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Abstract
Intraocular pressure (IOP) is the only modifiable risk factor for glaucoma progression, and many treatments target the trabecular meshwork (TM). Imaging this region in vivo is challenging due to optical limitations of imaging through the cornea at high angles. We propose a gonioscopic OCT approach using a custom goniolens and a commercially available OCT device to improve imaging of the TM, Schlemm’s canal (SC) and adjacent structures within the iridocorneal angle (ICA). The goniolens is modified with a plano-convex focusing lens and placed on the eye optically mated with goniogel and aided by a 3D adjustable mount. Gonioscopic OCT volume scans are acquired to image SC. Transverse enface images allowed measurements of SC over a 45° section of the ICA for the first time and revealed locations of SC narrowing. The band of extracanalicular limbal lamina and corneoscleral bands were imaged in most subjects and these bands were confirmed using exterior OCT imaging. The polarization dependence of the visibility of these structures is studied by polarization rotation the OCT beam with a half-wave plate, allowing increased contrast of SC. Gonioscopic OCT has successfully been used to image the human ICA in 3D in vivo. This approach provides more detailed characterization of the TM and SC, enhancing their contrast against their birefringent backgrounds.
© 2022 Optica Publishing Group under the terms of the Optica Open Access Publishing Agreement
Corrections
10 October 2022: A minor correction was made to the author list.
1. Introduction
Glaucoma is a progressive optic neuropathy [1,2] with approximately 64.3 million individuals affected worldwide and is a leading cause of irreversible vision loss and blindness in the world [1–4]. While glaucoma remains poorly understood, it clearly causes a loss of retinal ganglion cells leading to a progressive, characteristic visual field loss. The factors contributing to damage are thought to include biomechanical changes associated with elevated intraocular pressure (IOP) [5,6], cerebrospinal fluid pressure, and ocular hemodynamics. However, IOP is currently the only modifiable risk factor for glaucoma even though the correlation between elevated IOP and progressive glaucomatous damage is not high [7]. The low correlation presumably arises due to damage caused by interactions between IOP, blood pressure, cerebrospinal pressure, and other contributory factors which all show individual differences. Most strategies to delay or avoid vision loss in glaucoma patients, even those with normal IOP, are targeted towards lowering IOP.
The IOP represents a balance between aqueous humor inflow and outflow. The outflow is primarily through the anterior segment of the eye, either through the trabecular meshwork (TM), the major outflow pathway, or flow through the uveoscleral pathway. The TM is a layered sieve-like structure lying at the junction of the iris and cornea, the iridocorneal angle or ICA. Aqueous fluid, which is pumped into the eye by the ciliary body, drains through the TM then crosses the juxtacanalicular tissue into Schlemm’s canal (SC), and from there continues into the episcleral veins. Anatomically, the TM is divided into three sections: the most proximal (adjacent to the iris) is the uveal meshwork; the middle is the corneoscleral meshwork, and the most distal is the juxtacanalicular tissue, lying adjacent to SC [8]. A major cause of elevated IOP is increased resistance to flow through the TM, probably in the juxtacanalicular area [8–11], as well as changes to the extracellular matrix of the TM [12–16].
Unfortunately, the TM is not readily accessible to in vivo study. Because the TM lies at the apex of the ICA, light cannot be used to image obliquely through the cornea to the TM. This is due to the near total internal reflection of light returning from the TM by the cornea. Typical clinical imaging uses a slit lamp and a gonioscopy lens to overcome this. A typical goniolens consists of a PMMA conical frustum body with an interior flat mirror positioned at 59° to 64°, and a back surface with a radius of curvature similar to that of the cornea, as seen in Fig. 1(A). The lens is placed in direct contact with the cornea, optically mating the lens to the cornea using a viscous fluid, such as goniogel. The optical mating reduces the index difference at the cornea, and thus, avoids total internal reflection of light returning from the ICA. The ICA, including the TM is then visualized using a slit lamp. The resulting 2D enface image is inherently limited in the achievable resolution (Fig. 1(B) as the angles at which the light crosses the cornea and other surfaces are high. Light is reflected from the gonioscopy mirror at high angles and sequentially crosses the back surface of the goniolens, the goniogel layer, and the cornea, all of which have different refractive indices, causing optical aberrations that distort the image, and hence, limit the resolution. As a result of the limitation of gonioscopy, our knowledge of the anatomy of the TM and the impact of disease has been obtained mostly from either post-mortem studies or animal models such as mouse [9] or non-human primate [17,18], using both light and electron microscopy [14,16,19–21].
