Laboratories, Devers Eye Institute, Portland, Oregon; and the
  • Claude F. Burgoyne
    From the Optic Nerve Head Research Laboratory and the
Investigative Ophthalmology & Visual Science May 2009, Vol.50, 2165-2174. doi:10.1167/iovs.08-2786
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      Nicholas G. Strouthidis, Hongli Yang, J. Crawford Downs, Claude F. Burgoyne; Comparison of Clinical and Three-Dimensional Histomorphometric Optic Disc Margin Anatomy. Invest. Ophthalmol. Vis. Sci. 2009;50(5):2165-2174. doi: 10.1167/iovs.08-2786.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

purpose. To investigate the anatomic basis of the optic disc margin in the normal monkey eye by colocalizing optic disc photographs to three-dimensional (3D) histomorphometric reconstructions of the same optic nerve head.

methods. Optic disc photographs from 28 normal monkey eyes were overlaid onto 3D central retinal vessel reconstructions generated as part of postmortem optic nerve histomorphometric reconstructions for each eye. Within each reconstruction, the Bruch’s membrane opening (BMO) was delineated. Alignment was achieved by matching the clinical vessel outline to the vessel reconstruction with parallel viewing software. An experienced observer viewed stereophotographs and marked the disc margin onto clinical photographs with custom software. Alignment of the delineated disc margin to the histomorphometrically defined BMO was qualitatively assessed within each image.

results. In 20 eyes, BMO aligned well to the disc margin delineation. In four eyes, alignment improved after repeated colocalization. Careful review of the histomorphometric reconstructions identified that in most cases Bruch’s membrane extended beyond the termination of the border tissue of Elschnig, most substantially in the superior and nasal sectors. Misalignments could be explained by inaccurate BMO marking or where Bruch’s membrane terminated externally to the inferior edge of the border tissue; this latter structure aligned to the disc margin.

conclusions. BMO was a clinically detectable entity and represented the disc margin in most eyes in this study. The 3D architecture of the border tissue combined with the presence of an overhang of Bruch’s membrane makes an important contribution to disc margin anatomy.

