shows a representative 2D UHROCT tomogram of the healthy human corneo-scleral limbus acquired from a temporal location. The major corneal layers such as the epithelium (Ep), Bowman’s membrane (BM), stroma (S) and the Descemet’s – Endothelium complex (DEC) are visible on the left (corneal) side of the tomogram. Longitudinal and normal cross-sections of structural features resembling blood or lymph vessels are observed in the limbal (right) side of the image (red arrows). Because the ranges of vessel diameter and flow velocities in the blood and lymph vessels can overlap, structural and Doppler OCT cannot be used alone to differentiate between blood and lymph vessels in the limbus. Spectroscopic analysis that can utilize the wavelength dependent scattering and absorption properties of blood and lymph, maybe helpful in future studies that target to differential lymph from blood vasculature in the human corneo-scleral limbus. The limbus is also the location where the corneal epithelium transitions into the bulbar conjunctival epithelium, the most superficial layer of the transparent tissue overlying the white sclera. The image in shows differences in the structure and optical properties of the corneal epithelium and the bulbar conjunctiva that can be associated with difference in the tissue morphology. While the corneal epithelium (blue arrow) appears more transparent, the conjunctiva (green arrow) appears more optically scattering (darker shade of gray color). A magnified view of the region in the corneal epithelium, marked with the red square in , is presented in and compared with a representative histological cross-section [27
] (). The Bowman’s membrane boundaries are visible in and in (black solid arrows) and its thickness (~15 µm) compares well with the histological cross-section. Furthermore, a low reflective band is visible at the interface between the corneal epithelium and the Bowman’s membrane both in the original image () and the magnified view (, dashed black arrows). Comparison with histology () suggests that the band most likely corresponds to the basal cell layer of the corneal epithelium, comprised of columnar cells that are ~15µm to 20µm in length. Corneal basal epithelial cells have a large cytoplasm to nucleus ratio, which explains the lower scattering of this cell layer as compared to the upper epithelial layers. also shows the termination of the corneal epithelium basal layer in the limbus (orange arrow). Upper layers of the corneal epithelium were not individually resolved due to the smaller axial dimensions of the polyhedral and squamous cells comprising those layers, which are comparable to the 3µm axial resolution of the current UHROCT system.
Fig. 1 A representative cross-sectional UHROCT tomogram of the human corneo-scleral limbus acquired from a temporal location (A). The blue arrow marks the corneal epithelium; the orange, the termination of the basal cell layer of the corneal epithelium in the (more ...)
shows comparison between histology [28
] of the human corneo-scleral limbus (A) and a representative UHROCT tomogram of the human temporal limbus acquired in vivo
from a nasal location (B). The 3µm axial resolution of the UHROCT system is not sufficient to resolve the endothelium (En) and the Descemet’s membrane however a combination of the two layers is observed that we refer to as the Descemet’s Endothelium Complex (DEC) that can be distinguished from the corneal stroma (S). The high axial resolution of the optical imaging system also allows for clear visualization of the Schlemm’s canal (white arrows) and the trabecular meshwork (TM) (black arrows).
Fig. 2 Comparison between H&E histology of the human corneal limbus  (A) and a representative UHROCT tomogram of the human temporal limbus acquired in vivo (B). Red arrows mark at the corneal endothelium, white arrows the Schlemm’s canal, (more ...)
Multiple 3D stacks of UHROCT images were acquired from temporal and nasal locations in the eye in all subjects.
shows representative 2D tomograms of healthy, normal human limbus, acquired temporally (A-D) and nasally (E, F). The left side of the tomograms in show the cornea, where the corneal layers are clearly visualized. The right side of the same images shows the highly scattering sclera with networks of blood and lymph vessels. The corneal epithelium and the bulbar conjunctiva are marked with blue and green arrows respectively. shows another representative temporal scan of the limbus from the same subject. A highly reflective (dark grey color) fork-like feature, 15µm in diameter (inside the red rectangle) is clearly visible in the corneal region of the tomogram. According to a recent in vivo
study of the cornea conducted with a confocal microscope [13
], the size (~15µm diameter), location (mid stroma), shape (almost straight line with a bifurcating end) and high reflectivity of this structure suggests that it most likely corresponds to a corneal nerve fiber bundle. Notice that the feature extends well into sclera region of the tomogram (red arrow), but its appearance (color) changes from dark grey to almost white in the tomogram. This change is perhaps due to the fact that corneal tissue is far less scattering than the nerve fibers, therefore in the corneal part of the UHROCT tomogram, a nerve fiber bundle will appear dark grey in color. However, scleral tissue is highly scattering, therefore a nerve fiber bundle, which is less scattering than the scleral tissue, would appear in pale grey color in the scleral part of the UHROCT tomogram. Alternatively, the change in the apparent reflectivity of the nerve fiber bundle can be manifestation of the change in the nerve itself, since nerves lose their myelin sheaths as they transition into the cornea [29
Representative UHROCT tomograms of the normal human limbus acquired in vivo from temporal (A-D) and nasal (E,F) locations. Blue and green arrows mark the cornea and the bulbar conjunctiva, respectively. Red arrows mark corneal nerves.