Fig. 1. A) Schematic of a commercial goniolens coupled to the cornea with goniogel, where the yellow arrow indicates the direction of incident light. B) Clinical gonioscopy slit lamp image using a commercial goniolens, indicating with blue arrows Schwalbe’s line (SL), anterior TM (Ant. TM), posterior TM (Post. TM) and ciliary body band (CBB) (image reprinted with permission from B.J. King et al. High-resolution, adaptive optics imaging of the human trabecular meshwork in vivo, TVST. 8, 5–5; 2019). Commercial Heidelberg Spectralis C) SLO (subject with pterygium) and D) anterior segment OCT of the same subject taken perpendicular to the corneal apex, as marked by green arrow in the SLO image in C).
Recently our group performed high-resolution imaging of the TM in vivo by incorporating a modified gonioscopy lens into our adaptive optics scanning laser ophthalmoscope (AOSLO). A clinical goniolens was modified by cementing a 22 mm focal length button lens on the anterior surface of the goniolens body, centered above the 62° goniolens mirror. This allowed light from the AOSLO to be focused onto the TM and surrounding structures. With this adaptive optics gonioscopy (AOG) [22] approach we achieved a resolution of approximately 2.5 µm in situ, allowing visualizations of endothelial cells and macrophages along the trabecular beams. This technique successfully provided high-resolution enface images but lacked full 3D resolution of the iridocorneal angle.
Full 3D images have been made using lower lateral resolution Optical Coherence Tomography (OCT) [23–31] of the ICA. Thus, measurements of the TM through the cornea and sclera at the limbus [25,26,32] using OCT provides some details of the TM, as seen in Fig. 1(D). Crowell et al., using this technique, presented a novel landmark termed BELL (the band of extracanalicular limbal lamina) [25], previous identified as the TM shadow [33], located posteriorly to the TM and externally adjacent to SC, and defined BELL as a more condensed, avascular area of collagenous layers. However, OCT imaging of the TM is still limited by the scattering of light from the overlying scleral tissue. Thus, in typical anterior segment OCT, SC can only be seen in a limited number of subjects and locations around the ICA [34].
OCT systems have more recently been used in a gonioscopy approach [35,36], where the lateral resolution is similar to clinical imaging, but with added depth information and a 360° view. However, these studies used highly customized OCT systems having an optimal resolution of 24µm, and the custom goniolens incurred high cost and is not suitable for the clinical setting.
The current paper investigates measurements of the ICA using an alteration of commercial goniolenses and the use of a widely available clinical OCT imaging system (Heidelberg Spectralis). The goal is to allow more widespread imaging of the ICA, as well as relating the measurements to what is known about the anatomical structures. We also tested whether limitation in visibility of structures such as BELL could be due to interactions of the imaging beam with the birefringent nature of the corneoscleral collagen [37]. This simple modification allows fast, high-resolution 3D imaging of the angle and is suitable for clinical practice providing highly detailed images in comparison to the typical clinical 2D imaging using a slit lamp. Furthermore, gonioscopic OCT imaging may improve our understanding of the TM by serving as a critical tool for enhancing our understanding of glaucoma and its treatments.