An appreciation of the optic disc margin is centrally important in the examination of all patients with glaucoma because it defines the clinically visible boundary of neural tissue within the optic nerve head (ONH). With the advent of semiautomated ONH imaging devices, the ability to clearly identify and delineate the optic disc margin has assumed increasing importance. The disc margin serves as a basis for cross-sectional neural tissue quantification and as a structural anchor for a reference plane for longitudinal change detection. 1  
Understanding the anatomic basis of the clinical disc margin remains a significant challenge for all clinicians. 2 Although the optic disc is usually examined in a stereoscopic fashion, either by slit lamp biomicroscopy or by stereophotograph examination, it remains difficult to appreciate the location of the disc margin within the complex three-dimensional (3D) architecture of the ONH. The traditional view is that the clinical disc margin is defined by the scleral ring of Elschnig, which is the anteriormost extension of the border tissue of Elschnig. 3 The border tissue of Elschnig refers to densely compacted connective tissue that rises up from the sclera to join the Bruch’s membrane and thereby enclose the choroid. 4 5 6 7 We have previously proposed, however, that in monkey eyes, the clinically visible disc margin is the innermost opening in Bruch’s membrane (Bruch’s membrane opening [BMO]), which in this species often extends beyond the border tissue. 8  
In the present study, we explored the histologic basis of the monkey optic disc margin by colocalizing postmortem 3D ONH reconstructions to their clinical (in vivo) optic disc photographs in 28 normal monkey eyes. Our findings suggest that the 3D architecture of the border tissue of Elschnig, combined with the presence of a pigmented or an unpigmented overhang of Bruch’s membrane, underlies the clinical disc margin anatomy in the normal monkey eye. These findings in monkeys are important because they suggest a preliminary algorithm for clinically assessing the more complicated disc margin anatomy of the human eye. 
Materials and Methods
Animals
All animals were treated in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. All eyes were histomorphometrically reconstructed postmortem as part of other ongoing research studies. Twenty-eight normal eyes of twenty-one rhesus macaques and one cynomolgus monkey were reconstructed. 
Stereophotography
All animals included in the study had regular optic disc stereophotographs acquired as part of imaging protocols developed for other ongoing research studies. Photography was performed while the animal was under pentobarbital anesthesia. IOP in the imaged eye was set at 10 mm Hg through a manometer connected to a 27-gauge cannula inserted into the temporal anterior chamber. Pupils were dilated with 1 drop each of 1% tropicamide, 2.5% phenylephrine hydrochloride, and 2% cyclopentolate hydrochloride. Stereophotographs were acquired through a rigid plano contact lens placed on the corneal surface after at least 30 minutes of IOP stabilization. Stereophotographic pairs in 15 eyes were acquired with a retinal camera (TRC-WT; Topcon, Paramus, NJ). Later, this system was replaced with a fundus camera system (3DX; Nidek, Fremont, CA) that was used to acquire stereophotographs in the remaining 13 eyes. Images were captured onto 35-mm slide film, then developed and processed into color slides. 
For the purposes of the present study, a stereophotograph pair was selected from the day on which the animal was killed. If stereophotographs were not acquired on this day, then images acquired on the closest date were selected. Images had to be of sufficient quality to allow colocalization to the histomorphometric 3D vessel reconstruction, which meant that at least one photograph in the pair should have had clearly discernible central retinal vessels. A good stereo-effect and a clear focus at the disc margin were desirable secondary considerations but were not grounds for exclusion of a stereophotograph pair. Selected stereophotograph slides were digitized at a resolution of 4800 dpi using a color-calibrated slide scanner (ArtixScan M1; Microtek Laboratory, Inc., Fontana, CA). 
Perfusion Fixation
Study eyes were cannulated with a 27-gauge needle after the animals were anesthetized with pentobarbital, and the IOP was set to 10 mm Hg with an adjustable saline reservoir. After a minimum of 30 minutes, the animal was killed with perfusion fixation through the descending aorta with 1 L of 4% buffered hypertonic paraformaldehyde solution followed by 6 L of 5% buffered hypertonic glutaraldehyde solution. IOP was maintained at 10 mm Hg for 1 hour, after which each eye was enucleated (including a 0.5- to 1.5-cm section of the optic nerve), all extraorbital tissue was removed, and the anterior chamber was removed 2 to 3 mm posterior to the limbus. The posterior scleral shell with intact optic nerve head, choroid, and retina was placed in 5% glutaraldehyde solution for storage. 
3D Histomorphometric Reconstruction and BMO Delineation
Our initial technique for 3D optic nerve head reconstruction has been described in detail in a previous report. 9 For this study, 3 of 28 eyes were reconstructed with the use of this technique, and 25 eyes were serial sectioned with a second-generation device that increased axial and transverse image resolution to 1.5 × 1.5 × 1.5 μm voxels 1 10 rather than 2.5 × 2.5 × 3.0 μm voxels with the older device. 
Our technique for 3D delineation of BMO within the histomorphometric reconstructions has been described in detail elsewhere. 8 11 12 Briefly, the 3D ONH reconstruction was loaded into memory on a remote Linux server using a suite of custom software based on the Visualization Toolkit (VTK; Kitware, Inc., Clifton Park, NY). While serial digital transverse or radial sections of the volume were viewed, the delineator assigned the approximate center of the neural canal to be the center of rotation for 40 radial slices of the 3D reconstruction generated at 4.5° intervals. 
Within each radial section, the delineator performed full delineation of the seven landmark surfaces and six pairs of neural canal landmark points (one on each side of the canal), which we have described previously. 8 For the purposes of this study, however, only the BMO marks were relevant. Delineations were performed by one of three technicians as part of other ongoing studies. The delineator marked the termination of Bruch’s membrane on either side of the neural canal as the BMO. Delineation of each BMO point was 3D in that each radial section pixel was linked to its location within a serial transverse (en face) section through the same location (simultaneously displayed on a second monitor). Once the BMO points had been delineated for all 40 sections (80 points in total), the 3D Cartesian coordinates for each point were saved, allowing a 3D BMO point cloud to be generated. 
Overlay and Alignment of Histomorphometric Reconstructions onto Clinical Photographs
To establish the clinical orientation of each 3D histomorphometric reconstruction, a high-resolution 3D reconstruction of the central retinal vessels and BMO points was generated for each eye. The 3D vessel reconstruction was then overlaid onto a digitized clinical photograph (the best-focused photograph from the stereophotograph pair) with the use of parallel viewing software (Paraview; Kitware, Clifton Park, NY). All colocalizations were performed by a single operator (NGS). This parallel viewing software (Paraview; Kitware) enabled the operator to move the clinical photograph in 3D space for x-axis, y-axis, and z-axis shifts and allowed rotation about the z-axis, z-axis tilt, and magnification change. 
The 3D colocalization protocol is depicted in Figure 1 . Adjustments to the z-axis were made with the photograph viewed on its side, along the coronal plane, with the BMO point cloud visualized; this allowed the clinical photograph to be tilted in the same orientation as the BMO point cloud. BMO points were excluded from view for x- and y-axis manipulations and for z-axis rotation and magnification changes so that the final colocalization was based on the alignment of the central retinal vessels rather than on the alignment of the BMO points to the clinically visible disc margin. 
Systematic Review of Stereophotographs and Delineation of the Clinical Disc Margin
Once the operator achieved satisfactory colocalization of the clinical vessels to the 3D vessel reconstruction, screen captures were saved of the clinical photographs with and without the colocalized BMO points. The two photographs were then overlaid (Adobe Photoshop CS3; Adobe Systems, San Jose, CA) and were saved as a two-layer image file. 
An experienced clinical observer (CFB) viewed each eye’s digitized stereophotograph pair on a computer monitor with a stereoscope (Screen-Vu; PS Mfg., Portland, OR). If the disc margin structures were not clearly discernible, the observer also had access to the original stereophotograph slides, which could be viewed with a hand-held stereo viewer (Asahi-Pentax, Englewood, CO) if the stereophotographs were acquired with the Nidek system or with a light box and mounted stereo viewer (Luminos Photograph, Yonkers, NY) if the stereophotographs were acquired with the Topcon system. 
For each eye, the observer marked the disc margin on the clinical photograph with a custom application (JavaScript; Jupitermedia Corporation, Darien, CT). The clinical photograph used for this purpose was the colocalized image layer with the BMO excluded from view. The observer could discern two categories of marks: blue where the observer was “certain” of the disc margin and green where the observer was “uncertain” and made a “forced” choice. 
For this study the operator marked the innermost clinically visible disc margin structure, which was usually the internal edge of an unpigmented, whitish halo, or crescent. Where this unpigmented structure was not visible, the termination of the variably mottled disc margin pigment was delineated. In some eyes, the observer could stereoscopically discern two different levels of pigment, an outer superficial layer and an inner deeper layer, with a portion, or lip, of unpigmented tissue variably present at the termination of either pigmented tissue. In these circumstances, two rows of marks were made; an outer row of single marks demarcated the internal edge of the superficial tissue (pigmented or unpigmented), and an inner row of double marks (a green mark touching a blue mark) demarcated the termination of the deeper tissue (pigmented or unpigmented). 
Once the observer completed the demarcation of the clinically visible disc margin, the coordinates of the marked points were saved, and the points were transferred to the image layer in which the BMO points were also shown. An image of the clinical photograph incorporating the colocalized BMO points and the clinical demarcations was then saved for each eye. 
Systematic Review of Histomorphometric Reconstructions
Before assessment of the alignment of the BMO to the clinical disc margin, the 3D histomorphometric volumes of all 28 eyes were reviewed by two observers (CFB and NGS). For each eye, the observers documented the quality of the connective tissue staining, the degree of pigment in the reconstruction, and the presence or absence of artifacts such as choroidal or retinal detachments. Choroidal and retinal detachments were assumed to be postmortem artifacts because they were not present in optic disc images/disc photographs acquired on the day of kill. The perfusion fixation process was assumed to provide sufficient hydrostatic pressure to cause expansion of the choroidal space in most eyes. Sections at 0°, 45°, 90°, and 135° were loaded, enabling the histology in the superior-inferior, superotemporal-inferonasal, temporal-nasal, and inferotemporal-superonasal regions to be inspected. To establish the histologic underpinnings of the clinical disc margin, the observers carefully reviewed the configurations of Bruch’s membrane, BMO, and border tissue of Elschnig in each sector of the disc. The observers noted how far the Bruch’s membrane extended beyond the termination of the border tissue, the presence or absence of an unpigmented portion of Bruch’s membrane, the configuration of the border tissue in relation to where it met the Bruch’s membrane, and whether the histomorphometric BMO delineation was felt to be accurate. 
Comparison of Histomorphometrically Defined BMO with Clinical Disc Margin
Once delineation of the clinical photographs was completed, the images incorporating the histomorphometric BMO marks and disc margin marks were qualitatively reviewed by two observers (CFB and NGS) to assess how well the colocalized histomorphometric BMO marks aligned with the clinical disc margin delineations. An eye was classified as well aligned if there was less than a glyph diameter separation between adjacent BMO and clinical disc margin glyphs in all disc sectors, with disc sectors defined according to the systematic histomorphometric review. In eyes with misalignment between the histomorphometric BMO and disc margin marks consistent with this definition, the following investigations were performed to identify causes for the observed discrepancies. First, the colocalization process was repeated, leading to a second-pass review of the colocalized BMO and clinical marks. In eyes in which the alignment between BMO and clinical marks could not be improved by repeated colocalization, a second systematic review of the relevant stereophotographs and 3D histomorphometric reconstructions was carried out so as to identify the salient clinical and histologic features that might explain the misalignment. 
Results
Systematic Review of 3D Histomorphometric Reconstructions
Bruch’s Membrane Extension, Pigment, and Opening.
In most eyes, an extension of Bruch’s membrane with mottled pigment on its surface, which we defined as pigmented Bruch’s membrane, was usually present beyond the histomorphometric termination of the border tissue of Elschnig and choroid. A further extension of Bruch’s membrane without surface pigment (unpigmented Bruch’s membrane) could be detected internal to pigmented Bruch’s membrane in most high-resolution 3D histomorphometric reconstructions. Figure 2illustrates the distinction between unpigmented and pigmented Bruch’s membrane in a histomorphometric section. An extension of Bruch’s membrane was visible in at least one disc region of every eye examined in this study. A substantial extension of Bruch’s membrane was usually observed in the superior and nasal sectors (both 43% of eyes), followed by the inferonasal (39%), superonasal (36%), temporal (29%), inferotemporal (25%), and superotemporal (21%) sectors. Unpigmented Bruch’s membrane was usually visualized despite the presence of darker tissues or poor tissue staining. However, its detection in the three low-resolution volumes was less consistent because this resolution was perhaps insufficient to allow the reconstruction of unpigmented Bruch’s membrane. 
Accurate delineation of BMO was made difficult where pigment was present on the lamina or scleral surface because the “shadow” cast by this pigment could obscure the termination of Bruch’s membrane (Fig. 3) . Rarely, the border tissue met the termination of Bruch’s membrane, and no extension was present. Artifactual choroidal or retinal detachments were present in three eyes (Fig. 3) , but these did not involve the Bruch’s membrane/border tissue junction and, hence, did not affect the configuration of the disc margin anatomy. 
Border Tissue of Elschnig Configuration.
Four principal border tissue configurations were recognized. In the most common form, the superior edge of the border tissue extended internally to the border tissue/scleral junction. We defined this configuration as internally oblique (Figs. 4A 5A) . Less commonly, the superior edge of the border tissue was external to the border tissue/scleral junction. We defined this configuration as externally oblique (Figs. 4B 5B) . The border tissue also manifested vertical (no obliqueness) and horizontal (extreme form of internal obliqueness) configurations (Figs. 4C 4D , respectively). These configurations could vary regionally within an individual eye. In rare instances the border tissue was regionally not discernible. As noted, regardless of border tissue configuration, pigmented or unpigmented extension of Bruch’s membrane beyond its termination was commonly present. 
