Figure 1: Screenshot of the Triton swept-source optical coherence tomography angiography at the time of image acquisition of the optic nerve in a left eye. The circles in the right image provide alignment guidance so that the photographer can focus and center the image on the optic nerve head. Manual adjustment to center the image on the optic nerve head can also be performed.
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Figure 2: Screenshot of the viewing software (IMAGEnet® 6) provided by Triton optical coherence tomography angiography (OCTA) showing that the structural scans from Triton are linked to the angiogram, and an automated segmentation algorithm allows selection of various retinal layers for the angiogram. The Triton software detects the anterior surface of the optic nerve head (ONH), which is labeled “ILM”. It also detects Bruch’s membrane (BM) in the parapapillary region, immediately adjacent to the ONH, and the segmentation within the ONH is completed by maintaining the distance between the ILM and BM lines (bottom left square). Other structural landmarks are also available for software segmentation (drop-down menu in the bottom left square). The superficial angiogram is from the ILM to BM (square with orange outline on the top left) and the deep layer is from BM to 390 μm below BM (square with green outline top middle left). Furthermore, the viewing software co-localizes the manual marking on structural imaging to the superficial and deep angiograms. The vertical pink line in the bottom left image indicates Bruch’s membrane opening on the inferonasal part of the optic nerve head. This location is shown as the intersection of the pink and blue lines on the structuralen face image (bottom middle right), and all the angiograms in the top row.
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Figure 3: The viewing software does not allow the actual marking of the boundary of optic nerve head (ONH) onto the superficial and deep angiograms or removal of optical coherence tomography angiography (OCTA) signals outside the ONH boundary. Hence, we exported all the structural scans (320 horizontal scans per eye) into ImageJ and created a customized plug-in to perform these functions. First, the exported structural scans are re-sliced along the plane perpendicular to the structural scans and in the same orientation as the angiogram to confirm the alignment between structural scans and angiograms based on the location of large blood vessels (A). Then, two reviewers independently mark Bruch’s membrane opening on either side of the ONH on every 5th structural scan, which are spaced 47 μm apart. These markings are then compiled and projected onto the angiogram to show the boundary of the ONH. (B) shows this step with two structural scans spaced 675 μm apart for easier visualization. Both AandB in Figure 3 were generated using Amira™ Software 6.4 (Thermo Fisher Scientific).
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A B
Vessel density =
D. Deep angiogram
C. Superficial angiogram
VD =
Large Vessels Subtraction
20338 - 4744
____________ = 0.468
VD = ____________ = 0.36520338 - 4744 __________________________
ONH area – large vessels area Vessel density =
ONH area
Area occupied by microvasculature
VD =
203389703 = 0.477 A. Superficial angiogram
B. Deep angiogram
VD = 203386793 = 0.334
Large Vessels Inclusion
Figure 4: Details of vessel density calculation with and without large vessel removal are shown for the same eye used in Figure 1 of the manuscript. Vessel density is derived by dividing the area occupied by all blood vessels over the total area of the region of interest, either the optic nerve head (ONH) or the 0.70 mm elliptical annulus extending around the ONH for the peripapillary region. When large vessels are excluded, the area occupied by large vessels is subtracted from the total area of the region of interest. In the superficial ONH angiogram, inclusion of the large vessels increases both the area occupied by all blood vessels (the numerator) and the area of the region of interest (denominator). The final result is an increase in the vessel density value (A). In the deep ONH angiogram, two important artifacts are eliminated by removal of large vessels. Projection artifact, which is the image of a superficial vessel in the deep layer (red arrow in B), increases the measurement of the area occupied by all blood vessels, while shadowing artifact, which is the blockage of deep layer signal by a shadow casted by a superficial structure (blue arrow in B), decreases the measurement of the area occupied by all blood vessels. Furthermore, the area of the region of interest (denominator) is increased by inclusion of the large vessels. By including large vessels in the deep ONH angiogram, the combined result is a slight increase of the numerator with a larger increase of the denominator, resulting in a smaller vessel density value (B). For comparison, C and D show vessel density values in the superficial and deep ONH layers with large vessels removed.
7299
5695 _______
_______
__________________________ Area occupied by microvasculature
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Figure 5: Optic disc photo (A) and peripapillary retinal nerve fiber layer (RNFL) thickness profile (C) from the left eye of a control subject. 3 x 3 mm en face swept source OCT angiograms were generated for the superficial layer (D) and deep layer (E) of the optic nerve head (ONH), with red circles indicating the disc margin generated by manual identification of Bruch’s membrane (BM) opening. The BM opening (red dots) was marked in the set of cross-sectional OCT scans obtained during imaging (G, H). The superficial layer of the ONH extends from the internal limiting membrane
membrane (ILM) to BM (G, dark blue band), while the deep layer extends from BM to 390 µm below BM (H, orange band). Areas occupied by large vessels were excluded semi-automatically from the superficial layer and superimposed on the deep layer to limit projection artifact. The regions of the ONH analyzed after removal of large blood vessels are shown for the superficial layer (J) and deep layer (K). For this control eye, the vessel density and integrated OCTA by ratio analysis signal (IOS) in the superficial ONH layer were found to be 41.0% and 38.9% respectively, and 51.1%
and
and 42.6% in the deep ONH layer, respectively. The 4.5 x 4.5 mm angiogram of the superficial retinal layer (F) was used in analysis of the peripapillary region, with red lines indicating a 0.70 mm wide elliptical annulus extending from the disc margin generated from manually marking the BM opening on cross-sectional OCT scans. The superficial retinal layer (I, green band) extends from the ILM to the interface of RNFL and ganglion cell layer. Large vessel exclusion was performed automatically, and the peripapillary region analyzed after removal of large vessels is shown (L). For this control eye, the vessel density and IOS in the peripapillary region were 42.8% and 52.1%, respectively.