2. Methods
2.1 OCT imaging
2.1.1 Gonioscopic OCT
A commercial single-mirrored gonioscopy lens (OSMG; Ocular Instruments Inc., Bellevue, WA) was modified by cementing a 12mm diameter plano-convex lens (22mm focal length) onto its anterior surface to provide an image plane at the approximate distance of the ICA structures, as described in detail in [22]. The goniolens body was 15mm long, 22.92mm in diameter at its widest and 11mm diameter at its narrowest with a 7.45mm radius of curvature, where it interfaces with the cornea. A diagram of the goniolens used can be found in [22], and an image of the goniolens and its ray trace and spot diagram (Zemax software) can be seen in Fig. 2. In order to place the exit pupil of the OCT onto the mirror of the goniolens, the total body length of the goniolens was limited to 22mm. The goniolens was placed on the eye after coating the interface with goniogel (GONAK Hypromellose Ophthalmic Demulcent Solution 2.5%, Akorn Operating Company LLC., Lake Forest, Illinois, USA). For subjects with deep set eyes an eyelid speculum was optionally used (2 of 9 subjects) to retract the eyelids facilitating the placement of the goniolens on the eye and to improve stability of the goniolens on the cornea. To position the goniolens stably on the eye an adjustable mount was fixed to the head mount of the OCT imaging system. This mount allowed for vertical, horizontal and depth adjustments of the goniolens, and also incorporated a 360° ball joint to allow adjustment of the rotational degrees of freedom. Pilot experiments established the need for an adjustable but stable base for the goniolens. An image of a subject being imaged with our OCT gonioscopy can be observed in Fig. 2 pointing out the main elements of the setup.
Fig. 2. A) Picture of the modified goniolens, B) its ray trace diagram and C) its corresponding spot diagram at the ICA structures (using Zemax software, RMS radius of 11.73 µm). D) and E) show pictures of a subject being imaged by OCT gonioscopy using the Heidelberg Spectralis OCT with a rotatable λ/2 plate placed posteriorly to the last lens of the OCT device. The modified goniolens is mounted on a ball joint attached to an XYZ holder clamped to the headrest and positioned by a clinician. The OCT device can be moved independently from the goniolens.
For the current data, the goniolens was aimed at the inferior-temporal and inferior-nasal sectors of the eye. The scanning mirrors of the OCT beam were placed optically conjugate to the goniolens mirror. This allowed scanning of approximately 60 degrees of the ICA. The beam was also steered within the ICA by tilting the OCT device using its external mount. All gonioscopic OCT imaging is performed using the retinal imaging mode of the OCT device, and the scan location and orientation was adjusted using the Heidelberg Spectralis software. A model eye (SimulEye InsEYEt, LLC., Westlake Village California, USA) was initially used to test the goniolens mount. This allowed determination of optimal positioning to couple the goniolens to the eye, as well as testing the imaging protocol. The model eye was also used with an inserted sharp surface to ascertain that the goniolens did not greatly increase the axial resolution due to dispersion.
The axial scale of the gonioscopic OCT images was 3.87µm/px as provided by the specifications of the instrument, and the optical axial resolution is 8.35µm as calculated by the bandwidth of the superluminescent diode (SLD, λ=870µm, Δλ=40µm). To ensure that dispersion of the goniolens did not significantly degrade the axial resolution, we used a reflective target in a water-filled model eye. The estimated lateral scale for the gonioscopic OCT images was 8.02 µm/px. This was calculated using the measured corneal thickness at the limbus of the same eye (Anterior Segment OCT Visante Pachymeter (Carl Zeiss, Germany)), and the axial scale of the gonioscopic OCT images. Having two known distances at an angle, trigonometry was used to calculate the lateral scale. The newly acquired OCT/goniolens system data are based on these axial and lateral scales.
Volumetric OCT scans of maximum 15×5° with 12µm spacing between B-scans were performed. At least 2 OCT volume scans were performed per subject, although some volume scans covered less than 5° if movement or alignment impeded the acquisition of the full set of b-scans. Visibility of the ICA structures varied with the orientation of the b-scan. B-scan orientation was chosen to maximize the contrast of the TM and BELL in each case, which typically occurred when the scan formed a 45° angle from the radial orientation of the eye, as can be observed in Fig. 4(a) marked by the green arrow on the SLO image.
2.1.2 Anterior segment OCT
The Heidelberg Spectralis Anterior Segment module enables high-resolution OCT imaging of cornea, sclera, and anterior chamber angles from the exterior of the eye, that is, without contact of the lens with the eye. To image the limbus of the eye, we placed the OCT device such that the incident beam is nearly tangential to the corneal curvature at the limbus. Single OCT b-scans were captured at the same location when comparing different polarization rotations, and OCT volumes of 15°×10° or 15°×5° depending on the subject, were captured when imaging corneoscleral bands. Acquisition times in both cases ranged between 1 and 3 minutes, for a total of approximately 20 minutes including image acquisition at different locations and breaks between the acquisitions as needed. Images under this modality had a lateral scale of 6.57 µm/px, and an axial scale of 3.87µm/px. Anterior segment imaging was used to validate some of the findings of the current study, but in general could not be used for many of the measurements of the ICA.