Comparison of Clinical Disc Margin and BMO Alignment
Good Alignment.
Twenty eyes demonstrated good alignment between histomorphometric BMO marks and clinical disc margin marks (examples are shown in Fig. 6 ) within all sectors of the disc. In 16 of these 20 eyes, the disc margin was marked where an innermost unpigmented region was present at the disc margin (Fig. 6C) . In these eyes, the termination of unpigmented Bruch’s membrane corresponded to the delineated disc margin, and the clinically visible mottled pigment external to it corresponded to pigmented Bruch’s membrane. 
Of the four remaining eyes with good agreement between the clinical markings and histomorphometric BMO, two did not have a clinically visible unpigmented disc margin structure; therefore, the termination of mottled pigment was marked instead, which coincided with the histomorphometric BMO. In two eyes, the clinical observer felt that there was pigment at two different planes, with an internal unpigmented stripe at the internal border of the superficial outer pigment. In these eyes, the inner edge of the unpigmented stripe and the termination of the deeper pigment were marked. After colocalization, however, the BMO was found to be aligned with the innermost clinical markings. Systematic review of the relevant histomorphometric sections confirmed that Bruch’s membrane was the innermost disc margin structure, not the border tissue, within these areas of the disc. No histomorphometric correlate, however, could explain the pale stripe seen within the pigment or the clinically perceived different levels of pigment. It is possible that the pale stripe represented an area of vitreous attachment to the optic disc, but this could not be confirmed in the absence of access to in vivo examination of these eyes. 
Sources of Misalignment.
Eight eyes had substantial regions in which the clinical disc margin and histomorphometric BMO points were poorly matched. Alignment improved considerably in four of these eyes after repeat colocalization. 
Four eyes did not achieve a good match between clinical marks and BMO marks, despite satisfactory colocalization at the second attempt. In each of these four eyes, however, the discrepancy between marks was confined to two or fewer sectors. In the first of these eyes, the clinical disc margin marks were internal to the histomorphometric BMO marks in the superonasal and nasal sectors. Careful review of the histomorphometric sections within the poorly matched regions revealed that a substantial portion of unpigmented Bruch’s membrane was present and extended internally into the neural canal, beyond where the BMO had been originally delineated. Thus, in this eye, the clinical disc margin and the histomorphometric BMO marks would have been more closely matched if the full extension of unpigmented Bruch’s membrane had been histomorphometrically delineated. 
In the second eye, the clinical disc margin marks were again internal to the histomorphometric BMO marks in the temporal sector only. Histomorphometric review confirmed that an extension of unpigmented Bruch’s membrane was not histomorphometrically detectable in this region of the disc. When a review of the clinical photographs again confirmed the presence of a reflective inner halo within this region, we assumed our histomorphometric technique failed to reconstruct unpigmented Bruch’s membrane within this region. Given that this is one of the low-resolution (2.5 × 2.5 × 3.0-μm voxel) histomorphometric reconstructions, this finding may confirm a relative inability to resolve unpigmented Bruch’s membrane. 
In the third eye, the clinical disc margin marks were external to the histomorphometric BMO marks in the nasal and superonasal sectors. In this eye, histomorphometric review revealed that, as in the first eye, a narrow extension of unpigmented Bruch’s membrane had been missed in the original delineation. However, in this instance, correcting the histomorphometric BMO points would have located them even more internal to the clinical disc margin points, enhancing rather than resolving the discrepancy. It should be noted that the fundus photograph used for this colocalization (taken from the only available stereophotograph pair for this eye) was of poor quality, making accurate colocalization difficult. 
In the remaining eye, there was a marked misalignment in the temporal and inferotemporal sectors, with the clinical disc margin marks coinciding with an internal pigment border and the histomorphometric BMO marks coinciding with a more external pale stripe (defined by crescents of mottled pigment on either side). Histomorphometric review confirmed an externally oblique border tissue configuration within this region accompanied by no extension of Bruch’s membrane beyond the termination of the border tissue (Fig. 4B) . Here, because the border tissue was externally oblique to its junction with the sclera and because there was no extension of Bruch’s membrane beyond its termination, pigment on the surface of the border tissue was clinically visible beyond the termination of Bruch’s membrane but in a plane that was clearly deeper to it. In this instance, the disc margin was the border tissue/scleral junction, and the lack of a true unpigmented scleral lip on subsequent repeat stereophotographic examination could be explained as the result of a shadow cast by dense pigment present on the surface of the border tissue and within the scleral canal wall tissues themselves (Fig. 4B)
Discussion
In the present study we colocalized postmortem 3D ONH reconstructions to the clinical (in vivo) optic disc photographs of 28 normal monkey eyes. The principal findings of this report are as follows. First, in most eyes, the histomorphometrically delineated BMO aligned with the clinically defined innermost disc margin structure. Thus the innermost termination of BMO is a clinically discernible structure in the normal monkey eye. Second, two core components of optic disc margin architecture could be histomorphometrically identified, and their multiple combinations could be invoked to explain clinical disc margin anatomy in most normal monkey eyes. The first component is border tissue of Elschnig obliqueness and pigmentation relative to the sclera. The second component is Bruch’s membrane extension and pigmentation beyond the border tissue termination. 
In most eyes, a narrow halo (if present for 360°) or crescent (if present for <360°) of unpigmented Bruch’s membrane was visible as a pale discrete structure internal to, concentric with, and in the same plane as the termination of pigment at the disc margin. In regions in which unpigmented Bruch’s membrane was not clinically visible, histomorphometrically delineated BMO usually coincided with the termination of the pigmented tissues at the disc margin. 
The disc margin pigmented tissues themselves assumed two principal regions, an inner halo or crescent of mottled pigment followed by an outer, dense, and dark gray pigment traditionally thought to correspond to the termination of the viable retinal pigment epithelium (and underlying choroid). 6 It is important to emphasize that in the overwhelming majority of eyes, the pale halo, mottled pigment, and dense pigment border were all in the same plane, as observed during stereoscopic examination, and were colocalized (respectively) to histomorphometrically delineated unpigmented Bruch’s membrane, pigmented Bruch’s membrane, and the shadow associated with the termination of the choroid (and presumed overlying retinal pigment epithelium). The fact that these structures appear clinically to be on the same plane is especially important because it is the presence of pigmented structures deeper to the plane of the retinal pigment epithelium/Bruch’s membrane complex that underlies the clinical recognition of an externally oblique border tissue. 
These findings are contrary to previous reports in human eyes. 4 5 6 7 Hogan et al. 6 defined the traditional view that the ophthalmoscopically visible optic nerve begins at the termination of the retinal pigment epithelium. Where the edge of the choroid and the edge of the retinal pigment epithelium align, an extension of densely packed collagenous tissue arising from the sclera is usually interposed between the choroid and the ONH. 5 This structure, defined as the border tissue of Elschnig, is visible ophthalmoscopically as a white halo bounding the disc, terminating as a scleral lip. 
The clinical appearance of the scleral ring has been referred to as the scleral ring of Elschnig and has been the traditional clinical 3 and stereophotographic 13 14 15 16 17 18 definition of the disc margin. Although Bruch’s membrane has been observed to extend beyond the termination of the retinal pigment epithelium and to cover the border tissue, this has not been described as a phenomenon detectable by ophthalmoscopic examination. 5  
The results of our study suggest that in those normal monkey eyes in which the border tissue had an internally oblique configuration and Bruch’s membrane extended beyond the border tissue termination (by far the most common orientation), it was the termination of the BMO that was the clinically visible disc boundary. In these eyes, the white crescent observed at the disc margin was attributed to a rim of unpigmented Bruch’s membrane rather than to a manifestation of a scleral ring that, in fact, was shielded from clinical view (Fig. 5A) . Figure 7further illustrates this point. The ring of Elschnig (Figs. 7C 7E , white glyphs) does not colocalize to the innermost white reflective disc margin structure (indeed it is at a considerable distance external to it). The termination of unpigmented Bruch’s membrane (Figs. 7C 7E , red glyphs) does in fact colocalize to the white crescent, indicating that in this region of the disc, BMO is the disc margin. We have previously demonstrated that the termination of lightly pigmented Bruch’s membrane colocalizes to the disc margin. 8 In Figure 7 , however, we clearly demonstrate that unpigmented Bruch’s membrane, effectively a transparent structure, is an ophthalmoscopically visible structure that colocalizes to the disc margin. 
Where the border tissue had an externally oblique configuration and the Bruch’s membrane terminated before the edge of the border tissue, the clinical disc margin was indeed the edge of the border tissue. Unfortunately, in the example in which this was apparent, the histomorphometric presence of pigment in the underlying sclera combined with poorly focused stereophotographs did not allow for a true scleral lip to be detected in the stereophotograph. Rather, the pigment observed clinically was composed of two zones. The first was a superficial external pigment rim within the plane of the retinal pigment epithelium and histomorphometrically colocalized to pigmented Bruch’s membrane. The second was an internal pigment rim deep to the plane of the retinal pigment epithelium and histomorphometrically colocalized to pigment on the surface of the border tissue. The latter pigment may be seen stereoscopically to slope downward and inward, following the angle of incidence of the border tissue as it slopes down from Bruch’s membrane to the sclera. 
Two clinical reports have been published of gray optic disc crescents in human eyes. 19 20 A gray crescent is defined as a crescent-shaped, slate gray pigmentation in the periphery of the neuroretinal rim that is completely inside the scleral crescent. In these reports, the authors used the existing scleral lip definition of the disc margin to define the crescent and were uncertain of its histologic derivation. They did not specifically comment on whether the gray crescent appeared posterior to (and sloping away from) the Bruch’s membrane/retinal pigment epithelium plane. We now propose that the gray crescent is likely to be pigmented on the surface of an externally oblique border tissue of Elschnig. Clinical awareness of the plane in which the crescent occurs, perhaps clarified by spectral-domain optical coherence tomography (SD-OCT) ONH imaging, may provide further insight into this form of pigmentary crescent. 
The colocalization method used in this study is prone to several inherent sources of error. The caliber and size of the photographed vessels and the 3D reconstructed vessels may differ because of tissue shrinkage during processing, cardiac pulse, and distention during the perfusion fixation process. Colocalization is unlikely to be 100% accurate because the process requires the 2D photographic plane to be aligned to a three-dimensional structure. It was, therefore, felt reasonable to explore colocalization error as a source of misalignment and therefore to repeat the colocalization process in those eyes in which poor alignment between the BMO and the clinical marks was observed. It also should be noted that quantification of the accuracy of alignment would be rendered largely meaningless because of the difference in size between the in vivo imaged eye and the postperfusion-fixation histomorphometrically reconstructed eye. It is for these reasons that a qualitative approach to assessing alignment was adopted. 
Our work in monkeys is pertinent to humans for two important reasons. First, we believe that the anatomic relationships we describe will contribute to the histologic explanation of most forms of human disc margin anatomy. Second, by applying these concepts at the slit lamp and when interpreting SD-OCT ONH images, clinicians who have previously understood the disc margin to be the ring of Elschnig will recognize the histologic origin of the disc margin to be the termination of Bruch’s membrane, at the very least in the nasal region of most human optic discs. The principal explanation for this is that the nasal disc margin in monkeys and humans is prone to an internally oblique border tissue configuration to enable the optic nerve to pass obliquely through the sclera to reach the more midline optic chiasm. These clinical implications of neural canal anatomy have been explained in detail in our previous report. 8  
The results of this study have additional clinical implications. Recognition of the disc margin plays an important role in examination of the neuroretinal rim (or indeed assigning a focal cup-to-disc ratio). The ability to recognize a perhaps subtle rim of unpigmented Bruch’s membrane as the disc margin may yield a more accurate assessment of the neuroretinal rim. Similarly, ONH imaging modalities, such as scanning laser tomography (Heidelberg Retina Tomograph; Heidelberg Engineering GmbH, Heidelberg, Germany), require the placement of a contour line around the disc margin. As disc margin anatomy is more clearly defined, this may be applied to imaging modalities, enabling the reliability of contour line placement to be improved. 
We have previously proposed BMO as a source structure for a reference plane for quantification of 3D histomorphometric volumes. 8 An expert consensus group has also previously proposed this concept. 3 More recently, we have confirmed that BMO is an anatomically continuous, relatively planar structure in 3D histomorphometric volumes and in 3D SD-OCT volumes. 1 Results of the present study further support the adoption of the BMO as a reference plane in clinical ONH imaging. The fact that the BMO is usually the innermost disc margin structure in the monkey eye suggests that it will be easily and reliably delineated by automated segmentation. It is necessary, however, to first confirm that the SD-OCT-defined neural canal opening aligns with the clinical disc margin. 2 This work will be the subject of a future report from our group. 
In summary, the innermost edge of Bruch’s membrane, the BMO, is a clinically visible structure in the monkey eye. It is usually the innermost disc margin structure and hence constitutes the disc margin boundary in most monkey eyes. Border tissue may be clinically visible in regions in which it demonstrates an externally oblique configuration, and the border tissue/scleral junction is internal to the termination of Bruch’s membrane. We are beginning to study the relationship between BMO internal versus external oblique border tissue configuration and the disc margin in monkey and human eyes with the use of postmortem histomorphometric and clinical SD-OCT ONH reconstructions. 
 