To highlight subject-related differences in the morphology and optical properties of the limbal tissue as imaged with UHROCT, representative temporal UHROCT images of the limbus of the second subject are shown (). In general, the limbus structure appears similar in both cases; however, the difference in the optical properties of the corneal epithelium and the bulbar conjunctiva, as well as the transition between these two tissues appears more pronounced in the second illustration. Furthermore, a number of finer nerve fibre bundles are visible in (red arrows) and , which appear thinner (~10µm in diameter) than the nerve fibre observed in subject one (). Representative nasal scans of the limbus acquired from subject number two are shown in . Morphologically, the temporal and nasal scans appear very similar except that the tissue below the bulbar conjunctiva observed in the nasal scans shows discrete peaks and valleys. The nasal scans of the limbus also show the termination of the corneal Descemet’s-endothelium complex (), which was not observed in the temporal scans due to the short scanning range of the UHROCT system (~1.3mm in air).
A series of three-dimensional scans (1000 x 1024 x 256) were also acquired from rectangular areas (~4mm x 2mm) of the limbus of each subject. Two movies, presented in
show in-depth, en-face views of the limbus in two different orientations: parallel to the limbus surface (A) and parallel to the pupil plane (B). The red lines in the human eye diagrams above the movie frames indicate the orientation of the en-face scans.
Fig. 4 Frames from movies (Media 1 and Media 2) based on volumetric temporal scans of the human limbus. Dimensions: 1000 x 350x256 (A-scans x pixels x B-scans). The orientation of the en-face sections form the 3D stack are marked with red lines in the human (more ...)
The black arrows in the frame shown in mark what appear to be the highly scattering collagen ridges of the Vogt palisades. The white arrow in the frame in marks the transition from the corneal epithelium to the bulbar conjunctiva. Because the movies are rich in structural details, selected frames from the movie in are shown in
to highlight and discuss specific morphological features. The left side of the images shows the corneal region, while the right side corresponds to the sclera.
Fig. 5 Selected en-face frames from Media 1 (). White arrows in (A) mark the low scattering locations in the limbus between the ridges of the Vogt palisades that house corneal stem cells. The black arrows in (A-D) mark a network of micro-channels that (more ...)
shows an en-face scan acquired ~70 µm below the corneal/conjunctiva surface. Several elliptically shaped, low reflective structural features (white arrows) are observed in between the highly scattering collagen ridges of the putative Vogt palisades. By comparing the size (50µm to 200 µm in diameter, ~500 µm in length), location (50 µm to 100 µm below the tissue surface) and reduced scattering properties of these structures with similar morphological features observed in the limbus with LSCM [11
] we can conclude that most likely these features correspond to the locations in the Vogt palisades that house the stem cells responsible for the regeneration of the corneal epithelium. Because the stem cells have large cytoplasm-to-nucleus ratio, these regions appear less scattering in the OCT tomograms as compared to the fibrous ridges of the palisades.
A network of radially oriented, channel-like, low reflective features are observed at different depths in the limbus (black arrows, A-D), that appear to extend from the cornea toward the sclera. These channels appear to connect to the blood vasculature in the sclera (white arrows, C and D) at depths of 100 µm–150 µm below the tissue surface. The image frames E and F were acquired at depths of ~500µm below the tissue surface and show another set of channel like features that most likely are part of the trabecular meshwork or the drainage system. These channel-like structures bare close resemblance to the microchannels observed in vivo
in the human limbus with LSCM [11
The images presented in
are selected frames from the movie in , which is based on the same data set, with different orientation of the en-face cross-sections. The image in was acquired at a depth ~50µm below the tissue surface and shows the transition between the cornea and the bulbar conjunctiva in the limbus (black arrow). The images in show the circular cross-sections (black arrows) of the micro-channels ~10-20µm in diameter, that extend from the cornea toward the sclera, at different imaging depths within the limbus. The larger circular features (~20-50µm in diameter) in marked with white arrows and located within the highly scattering sclera are most likely cross-sections of the blood and lymph vessels. Smaller (~10-20µm in diameter) channel-like features were observed on the same frame closer to the cornea, forming what appears to be an arc from a circle (black arrows). Given the imaging depth (~500µm below the corneal surface), it is likely that these morphological features are elements of the aqueous humor drainage system. The image in was acquired ~50µm deeper than the frame in , and reveals both radial and arc channel-like features (white arrows), that could be parts of the trabecular meshwork.
Fig. 6 Selected frames Media 2 (). Black arrows (B-E) mark cross-sections of the micro-channels that extend from the cornea toward the sclera. White arrows mark cross-section of the blood and lymph vessels in the sclera (E) and the trabecular meshwork (more ...)