2.1.3 Polarization rotation
The Heidelberg Spectralis OCT device used linearly s-polarized light with the orientation of polarization in the vertical direction. To test the interaction of imaging of the birefringent cornea and sclera with the angle of polarization we used a zero-order polymer half-wave plate (Thorlabs WPH10E-830, Newton, NJ, USA) to rotate the linearly polarized light. A custom mount was designed to mount the λ/2 plate adjacent to the final lens of the OCT device, both for the default retinal lens and for the anterior segment lens. These cylindrical amounts fit over each of the OCT lenses (retinal and anterior chamber lens) and allowed for 360° rotation of the λ/2 plate and was mounted as close to the objective lens as possible. A λ/2 plate rotates linear polarization by twice the angle of rotation, such that a 15° rotation of the plate equated to a 30° rotation of the linear polarization. On return from the eye, the λ/2 plate again rotates the returning beam. The imaging beam of the OCT is essentially collimated travelling through the zero-order λ/2 plate, which is insensitive to small angles possibly caused by focus adjustments.
For gonioscopic OCT imaging, the goniolens is placed such that the inferior-nasal or inferior-temporal ICA can be imaged, while for the anterior segment OCT, the incident beam is directed tangential to the cornea near the limbus. In both cases the λ/2 plate is initially placed with its fast axis aligned with the direction of the linear polarization of the OCT beam. Single OCT b-scan images are acquired at each position of the λ/2 plate as it is rotated in steps of 15°, capturing a full set of b-scans at the same location with a full 180° polarization rotation in steps of 30° in approximately 2 minutes.
2.2. Transverse structural OCT images
Transverse structural enface images were obtained using the segmentation options available on the Heidelberg Spectralis software for volumetric OCT scans. Segmentation was performed manually on all the b-scan within a volumetric OCT in one of two ways: 1) along the anterior chamber facing surface of the uveal meshwork of TM, or 2) along the posterior edge of the TM limited by BELL [25]. In the latter case, transverse images were averaged to create slabs of 150µm depth centered at the posterior surface of the TM. This slab thickness was selected as best fit for most subjects.
2.3 Subjects
All individuals imaged (Table 1) received an anterior eye exam, including anterior segment OCT and gonioscopy with a slit lamp prior to imaging. The subjects for this study were 9 healthy adults (29-72 years old) with normal eye exam results and 20/20 or better best corrected visual acuity and were free to choose which eye they preferred for imaging. The eye was anesthetized with 1 drop of Proparacaine Hydrochloride USP 0.5% before starting the imaging session and every 15 minutes thereafter. Total gonioscopy imaging time was less than 30 minutes broken into several shorter intervals. The study protocol was approved by the Indiana University Institutional Review Board and adhered to the Declaration of Helsinki.
Table 1. Subject data imaged with OCT gonioscopy and landmark visibility
3. Results
3.1. Trabecular meshwork and Schlemm’s canal
The gonioscopic OCT imaging from the anterior chamber was able to penetrate the ICA. The average signal penetration depth was of 636 ± 116µm, based on the signal dropping to 2x the noise level as measured on an average a-scan.
The TM, including structures not typically resolved with clinical imaging, was successfully imaged in 8 of 9 subjects. The exception was a subject with a plateau iris, where the iris is shifted forwards in the anterior chamber and obscures the view of the ICA. We were able to image details of the uveal meshwork with tangentially oriented scans of the ICA. Fig. 3 compares a tangential scan in a control subject (S1) in panels a) and b) showing the SLO and OCT b-scan respectively, with results from a subject (S5) with pigment dispersion syndrome (PDS) in panels c) and d). In PDS, pigment granules are deposited in the TM as lens zonules rub against the posterior surface of the iris. In general, the highly pigmented TM in this PDS subject appears much brighter in comparison to other subjects. For subject S1, the tangential b-scan volume was manually segmented anteriorly along the uveal meshwork to construct a transverse OCT, revealing a highly detailed enface image of the uveal meshwork structure (Fig. 3(e)).