Figure 1.
 
Method for colocalizing the clinical disc photograph to the 3D vessel reconstruction using parallel viewing software. (A) The clinical photograph is viewed before colocalization at a distance from the vessel reconstruction. (B) Shifts in the x- and y-axes allow approximate colocalization in the horizontal plane. (C) Magnification of the clinical image has been increased to match the dimensions of the vessel reconstruction. (D) The clinical photograph and vessel reconstruction are viewed in the coronal plane, with the BMO point cloud visible (red glyphs). This maneuver is performed to facilitate z-axis adjustment. The clinical photograph can be seen to be tilted in a different orientation to the long axis of the BMO point cloud. (E) The z-axis tilt has been adjusted so that the clinical image (coronal profile, arrows) is now orientated in the same plane as the long axis of the BMO point cloud. (F) The clinical image has been moved vertically in the z-axis so that the coronal image profile (arrows) coincides with the BMO point cloud. Once this adjustment has been completed, the BMO points are switched off. (G) The clinical image is now viewed in the en face orientation after z-axis adjustment. Rotation about the image centroid was performed to accurately align the clinical vessel outline to the vessel reconstruction. (H) Final location of the BMO points in relation to the clinical disc photograph is displayed.
Figure 1.
 
Method for colocalizing the clinical disc photograph to the 3D vessel reconstruction using parallel viewing software. (A) The clinical photograph is viewed before colocalization at a distance from the vessel reconstruction. (B) Shifts in the x- and y-axes allow approximate colocalization in the horizontal plane. (C) Magnification of the clinical image has been increased to match the dimensions of the vessel reconstruction. (D) The clinical photograph and vessel reconstruction are viewed in the coronal plane, with the BMO point cloud visible (red glyphs). This maneuver is performed to facilitate z-axis adjustment. The clinical photograph can be seen to be tilted in a different orientation to the long axis of the BMO point cloud. (E) The z-axis tilt has been adjusted so that the clinical image (coronal profile, arrows) is now orientated in the same plane as the long axis of the BMO point cloud. (F) The clinical image has been moved vertically in the z-axis so that the coronal image profile (arrows) coincides with the BMO point cloud. Once this adjustment has been completed, the BMO points are switched off. (G) The clinical image is now viewed in the en face orientation after z-axis adjustment. Rotation about the image centroid was performed to accurately align the clinical vessel outline to the vessel reconstruction. (H) Final location of the BMO points in relation to the clinical disc photograph is displayed.
Figure 2.
 
The identification of Bruch’s membrane and BMO in a histomorphometric section. Top left panel: clinical disc photograph (OD) before disc margin delineation. Top right panel: clinical disc photograph displaying colocalized histomorphometric BMO points (red glyphs) and the clinical disc margin points (blue and green glyphs). The black line is the approximate location of the vertical histomorphometric section shown in the middle panel. The black circle is the approximate location of the histomorphometric region (white box in the middle panel) magnified in the two bottom panels. Middle panel: central vertical histomorphometric section shown as a black line in the top right panel. Superior is left and inferior is right. The area within the white box is magnified in the bottom panels. Note that because the tissues are sectioned from the vitreous (top) to the orbital optic nerve (bottom), a dark shadow is present until the serial sectioning plane passes through the dense pigment of the retinal pigment epithelium, choroid, and Bruch’s membrane. Bottom left panel: magnified view of the highlighted white box in the middle panel that demonstrates the superior disc margin anatomy. Bottom right panel: the same region, labeled as follows: A, sclera; B, choroid; C, Bruch’s membrane; D, commencement of pigmented Bruch’s membrane, which in this section appears to colocalize to the termination of the choroid; E, termination of pigmented Bruch’s membrane and the commencement of unpigmented Bruch’s membrane (note the presence of pigment shadows of variable density cast vertically along the course of the pigmented Bruch’s membrane but absent in the unpigmented Bruch’s membrane); F, termination of unpigmented Bruch’s membrane, which would be delineated as BMO in this section; G, border tissue of Elschnig. In this eye, Bruch’s membrane fuses with the superior edge of the border tissue and extends slightly beyond its termination.
Figure 2.
 
