Fig 1.
Application of the “Find Maxima”-function in the open source software FIJI on a full en-face image depicting photoreceptors.
Shown is an example of photoreceptors at 6.5° foveal eccentricity inferior in a 23yo healthy woman. The region within the white rectangle is magnified on the right half of the image. The red rectangle depicts the region of interest. The “Find Maxima”-function determines and counts local maxima of intensity, offering a fast and easy way to find bright signals within an image, here indicated by red crosshairs. The examiner can add and remove selections freely. Using this function, even weaker reflective signals of cones can be detected and then validated, resulting in a total count of 205 cones for this image.
Fig 2.
AO-OCT B-Scan with labelled photoreceptor layers.
Non-averaged image, linear brightness scale, location 6.5° superior eccentricity in a 26yo healthy male. The upper image shows the whole B-Scan. Note, how the inner retinal layers are barely visible due to their lower reflectivity in comparison to the outer retinal layers. Visualization of the inner retina can be improved by focusing the AO-OCT on the nerve fibre layer instead of the RPE, switching from a linear to a logarithmic brightness scale and averaging multiple B-Scans to a single image. The white rectangle shows the region of interest of the outer retina, which can be seen in the lower section of the image: The cone inner segment-outer segment junction (IS/OS) consists of bold dots (purple arrow) with hyper reflectivity and with oscillating intensity, ranging from grey to bright white. Below the IS/OS, the cone outer segments (OS) show mostly very faint and blurry signals; a few hyper-reflective spots can be seen (blue arrow), most likely defects in the packing density of the cone outer segment discs (Pircher et al. 2011). Below the OS, the cone outer segment tips (COST) show a similar pattern as the IS/OS, but the spots (red arrow) appear slightly smaller and better separated. The rod outer segment tips (ROST), are located directly below the COST; depiction in a non-averaged image is difficult due to their small size, however, some signals can be depicted (green arrows). Note: Due to its weak reflectivity, the external limiting membrane, which is located above the IS/OS, can barely be seen in linear brightness scale and non-averaged images.
Fig 3.
The different photoreceptor planes visualized with AO-OCT at 6.5°eccentricity.
The complete retina (CR plane) image integrates signals from all retinal layers, beginning with the ILM through the choroid. This unsegmented depiction is comparable to two-dimensional AO-FC and AO-SLO imaging and shows the cone mosaic with reduced contrast and strong background signals, in particular from the rods and the RPE. The inner segment outer segment junction (IS/OS plane) shows a dense pattern of hyper-reflective dots. The cone outer segments (OS plane) show only a few hyperreflective and some several weak signals without a distinct pattern. The signals of the cone outer segment tips (COST plane) appear smaller and better separable then the IS/OS. The signals of the rod outer segment tips (ROST plane) form a mottled network. The IS/OS+ plane combines signals from the IS/OS to the OS. While its appearance resembles mostly to the IS/OS, subtle difference between these two en-face projections can be found in S1 File. The full en-face image (FEF) projects all signals from the IS/OS to the COST and gives the most complete appearance of the cone mosaic, especially when compared to the CR plane. Additionally, three false-color composite images are shown: A false color-composite image of the COST (red) and IS/OS+ plane (blue) shows signal overlap (purple) in most but not all areas and noticeable variations of signal strength between the layers (blue and red signals). In contrast, false-color composite images of the of the ROST (green) and the IS/OS (purple) or the COST (red) show no relevant overlap (white or yellow) between cone and rod signals.
Fig 4.
The 5 different cone planes, depicted at 2.5°nasal in a 26yo healthy male.
Inner segment outer segment junction zone (IS/OS) shows a dense mosaic of hyper-reflective dots. Cone outer segments (OS) show significantly less hyperreflective dots beside several blurry and weak signals. Combination of IS/OS and OS (IS/OS+) resembles mainly the IS/OS mosaic with some additional signals from the OS below (see also S1 File). Dots from the mosaic of the cone outer segment tips (COST) are better distinguishable compared to the IS/OS. Through integration of all cone signals to a full en-face image (FEF), the most complete appearance of the cone mosaic is achieved. S1 Fig gives a complete overview of all 8 regions imaged in this eye.
Table 1.
Interclass correlation coefficients (two way mixed effect model, absolute agreement, three graders) for cone density measurements utilizing the Find Maxima function.
Three different sets of en-face images were used, each consisting of a random selection of 20 images.
Fig 5.
Mean cone density of all 4 planes, 4 meridians (blue = superior, purple = nasal, yellow = inferior, green = temporal) and 2 eccentricities (solid fill = ecc 2.5°, dashed fill = ecc 6.5°).
N = 20 eyes. Density is generally higher at a foveal eccentricity of 2.5° compared to 6.5°. In all 4 meridians and 2 eccentricities, density was highest in the FEF images, followed by COST, IS/OS+ and ISOS. Significant differences are marked in Table 2.
Table 2.
Mean differences of cone density between the IS/OS, IS/OS+, COST and FEF layers, shown for the 4 meridians and two eccentricities.
All numbers are given in cones/mm2.