Fig. 3. Comparison of Gonioscopy a) SLO and b) OCT b-scan oriented tangentially to the ICA (orientation of scan shown as green arrow in the SLO image) for S1, and similarly in panels c) and d) for S5 with PDS. e) Transverse enface image constructed from a dense OCT volume scan after manual segmentation along the anterior surface of the TM for S1. Yellow box demarcates the uveal meshwork. Yellow arrows show shadowing of the OCT beam created by iris folds.
Fig. 4 shows results for a right eye of subject S1 in two locations. The top row provides the gonioscopy SLO image (left) with the orientation of the OCT b-scan marked with a green line, and the corresponding OCT b-scan image (right) a) tangentially oriented to the ICA in the inferior-temporal region, and b) perpendicularly oriented to the ICA in the inferior-nasal region.
Fig. 4. Gonioscopic SLO (left) and OCT b-scan (right) of a) the inferior-temporal angle with the scan oriented tangentially (green arrow top-left) (see Visualization 1 video) and b) the inferior-nasal angle with the scan orthogonally oriented to the ICA (green arrow bottom-left) in the right eye of S1. Corneal thickness at the limbus is 850 µm as measured by the Anterior Segment OCT Visante (Carl Zeiss). Yellow labels point out the key features: the iris, trabecular meshwork (TM), band of extracanalicular limbal lamina (BELL), Schlemm's canal (SC) and corneoscleral bands (CSB). Blue arrows point at the dark line seen on the SLO coinciding with SC.
In both OCT b-scans, the main structures are indicated with yellow arrows, including the iris, the TM, SC, BELL, and cornea. In panel a) a slight wrapping and, thus, superposition of the OCT of the cornea is seen at the upper right side overlaying the more distal structures. The TM appears to end abruptly on the left side, which occurs due to shadowing from the iris as the imaging beam skims the iris surface when imaging this region. On the right side of Panel b) an inversion of the iris continuation can be seen caused by the Heidelberg Spectralis software. The BELL is seen as a dark curve posterior to the bright TM and was observed in 7 of 9 subject. Highest contrast between the TM and BELL was achieved with b-scans angled at approximately 45° from the radial orientation of the eye. Within the limbus regions of Fig. 4, alternating bright and dark bands are seen, hereafter called Corneoscleral Bands (CSB). These are explored further in the following section. A fly-through video of the full OCT volume acquired for subject 1, as well as a gonioscopic OCT b-scan image of the ICA for each subject can found in Fig. S1 of the supplementary document.
Fig. 5. Transverse OCT constructed from a segmented OCT volume for S1 at a) the anterior edge of the uveal meshwork posteriorly displaced by 320 µm, b) the posterior edge of the TM limiting with the BELL posteriorly displaced by 95um, c) the same as b) but averaging a slab of ±75 µm around the segmented line as marked by the red lines in figure d). Panel e) shows the transverse OCT (green box) superposed to the SLO image for S2 of an averaged slab of ±75 µm centered over the segmented line along the posterior surface of the TM, as shown by the red lines in panel f). Yellow arrow heads mark the darkened line in the transverse images coinciding with SC. Red arrows point to a narrowing of SC in subject 1. Scale bars are 200 µm.
SC was visible in 4 of the 7 subjects under gonioscopic OCT imaging. SC measurements were taken from the transverse OCT images after segmentation of a dense OCT volume scan. However, this required high stability and minimum eye and head movement which was only achieved with a limited number of subjects. Although the Heidelberg Spectralis has an alignment software feature to reduce movement artifact, it is designed for retinal imaging and is not optimized for the ICA and thus, interfered with the ICA imaging in some cases. All in vivo measurements of SC are an average of approximately 15 measurements performed on the transverse structural OCT images along the best focused region of the darkened line parallel to the ICA and is marked with yellow arrow heads in Fig. 5. Panel a) is a reconstruction from an OCT b-scan volume segmented along the anterior surface of the uveal meshwork and displaced posteriorly by 320 µm for subject 1, while b) is reconstructed from the same OCT volume segmented along the posterior surface of the TM limited by the BELL curve and displaced posteriorly by 95µm for subject 1. Panels c) and e) are constructed from a segmentation similar to b) but now averaging a slab 150µm thick centered around the segmented line (posterior TM) for S1 and S2, respectively. An example of the OCT b-scan is shown in panels d) and f) for each subject respectively, and the red lines delimit the slabs being averaged in each case.