The identification of Bruch’s membrane and BMO in a histomorphometric section. Top left panel: clinical disc photograph (OD) before disc margin delineation. Top right panel: clinical disc photograph displaying colocalized histomorphometric BMO points (red glyphs) and the clinical disc margin points (blue and green glyphs). The black line is the approximate location of the vertical histomorphometric section shown in the middle panel. The black circle is the approximate location of the histomorphometric region (white box in the middle panel) magnified in the two bottom panels. Middle panel: central vertical histomorphometric section shown as a black line in the top right panel. Superior is left and inferior is right. The area within the white box is magnified in the bottom panels. Note that because the tissues are sectioned from the vitreous (top) to the orbital optic nerve (bottom), a dark shadow is present until the serial sectioning plane passes through the dense pigment of the retinal pigment epithelium, choroid, and Bruch’s membrane. Bottom left panel: magnified view of the highlighted white box in the middle panel that demonstrates the superior disc margin anatomy. Bottom right panel: the same region, labeled as follows: A, sclera; B, choroid; C, Bruch’s membrane; D, commencement of pigmented Bruch’s membrane, which in this section appears to colocalize to the termination of the choroid; E, termination of pigmented Bruch’s membrane and the commencement of unpigmented Bruch’s membrane (note the presence of pigment shadows of variable density cast vertically along the course of the pigmented Bruch’s membrane but absent in the unpigmented Bruch’s membrane); F, termination of unpigmented Bruch’s membrane, which would be delineated as BMO in this section; G, border tissue of Elschnig. In this eye, Bruch’s membrane fuses with the superior edge of the border tissue and extends slightly beyond its termination.
Figure 3.
 
Pigment on the lamina surface causing obfuscation of BMO. Top left panel: clinical disc photograph (OS). Bottom left panel: en face view of the histomorphometric reconstruction of the same eye. Black line: orientation of a histomorphometric section image, a portion of which (the superior disc margin) is magnified in the right panels. Black circle: region viewed in the right panels; note the presence of pigment on the lamina surface. Top right panel: histomorphometric view of the superior part of the neural canal. Bottom right panel: Bruch’s membrane delineated (orange glyphs). The white rectangle highlights an area in which the view of Bruch’s membrane is obscured by a dark shadow cast from the lamina pigment below. Accurate delineation of BMO can be difficult. White arrowheads: extent of an artifactual choroidal detachment, most likely caused by the perfusion fixation process.
Figure 3.
 
Pigment on the lamina surface causing obfuscation of BMO. Top left panel: clinical disc photograph (OS). Bottom left panel: en face view of the histomorphometric reconstruction of the same eye. Black line: orientation of a histomorphometric section image, a portion of which (the superior disc margin) is magnified in the right panels. Black circle: region viewed in the right panels; note the presence of pigment on the lamina surface. Top right panel: histomorphometric view of the superior part of the neural canal. Bottom right panel: Bruch’s membrane delineated (orange glyphs). The white rectangle highlights an area in which the view of Bruch’s membrane is obscured by a dark shadow cast from the lamina pigment below. Accurate delineation of BMO can be difficult. White arrowheads: extent of an artifactual choroidal detachment, most likely caused by the perfusion fixation process.
Figure 4.
 
Two principal border tissue configurations with variations. (A) Internally oblique. Left: disc photograph (OS) showing the colocalized BMO (red glyphs) and disc margin delineations (blue and green glyphs). Black line: approximate orientation of the histomorphometric section from which the circular region is magnified in the middle. Middle: histomorphometric disc margin region. Bruch’s membrane (orange glyphs), BMO (red glyph), and border tissue (green glyphs) are delineated. The inferior edge of the border tissue communicates with sclera (the border tissue/scleral junction), and the superior edge extends into the neural canal fusing with Bruch’s membrane (the border tissue termination), which extends beyond this point and includes an unpigmented portion. Right: representative histologic section taken from a healthy monkey eye (perfusion fixed at IOP 10 mm Hg; midhorizontal sagittal section, hematoxylin and eosin stain) demonstrating an internally oblique configuration. White arrows: border tissue. Black arrows: extension of unpigmented Bruch’s membrane. In this case, there is no clear extension of Bruch’s membrane beyond the termination of the border tissue. (B) Externally oblique. Left: disc photograph (OD), demarcated as in (A). Note that within and around the circled region, the clinical disc margin has been marked internal to the histomorphometric BMO points. Middle: histomorphometric section showing the border tissue configuration, demarcated as in (A). Note that the inferior edge of the border tissue is internal to its termination at Bruch’s membrane. Bruch’s membrane does not extend beyond the border tissue termination. In this instance, dense pigment within the sclera immediately adjacent to the neural canal casts a shadow upward that probably explains the lack of a highly reflective scleral lip (white arrows) within this region of the clinical photograph. Right: representative histologic section taken from a healthy monkey eye (perfusion fixed at IOP 10 mm Hg; midhorizontal sagittal section, hematoxylin and eosin stain) demonstrating the externally oblique configuration. White arrows: border tissue. Black arrows: Bruch’s membrane (pigmented). (C) Vertical configuration. The border tissue extends vertically from the sclera to meet Bruch’s membrane. Bruch’s membrane extends beyond this point with pigmented (outer) and unpigmented (inner) portions. (D) Horizontal configuration. The border tissue extends horizontally to meet BMO. This configuration is an extreme form of an internally oblique border tissue configuration.
Figure 4.
 
Two principal border tissue configurations with variations. (A) Internally oblique. Left: disc photograph (OS) showing the colocalized BMO (red glyphs) and disc margin delineations (blue and green glyphs). Black line: approximate orientation of the histomorphometric section from which the circular region is magnified in the middle. Middle: histomorphometric disc margin region. Bruch’s membrane (orange glyphs), BMO (red glyph), and border tissue (green glyphs) are delineated. The inferior edge of the border tissue communicates with sclera (the border tissue/scleral junction), and the superior edge extends into the neural canal fusing with Bruch’s membrane (the border tissue termination), which extends beyond this point and includes an unpigmented portion. Right: representative histologic section taken from a healthy monkey eye (perfusion fixed at IOP 10 mm Hg; midhorizontal sagittal section, hematoxylin and eosin stain) demonstrating an internally oblique configuration. White arrows: border tissue. Black arrows: extension of unpigmented Bruch’s membrane. In this case, there is no clear extension of Bruch’s membrane beyond the termination of the border tissue. (B) Externally oblique. Left: disc photograph (OD), demarcated as in (A). Note that within and around the circled region, the clinical disc margin has been marked internal to the histomorphometric BMO points. Middle: histomorphometric section showing the border tissue configuration, demarcated as in (A). Note that the inferior edge of the border tissue is internal to its termination at Bruch’s membrane. Bruch’s membrane does not extend beyond the border tissue termination. In this instance, dense pigment within the sclera immediately adjacent to the neural canal casts a shadow upward that probably explains the lack of a highly reflective scleral lip (white arrows) within this region of the clinical photograph. Right: representative histologic section taken from a healthy monkey eye (perfusion fixed at IOP 10 mm Hg; midhorizontal sagittal section, hematoxylin and eosin stain) demonstrating the externally oblique configuration. White arrows: border tissue. Black arrows: Bruch’s membrane (pigmented). (C) Vertical configuration. The border tissue extends vertically from the sclera to meet Bruch’s membrane. Bruch’s membrane extends beyond this point with pigmented (outer) and unpigmented (inner) portions. (D) Horizontal configuration. The border tissue extends horizontally to meet BMO. This configuration is an extreme form of an internally oblique border tissue configuration.
Figure 5.
 
Two principal border tissue configurations, their relationship to a pigmented or unpigmented extension of Bruch’s membrane, and the resultant clinical disc margin anatomy. (A) Internally oblique. The diagram shows the clinical optic disc appearance (top) and a cross-section of the optic nerve head (bottom). Labeling is as follows: 1, sclera; 2, choriocapillaris; 3, retinal pigment epithelium with Bruch’s membrane; 4, border tissue; 5, neural canal boundary; 6, pigment on the surface of Bruch’s membrane; 7, Bruch’s membrane. Left inset: pigmented Bruch’s membrane corresponds to the halo of pigment on the left side of the disc margin. Right inset: region of unpigmented Bruch’s membrane is shown; this corresponds to a white crescent internal to the pigment halo at the disc margin, which corresponds to a portion of pigmented Bruch’s membrane. (B) Externally oblique. Labeling is according to the schematic in (A). Left inset: Bruch’s membrane is pigmented to its end and does not extend beyond the termination of the border tissue. This Bruch’s membrane extension corresponds to an external crescent of pigment at the disc margin that is internal to the termination of the retinal pigment epithelium. The portion of the border tissue that is internal to the end of Bruch’s membrane (BMO) may be clinically recognizable as an inner reflective (if there is no pigment on the border tissue surface) or a pigmented crescent (if there is pigment on the border tissue surface) that is posterior to the plane of the retinal pigment epithelium. An inner pigmented halo (lighter gray, stippled) is shown on both sides of the disc diagram. Right inset: unpigmented Bruch’s membrane extends internally to the border tissue termination, corresponding to a reflective crescent internal to the pigment crescent. Again, pigmented border tissue (lighter gray, stippled) extends internally to the reflective crescent. In left and right insets the border tissue/scleral junction is depicted without a true scleral lip, which, when present and visible, appears internal and deep to the other structures.
Figure 5.
 
