Multiple 3D image stacks with dimensions 1000 × 512 × 256 (A-scans x Pixels x B-scans) were acquired from the chicken retina at wide (~20°) and narrow (~7°) scanning angles.
shows representative UHROCT images of the chicken retina at locations away from (A) and near the optical nerve head (C), and corresponding H&E stained histological cross-sections of the same regions ( and , respectively). Scale bars provided in all images allow for determination of the size and spatial separation of morphological features in the chicken retina. All retinal layers are clearly visualized in the UHROCT tomograms and match well with the retinal layers as observed in the histological cross-sections (, and ). The ganglion cell layer (GCL) is clearly visible in the UHROCT images as a bright band located immediately below the dark, highly scattering nerve finer layer (NFL). The NFL thickness varies with location in the retina and is thickest at the optic nerve head (ONH). The chicken retina has a fairly thin outer nuclear layer compared to mammalian retinas, which is clear from the images in . The external limiting membrane appears on the UHROCT tomograms as a very distinct dark gray line above the low scattering photoreceptor layer.
Fig. 1 Representative UHROCT tomograms of the chicken retina acquired from locations away (A) and near (C) the ONH, compared with H&E stained histology (B and D). Individual retinal layers observed in the UHROCT tomograms match well with corresponding (more ...)
The chorio-scleral interface (CSI) is clearly visible on both UHROCT images. The chicken sclera contains a fairly thick cartilaginous layer (SC), separating the choroid from the fibrous sclera (S). The cartilaginous sclera is composed of cells with a large cytoplasm to nucleus ratio, thus rendering the cartilage almost transparent to infrared light. Termination of the cartilage is clearly observed near the ONH ( and , black arrows). The apparent difference in the choroidal thickness observed between the UHROCT and histological cross-sections is most likely a post-mortem artifact due to the choroid moving away from the retina during the cryo-sectioning.
shows rendered volumetric stacks from different locations in the chicken retina acquired at wide (A, B and C) and narrow (D) scanning angles. These volumes are comprised of 256 B-scans (1000 x 512), corresponding to ~3 mm x 3 mm (wide angle) and ~1mm x 1 mm (narrow scanning angle) area on the tissue surface. The volumetric images presented in , and , were acquired from a location in the retina close to the pecten (white arrows). Since the pecten is a highly vascular tissue, light absorption and scattering within the blood vessel in the pecten produces a shadowing effect in the UHROCT tomograms. The folds of the pecten can be observed clearly on the en face (
and ) and cross-sectional movies (). The volumetric images in and also show an area at the retinal surface with significantly thinner NFL. This area, located about 2 mm away from the ONH, appears darker, more transparent and round in shape ( and , yellow arrows), and is tentatively speculated to represent the area centralis of the chicken retina. Further examination of other retinal layers in this area is required for confirmation of this hypothesis. The movie frame in shows a view of the chicken retina choroidal vasculature.
Fig. 2 Selected frames from rendered volumetric image stacks of the chicken retina acquired at wide (A, B and C) and narrow (D) scanning angles. Different views of the pecten layer are presented in A (Media 1), B and C (Media 2) and marked with white arrows. (more ...)
Fig. 3 Selected B-scans from the volumetric data set presented in . Thickening of the NFL, thinning of the choroid and termination of the sclera cartilage are observed close to the pecten in the vicinity of the ONH (A). Progressive thinning of the NFL (more ...)
shows representative B-scans that were used to generate the volumetric image presented in . The image in shows a cross-sectional view of the pecten (black arrow). This image also shows thickening of the NFL, thinning of the choroid and termination
of the sclera cartilage near the ONH. The images shown in , and show progressive thinning of the NFL and thickening of both the GCL and the choroid in one location in the retina.
Morphometric analysis of the chicken retina could be very helpful in various chicken models of retinal diseases. Here we used a semi-automatic segmentation algorithm developed by our research group [16
] to segment and measure the thickness of individual layers in the retina. Although NFL and GCL thicknesses are presented in this paper (
) to highlight the usefulness of UHROCT with optic nerve-associated disease models [17
], the algorithm allows for segmentation of all layers of the retina. An original cross-sectional UHROCT tomogram of the chicken retina is shown in . The same image with selected and segmented NFL and GCL is shown in (red lines). En face
thickness maps of the NFL and the GCL are shown in and , respectively. A Gaussian blur algorithm (10 pixels) was used to smooth the jagged appearance of the thickness maps, which is related to the axial OCT resolution and the limited number of B-scans (256) in the 3D imaging stack of the retina, covering a square area of ~3 mm x 3 mm. In the NFL thickness map (), the area closer to the ONH and the pecten is significantly (~50 µm) thicker (black arrow), while an area located ~2 mm away has a thickness of ~10 µm to 15 µm (white arrow). Similar areas at approximately the same locations are observed in the GCL thickness map (). In this case, the area near the ONH and the pecten has a non-existent GCL, while the area marked with the white arrow has a GCL of ~15 µm average thickness. No significant spatial variation in the thickness of the remaining retinal layers was observed in the healthy chicken imaged in this study. It is expected that such thickness changes can occur in some retinal degenerative diseases, which will be the focus of future studies.
Fig. 4 Original UHROCT cross-section of the chicken retina (A); the same image with segmented NFL and GCL (C, red lines); thickness maps of the NFL (B) and the GCL (D). Black arrows mark locations close to the pecten and the ONH, while white arrows mark a location (more ...)