Average measurements of SC along the dark line marked with yellow arrow heads in Fig. 5 were 82 ± 25µm in a), 88 ± 18µm in b), and 70 ± 19µm in c) for S1. A region where SC narrows to 40µm is marked with a red arrow, which is only seen with gonioscopic OCT imaging. The presence of regions of narrowed SC increased the standard deviation of SC measurements. For S2, the average measurement of SC in e) was 85 ± 6µm, in a region with no narrowing of SC. These dark regions of SC seen in the transverse OCT images are also visible in the simultaneously acquired SLO images, as marked with blue arrows in Fig. 4. To measure SC, careful focusing of the device was required, as well as minimal eye movements.
While imaging of the ICA using an external approach is possible, it is generally possible in only some subjects [38]. This difficulty seems to arise from a reduced contrast between SC and BELL, seen as a dark line posteriorly adjacent to the TM. Due to the collagenous structure of
Fig. 6. Change in visible contrast of BELL with polarization orientation (180° in steps of 30°) for S1 using the anterior segment module with incident perpendicular to the corneal apex. Cross-sectional measurement of SC diameter was 48 ± 5 µm wide and 186 ± 13 µm in length, which are in good agreement with the literature values [38].
BELL and TM, and the birefringence of collagen, we examined the role of polarization on the visibility of these structures using the anterior segment lens. The λ/2 plate was first aligned with the polarization of the OCT beam as explained in section 2.1.3. By rotating the linear polarization of the incident light, we were able to enhance the brightness of BELL. The brightness of surrounding regions was polarization independent as seen in Fig. 6. As a result, the contrast of SC versus BELL varies by up to 50% with polarization angle.
3.2. Corneoscleral bands
The CSB were visible in all subjects. Clarity and contrast of the CSB depended systematically on the location within the eye and orientation of the b-scan. Highest contrast of the bands in most subjects was achieved in the inferior-nasal or inferior-temporal ICA with a b-scan oriented approximately at 45 degrees to radial direction (Fig. 4(a). The CSB are visible starting posteriorly to the BELL curve and continue into the peripheral cornea. The corneal thickness near the limbus for S1 (Fig. 4(a) was 850µm, and the number of CSB range from 3 to 5 bright bands depending on location and orientation of the b-scan. The diameter of the bright CSB, as measured on a gonioscopic OCT b-scan with highest CSB contrast, on 3 subjects was on average 96 ± 14µm for S1, 48 ± 7µm for S2, and 70 ± 16µm for S3.
Fig. 7. ICA SLO (left) and OCT cropped b-scan (right) acquired using the Anterior Segment Module (Heidelberg Spectralis) showing the corneoscleral bands at a) inferior-temporal angle of OS for S1, b) inferior-temporal angle of OD for S1, and c) inferior-nasal of OS for S3. Very wide ICA in the latter case is a consequence of cataract surgery [39].
The CSB were also visible using the Heidelberg Spectralis Anterior Chamber lens, without the need for a goniolens. The CSB visibility was achieved when incident light from the OCT is nearly tangential to the cornea at the limbus. These bands were easiest to image at the inferior-nasal or inferior-temporal sector of the limbus, as seen in Fig. 7 for S1 in the a) inferior-temporal angle of the left eye, and b) in the inferior-nasal angle in the right eye, as well as for S3 c) in the inferior-temporal angles of the left eye. In the latter case, a wide ICA is seen for S3 probably due to cataract surgery [39]. The inferior or superior ICA was the most difficult to image due to obstruction by eye lids and the high angle of incidence of the OCT beam needed which is limited by the range of tilt of the OCT device in its vertical direction. As in OCT gonioscopy, the bands are seen to start posteriorly to the TM and with a similar shape to the BELL curve extending further into the cornea as they fan out. These bands were not able to be seen in the central region of the cornea, likely due to thinning and random orientation of the collagen lamellae [40].