Two principal border tissue configurations, their relationship to a pigmented or unpigmented extension of Bruch’s membrane, and the resultant clinical disc margin anatomy. (A) Internally oblique. The diagram shows the clinical optic disc appearance (top) and a cross-section of the optic nerve head (bottom). Labeling is as follows: 1, sclera; 2, choriocapillaris; 3, retinal pigment epithelium with Bruch’s membrane; 4, border tissue; 5, neural canal boundary; 6, pigment on the surface of Bruch’s membrane; 7, Bruch’s membrane. Left inset: pigmented Bruch’s membrane corresponds to the halo of pigment on the left side of the disc margin. Right inset: region of unpigmented Bruch’s membrane is shown; this corresponds to a white crescent internal to the pigment halo at the disc margin, which corresponds to a portion of pigmented Bruch’s membrane. (B) Externally oblique. Labeling is according to the schematic in (A). Left inset: Bruch’s membrane is pigmented to its end and does not extend beyond the termination of the border tissue. This Bruch’s membrane extension corresponds to an external crescent of pigment at the disc margin that is internal to the termination of the retinal pigment epithelium. The portion of the border tissue that is internal to the end of Bruch’s membrane (BMO) may be clinically recognizable as an inner reflective (if there is no pigment on the border tissue surface) or a pigmented crescent (if there is pigment on the border tissue surface) that is posterior to the plane of the retinal pigment epithelium. An inner pigmented halo (lighter gray, stippled) is shown on both sides of the disc diagram. Right inset: unpigmented Bruch’s membrane extends internally to the border tissue termination, corresponding to a reflective crescent internal to the pigment crescent. Again, pigmented border tissue (lighter gray, stippled) extends internally to the reflective crescent. In left and right insets the border tissue/scleral junction is depicted without a true scleral lip, which, when present and visible, appears internal and deep to the other structures.
Figure 6.
 
Five examples (AE) of good alignment between BMO points and the clinical disc margin, after first-pass colocalization. Left: clinical images. Colocalizations of the vessel reconstructions to the clinical images are shown in the middle. Right: colocalized BMO points (red glyphs) and clinical disc margin delineations (blue and green glyphs).
Figure 6.
 
Five examples (AE) of good alignment between BMO points and the clinical disc margin, after first-pass colocalization. Left: clinical images. Colocalizations of the vessel reconstructions to the clinical images are shown in the middle. Right: colocalized BMO points (red glyphs) and clinical disc margin delineations (blue and green glyphs).
Figure 7.
 
Termination of unpigmented Bruch’s membrane is clinically visible and aligns to the disc margin. (A) Histomorphometric section taken at 67.5° from a left eye. White box: area of interest in the nasal region of the optic nerve head. (B) Area within the white box has been magnified to highlight the structures composing the disc margin. In this section, there is a substantial overhang of Bruch’s membrane (pigmented and unpigmented) beyond the termination of the border tissue of Elschnig. (C) Structures composing the disc margin have been delineated; termination of unpigmented Bruch’s membrane, or BMO (red glyph), termination of pigmented Bruch’s membrane (light blue glyph), junction of border tissue of Elschnig with Bruch’s membrane (scleral ring of Elschnig; white glyph), and anterior scleral canal opening (dark blue glyph). (D) In vivo disc photograph of the eye from which the histomorphometric reconstruction was obtained. (E) After colocalization of the 3D vessel reconstruction to the disc photograph, the termination of unpigmented Bruch’s membrane (red glyph) coincides with the innermost white reflective halo at the disc margin. The termination of pigmented Bruch’s membrane (light blue glyph) coincides with the inner edge of the pigment at the disc margin. The border tissue/Bruch’s membrane junction (scleral ring, white glyph) coincides with a white reflective stripe within the mottled disc margin pigment; the anterior scleral canal opening (dark blue glyph) is external to this. It is apparent in this eye that the innermost white reflective structure at the disc margin is not the scleral ring of Elschnig but the edge of unpigmented Bruch’s membrane. In this region of the disc, the scleral ring is considerably external to what the clinician perceives as the disc margin.
Figure 7.
 
Termination of unpigmented Bruch’s membrane is clinically visible and aligns to the disc margin. (A) Histomorphometric section taken at 67.5° from a left eye. White box: area of interest in the nasal region of the optic nerve head. (B) Area within the white box has been magnified to highlight the structures composing the disc margin. In this section, there is a substantial overhang of Bruch’s membrane (pigmented and unpigmented) beyond the termination of the border tissue of Elschnig. (C) Structures composing the disc margin have been delineated; termination of unpigmented Bruch’s membrane, or BMO (red glyph), termination of pigmented Bruch’s membrane (light blue glyph), junction of border tissue of Elschnig with Bruch’s membrane (scleral ring of Elschnig; white glyph), and anterior scleral canal opening (dark blue glyph). (D) In vivo disc photograph of the eye from which the histomorphometric reconstruction was obtained. (E) After colocalization of the 3D vessel reconstruction to the disc photograph, the termination of unpigmented Bruch’s membrane (red glyph) coincides with the innermost white reflective halo at the disc margin. The termination of pigmented Bruch’s membrane (light blue glyph) coincides with the inner edge of the pigment at the disc margin. The border tissue/Bruch’s membrane junction (scleral ring, white glyph) coincides with a white reflective stripe within the mottled disc margin pigment; the anterior scleral canal opening (dark blue glyph) is external to this. It is apparent in this eye that the innermost white reflective structure at the disc margin is not the scleral ring of Elschnig but the edge of unpigmented Bruch’s membrane. In this region of the disc, the scleral ring is considerably external to what the clinician perceives as the disc margin.
The authors thank Galen Williams, Erica Dyrud, and Wenxia Wang for the delineation of histomorphometric reconstructions; Jonathan Grimm for software support; Juan Reynaud for software and hardware support; and Joanne Couchman for assistance with the figures. 
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Figure 1.
 
Method for colocalizing the clinical disc photograph to the 3D vessel reconstruction using parallel viewing software. (A) The clinical photograph is viewed before colocalization at a distance from the vessel reconstruction. (B) Shifts in the x- and y-axes allow approximate colocalization in the horizontal plane. (C) Magnification of the clinical image has been increased to match the dimensions of the vessel reconstruction. (D) The clinical photograph and vessel reconstruction are viewed in the coronal plane, with the BMO point cloud visible (red glyphs). This maneuver is performed to facilitate z-axis adjustment. The clinical photograph can be seen to be tilted in a different orientation to the long axis of the BMO point cloud. (E) The z-axis tilt has been adjusted so that the clinical image (coronal profile, arrows) is now orientated in the same plane as the long axis of the BMO point cloud. (F) The clinical image has been moved vertically in the z-axis so that the coronal image profile (arrows) coincides with the BMO point cloud. Once this adjustment has been completed, the BMO points are switched off. (G) The clinical image is now viewed in the en face orientation after z-axis adjustment. Rotation about the image centroid was performed to accurately align the clinical vessel outline to the vessel reconstruction. (H) Final location of the BMO points in relation to the clinical disc photograph is displayed.
Figure 1.
 
Method for colocalizing the clinical disc photograph to the 3D vessel reconstruction using parallel viewing software. (A) The clinical photograph is viewed before colocalization at a distance from the vessel reconstruction. (B) Shifts in the x- and y-axes allow approximate colocalization in the horizontal plane. (C) Magnification of the clinical image has been increased to match the dimensions of the vessel reconstruction. (D) The clinical photograph and vessel reconstruction are viewed in the coronal plane, with the BMO point cloud visible (red glyphs). This maneuver is performed to facilitate z-axis adjustment. The clinical photograph can be seen to be tilted in a different orientation to the long axis of the BMO point cloud. (E) The z-axis tilt has been adjusted so that the clinical image (coronal profile, arrows) is now orientated in the same plane as the long axis of the BMO point cloud. (F) The clinical image has been moved vertically in the z-axis so that the coronal image profile (arrows) coincides with the BMO point cloud. Once this adjustment has been completed, the BMO points are switched off. (G) The clinical image is now viewed in the en face orientation after z-axis adjustment. Rotation about the image centroid was performed to accurately align the clinical vessel outline to the vessel reconstruction. (H) Final location of the BMO points in relation to the clinical disc photograph is displayed.
Figure 2.
 
The identification of Bruch’s membrane and BMO in a histomorphometric section. Top left panel: clinical disc photograph (OD) before disc margin delineation. Top right panel: clinical disc photograph displaying colocalized histomorphometric BMO points (red glyphs) and the clinical disc margin points (blue and green glyphs). The black line is the approximate location of the vertical histomorphometric section shown in the middle panel. The black circle is the approximate location of the histomorphometric region (white box in the middle panel) magnified in the two bottom panels. Middle panel: central vertical histomorphometric section shown as a black line in the top right panel. Superior is left and inferior is right. The area within the white box is magnified in the bottom panels. Note that because the tissues are sectioned from the vitreous (top) to the orbital optic nerve (bottom), a dark shadow is present until the serial sectioning plane passes through the dense pigment of the retinal pigment epithelium, choroid, and Bruch’s membrane. Bottom left panel: magnified view of the highlighted white box in the middle panel that demonstrates the superior disc margin anatomy. Bottom right panel: the same region, labeled as follows: A, sclera; B, choroid; C, Bruch’s membrane; D, commencement of pigmented Bruch’s membrane, which in this section appears to colocalize to the termination of the choroid; E, termination of pigmented Bruch’s membrane and the commencement of unpigmented Bruch’s membrane (note the presence of pigment shadows of variable density cast vertically along the course of the pigmented Bruch’s membrane but absent in the unpigmented Bruch’s membrane); F, termination of unpigmented Bruch’s membrane, which would be delineated as BMO in this section; G, border tissue of Elschnig. In this eye, Bruch’s membrane fuses with the superior edge of the border tissue and extends slightly beyond its termination.
Figure 2.
 
The identification of Bruch’s membrane and BMO in a histomorphometric section. Top left panel: clinical disc photograph (OD) before disc margin delineation. Top right panel: clinical disc photograph displaying colocalized histomorphometric BMO points (red glyphs) and the clinical disc margin points (blue and green glyphs). The black line is the approximate location of the vertical histomorphometric section shown in the middle panel. The black circle is the approximate location of the histomorphometric region (white box in the middle panel) magnified in the two bottom panels. Middle panel: central vertical histomorphometric section shown as a black line in the top right panel. Superior is left and inferior is right. The area within the white box is magnified in the bottom panels. Note that because the tissues are sectioned from the vitreous (top) to the orbital optic nerve (bottom), a dark shadow is present until the serial sectioning plane passes through the dense pigment of the retinal pigment epithelium, choroid, and Bruch’s membrane. Bottom left panel: magnified view of the highlighted white box in the middle panel that demonstrates the superior disc margin anatomy. Bottom right panel: the same region, labeled as follows: A, sclera; B, choroid; C, Bruch’s membrane; D, commencement of pigmented Bruch’s membrane, which in this section appears to colocalize to the termination of the choroid; E, termination of pigmented Bruch’s membrane and the commencement of unpigmented Bruch’s membrane (note the presence of pigment shadows of variable density cast vertically along the course of the pigmented Bruch’s membrane but absent in the unpigmented Bruch’s membrane); F, termination of unpigmented Bruch’s membrane, which would be delineated as BMO in this section; G, border tissue of Elschnig. In this eye, Bruch’s membrane fuses with the superior edge of the border tissue and extends slightly beyond its termination.
Figure 3.
 
Pigment on the lamina surface causing obfuscation of BMO. Top left panel: clinical disc photograph (OS). Bottom left panel: en face view of the histomorphometric reconstruction of the same eye. Black line: orientation of a histomorphometric section image, a portion of which (the superior disc margin) is magnified in the right panels. Black circle: region viewed in the right panels; note the presence of pigment on the lamina surface. Top right panel: histomorphometric view of the superior part of the neural canal. Bottom right panel: Bruch’s membrane delineated (orange glyphs). The white rectangle highlights an area in which the view of Bruch’s membrane is obscured by a dark shadow cast from the lamina pigment below. Accurate delineation of BMO can be difficult. White arrowheads: extent of an artifactual choroidal detachment, most likely caused by the perfusion fixation process.
Figure 3.
 
Pigment on the lamina surface causing obfuscation of BMO. Top left panel: clinical disc photograph (OS). Bottom left panel: en face view of the histomorphometric reconstruction of the same eye. Black line: orientation of a histomorphometric section image, a portion of which (the superior disc margin) is magnified in the right panels. Black circle: region viewed in the right panels; note the presence of pigment on the lamina surface. Top right panel: histomorphometric view of the superior part of the neural canal. Bottom right panel: Bruch’s membrane delineated (orange glyphs). The white rectangle highlights an area in which the view of Bruch’s membrane is obscured by a dark shadow cast from the lamina pigment below. Accurate delineation of BMO can be difficult. White arrowheads: extent of an artifactual choroidal detachment, most likely caused by the perfusion fixation process.
Figure 4.
 
Two principal border tissue configurations with variations. (A) Internally oblique. Left: disc photograph (OS) showing the colocalized BMO (red glyphs) and disc margin delineations (blue and green glyphs). Black line: approximate orientation of the histomorphometric section from which the circular region is magnified in the middle. Middle: histomorphometric disc margin region. Bruch’s membrane (orange glyphs), BMO (red glyph), and border tissue (green glyphs) are delineated. The inferior edge of the border tissue communicates with sclera (the border tissue/scleral junction), and the superior edge extends into the neural canal fusing with Bruch’s membrane (the border tissue termination), which extends beyond this point and includes an unpigmented portion. Right: representative histologic section taken from a healthy monkey eye (perfusion fixed at IOP 10 mm Hg; midhorizontal sagittal section, hematoxylin and eosin stain) demonstrating an internally oblique configuration. White arrows: border tissue. Black arrows: extension of unpigmented Bruch’s membrane. In this case, there is no clear extension of Bruch’s membrane beyond the termination of the border tissue. (B) Externally oblique. Left: disc photograph (OD), demarcated as in (A). Note that within and around the circled region, the clinical disc margin has been marked internal to the histomorphometric BMO points. Middle: histomorphometric section showing the border tissue configuration, demarcated as in (A). Note that the inferior edge of the border tissue is internal to its termination at Bruch’s membrane. Bruch’s membrane does not extend beyond the border tissue termination. In this instance, dense pigment within the sclera immediately adjacent to the neural canal casts a shadow upward that probably explains the lack of a highly reflective scleral lip (white arrows) within this region of the clinical photograph. Right: representative histologic section taken from a healthy monkey eye (perfusion fixed at IOP 10 mm Hg; midhorizontal sagittal section, hematoxylin and eosin stain) demonstrating the externally oblique configuration. White arrows: border tissue. Black arrows: Bruch’s membrane (pigmented). (C) Vertical configuration. The border tissue extends vertically from the sclera to meet Bruch’s membrane. Bruch’s membrane extends beyond this point with pigmented (outer) and unpigmented (inner) portions. (D) Horizontal configuration. The border tissue extends horizontally to meet BMO. This configuration is an extreme form of an internally oblique border tissue configuration.
Figure 4.
 
Two principal border tissue configurations with variations. (A) Internally oblique. Left: disc photograph (OS) showing the colocalized BMO (red glyphs) and disc margin delineations (blue and green glyphs). Black line: approximate orientation of the histomorphometric section from which the circular region is magnified in the middle. Middle: histomorphometric disc margin region. Bruch’s membrane (orange glyphs), BMO (red glyph), and border tissue (green glyphs) are delineated. The inferior edge of the border tissue communicates with sclera (the border tissue/scleral junction), and the superior edge extends into the neural canal fusing with Bruch’s membrane (the border tissue termination), which extends beyond this point and includes an unpigmented portion. Right: representative histologic section taken from a healthy monkey eye (perfusion fixed at IOP 10 mm Hg; midhorizontal sagittal section, hematoxylin and eosin stain) demonstrating an internally oblique configuration. White arrows: border tissue. Black arrows: extension of unpigmented Bruch’s membrane. In this case, there is no clear extension of Bruch’s membrane beyond the termination of the border tissue. (B) Externally oblique. Left: disc photograph (OD), demarcated as in (A). Note that within and around the circled region, the clinical disc margin has been marked internal to the histomorphometric BMO points. Middle: histomorphometric section showing the border tissue configuration, demarcated as in (A). Note that the inferior edge of the border tissue is internal to its termination at Bruch’s membrane. Bruch’s membrane does not extend beyond the border tissue termination. In this instance, dense pigment within the sclera immediately adjacent to the neural canal casts a shadow upward that probably explains the lack of a highly reflective scleral lip (white arrows) within this region of the clinical photograph. Right: representative histologic section taken from a healthy monkey eye (perfusion fixed at IOP 10 mm Hg; midhorizontal sagittal section, hematoxylin and eosin stain) demonstrating the externally oblique configuration. White arrows: border tissue. Black arrows: Bruch’s membrane (pigmented). (C) Vertical configuration. The border tissue extends vertically from the sclera to meet Bruch’s membrane. Bruch’s membrane extends beyond this point with pigmented (outer) and unpigmented (inner) portions. (D) Horizontal configuration. The border tissue extends horizontally to meet BMO. This configuration is an extreme form of an internally oblique border tissue configuration.
Figure 5.
 
Two principal border tissue configurations, their relationship to a pigmented or unpigmented extension of Bruch’s membrane, and the resultant clinical disc margin anatomy. (A) Internally oblique. The diagram shows the clinical optic disc appearance (top) and a cross-section of the optic nerve head (bottom). Labeling is as follows: 1, sclera; 2, choriocapillaris; 3, retinal pigment epithelium with Bruch’s membrane; 4, border tissue; 5, neural canal boundary; 6, pigment on the surface of Bruch’s membrane; 7, Bruch’s membrane. Left inset: pigmented Bruch’s membrane corresponds to the halo of pigment on the left side of the disc margin. Right inset: region of unpigmented Bruch’s membrane is shown; this corresponds to a white crescent internal to the pigment halo at the disc margin, which corresponds to a portion of pigmented Bruch’s membrane. (B) Externally oblique. Labeling is according to the schematic in (A). Left inset: Bruch’s membrane is pigmented to its end and does not extend beyond the termination of the border tissue. This Bruch’s membrane extension corresponds to an external crescent of pigment at the disc margin that is internal to the termination of the retinal pigment epithelium. The portion of the border tissue that is internal to the end of Bruch’s membrane (BMO) may be clinically recognizable as an inner reflective (if there is no pigment on the border tissue surface) or a pigmented crescent (if there is pigment on the border tissue surface) that is posterior to the plane of the retinal pigment epithelium. An inner pigmented halo (lighter gray, stippled) is shown on both sides of the disc diagram. Right inset: unpigmented Bruch’s membrane extends internally to the border tissue termination, corresponding to a reflective crescent internal to the pigment crescent. Again, pigmented border tissue (lighter gray, stippled) extends internally to the reflective crescent. In left and right insets the border tissue/scleral junction is depicted without a true scleral lip, which, when present and visible, appears internal and deep to the other structures.
Figure 5.
 
Two principal border tissue configurations, their relationship to a pigmented or unpigmented extension of Bruch’s membrane, and the resultant clinical disc margin anatomy. (A) Internally oblique. The diagram shows the clinical optic disc appearance (top) and a cross-section of the optic nerve head (bottom). Labeling is as follows: 1, sclera; 2, choriocapillaris; 3, retinal pigment epithelium with Bruch’s membrane; 4, border tissue; 5, neural canal boundary; 6, pigment on the surface of Bruch’s membrane; 7, Bruch’s membrane. Left inset: pigmented Bruch’s membrane corresponds to the halo of pigment on the left side of the disc margin. Right inset: region of unpigmented Bruch’s membrane is shown; this corresponds to a white crescent internal to the pigment halo at the disc margin, which corresponds to a portion of pigmented Bruch’s membrane. (B) Externally oblique. Labeling is according to the schematic in (A). Left inset: Bruch’s membrane is pigmented to its end and does not extend beyond the termination of the border tissue. This Bruch’s membrane extension corresponds to an external crescent of pigment at the disc margin that is internal to the termination of the retinal pigment epithelium. The portion of the border tissue that is internal to the end of Bruch’s membrane (BMO) may be clinically recognizable as an inner reflective (if there is no pigment on the border tissue surface) or a pigmented crescent (if there is pigment on the border tissue surface) that is posterior to the plane of the retinal pigment epithelium. An inner pigmented halo (lighter gray, stippled) is shown on both sides of the disc diagram. Right inset: unpigmented Bruch’s membrane extends internally to the border tissue termination, corresponding to a reflective crescent internal to the pigment crescent. Again, pigmented border tissue (lighter gray, stippled) extends internally to the reflective crescent. In left and right insets the border tissue/scleral junction is depicted without a true scleral lip, which, when present and visible, appears internal and deep to the other structures.
Figure 6.
 
Five examples (AE) of good alignment between BMO points and the clinical disc margin, after first-pass colocalization. Left: clinical images. Colocalizations of the vessel reconstructions to the clinical images are shown in the middle. Right: colocalized BMO points (red glyphs) and clinical disc margin delineations (blue and green glyphs).
Figure 6.
 
Five examples (AE) of good alignment between BMO points and the clinical disc margin, after first-pass colocalization. Left: clinical images. Colocalizations of the vessel reconstructions to the clinical images are shown in the middle. Right: colocalized BMO points (red glyphs) and clinical disc margin delineations (blue and green glyphs).
Figure 7.
 
Termination of unpigmented Bruch’s membrane is clinically visible and aligns to the disc margin. (A) Histomorphometric section taken at 67.5° from a left eye. White box: area of interest in the nasal region of the optic nerve head. (B) Area within the white box has been magnified to highlight the structures composing the disc margin. In this section, there is a substantial overhang of Bruch’s membrane (pigmented and unpigmented) beyond the termination of the border tissue of Elschnig. (C) Structures composing the disc margin have been delineated; termination of unpigmented Bruch’s membrane, or BMO (red glyph), termination of pigmented Bruch’s membrane (light blue glyph), junction of border tissue of Elschnig with Bruch’s membrane (scleral ring of Elschnig; white glyph), and anterior scleral canal opening (dark blue glyph). (D) In vivo disc photograph of the eye from which the histomorphometric reconstruction was obtained. (E) After colocalization of the 3D vessel reconstruction to the disc photograph, the termination of unpigmented Bruch’s membrane (red glyph) coincides with the innermost white reflective halo at the disc margin. The termination of pigmented Bruch’s membrane (light blue glyph) coincides with the inner edge of the pigment at the disc margin. The border tissue/Bruch’s membrane junction (scleral ring, white glyph) coincides with a white reflective stripe within the mottled disc margin pigment; the anterior scleral canal opening (dark blue glyph) is external to this. It is apparent in this eye that the innermost white reflective structure at the disc margin is not the scleral ring of Elschnig but the edge of unpigmented Bruch’s membrane. In this region of the disc, the scleral ring is considerably external to what the clinician perceives as the disc margin.
Figure 7.
 
Termination of unpigmented Bruch’s membrane is clinically visible and aligns to the disc margin. (A) Histomorphometric section taken at 67.5° from a left eye. White box: area of interest in the nasal region of the optic nerve head. (B) Area within the white box has been magnified to highlight the structures composing the disc margin. In this section, there is a substantial overhang of Bruch’s membrane (pigmented and unpigmented) beyond the termination of the border tissue of Elschnig. (C) Structures composing the disc margin have been delineated; termination of unpigmented Bruch’s membrane, or BMO (red glyph), termination of pigmented Bruch’s membrane (light blue glyph), junction of border tissue of Elschnig with Bruch’s membrane (scleral ring of Elschnig; white glyph), and anterior scleral canal opening (dark blue glyph). (D) In vivo disc photograph of the eye from which the histomorphometric reconstruction was obtained. (E) After colocalization of the 3D vessel reconstruction to the disc photograph, the termination of unpigmented Bruch’s membrane (red glyph) coincides with the innermost white reflective halo at the disc margin. The termination of pigmented Bruch’s membrane (light blue glyph) coincides with the inner edge of the pigment at the disc margin. The border tissue/Bruch’s membrane junction (scleral ring, white glyph) coincides with a white reflective stripe within the mottled disc margin pigment; the anterior scleral canal opening (dark blue glyph) is external to this. It is apparent in this eye that the innermost white reflective structure at the disc margin is not the scleral ring of Elschnig but the edge of unpigmented Bruch’s membrane. In this region of the disc, the scleral ring is considerably external to what the clinician perceives as the disc margin.
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