Fig. 8. Corneoscleral band changes seen in a vertically oriented gonioscopy OCT b-scan with linear polarization rotation of over 180 degrees in 30-degree steps for S9 at the inferior-temporal angle.
The relation between corneoscleral collagen fibers and polarization orientation of the OCT beams has been studied further. The linear polarization of the OCT beam was rotated using a λ/2 plate to study the changes in the CSB with polarization rotation, as explained in section 2.1.3. As the λ/2 plate was rotated, the bands disappeared such that the darker CSB appeared brighter, fading out the band-like structure. The CSB reappeared at 90° and 180° rotation of the s-polarization, as the darker bands reemerged. This occurred both with gonioscopy (Fig. 8) and anterior segment OCT imaging.
4. Discussion
For the first time to our knowledge, we have introduced a set of modifications to a frequently utilized goniolens used together with a commercially available OCT imaging system, to allow improved, non-invasive in vivo gonioscopic OCT imaging. Using these modifications, we successfully imaged the ICA in healthy adults. This imaging technique is straight-forward, cost-efficient, and allows identification of the TM, BELL, SC, and adjacent structures at higher contrast than most other methods of ICA imaging. Using a commercial device and a low-cost modified goniolens, makes this imaging technique feasible to be used in the clinical setting.
Previous OCT approaches typically provided limited depth penetration and resolution when imaging exteriorly, due to shadowing of the TM region and light scattering from overlying sclera, respectively, or required highly customized OCT systems that are not easily accessible and can be very costly Our gonioscopic OCT images allow sufficient light penetration to image the ICA landmarks even behind the TM. Clinical slit lamp gonioscopy only provides a superficial low-resolution image of the angle structures. The gonioscopic OCT volume scans oriented tangential to the ICA, with manual segmentation, enable highly detailed enface images of the uveal meshwork. Like other enface projection approaches with OCT, the depth and averaging of the enface visualization is adjustable, obtaining enface images at different depths. Furthermore, segmentation of OCT volume scans oriented approximately at 45° from the radial direction allowed the construction of a transverse enface type image of the ICA which allowed for visualization and measurement of SC, including identification of narrowed regions. Slightly different segmentation procedures either along the anterior TM, posterior TM, or averaging a slab of b-scans centered along the BELL curve, gave similar values of SC measurements. However, gonioscopic OCT dense volumes required high lens stability, and minimal eye movement by the subjects. Our current measurements (on average 80 ± 12µm) represent a non-radial cross-section of SC, thus these measurements are slightly larger than in previous reports (coronal diameter of 44.5 ± 12.6µm and meridional canal diameter of 233.0 ± 34.5µm [38]).
Both gonioscopic and anterior segment OCT imaging allowed visualization of CSB located within the limbus. While images from previous studies show evidence of these bands [35,41], they are not discussed further than mentioning the TM shadow or BELL. However, in the current study we were able to visualize multiple bands in all subjects and perform detailed measurements on 3 subjects. We believe the bright CSB to be bundles of collagen fibers oriented largely in the same direction. The polarization dependence of the visibility of the CSB presumably arises from the birefringence of the collagen, and thus there is a need to image the cornea at an appropriate angle for scattering and with the appropriate polarization orientation to increase the contrast of the bands as shown in Fig. 8. Both the cornea and sclera are mainly composed of type I collagen fibers (50-55% in the cornea [40] and 90% in the sclera [42]). Collagen fiber structures and orientations have been thoroughly studied ex vivo at the center of the cornea [43]. X-ray scattering techniques [44,45] have been used to quantify the preferred lamellar orientations in the human cornea, but do not allow for direct imaging. On the other hand, second harmonic generation non-linear imaging techniques have more recently been used to provide qualitative [46–48] and quantitative [49–51] information about lamellar organization, including in vivo measurements on animals [52].
For the central cornea collagen fibers are arranged in thin lamellae with fibrils oriented parallel to one another within the lamellae and oriented at almost right angles to those in consecutive lamellae [40]. The anterior third of the stroma is seen to have a less precise arrangement of collagen lamellae with some inserting into Bowman’s layer. The deeper stroma (approximately the posterior 2/3 of the corneal thickness) has ribbon shaped lamellae with collagen fibers having preferred directions, forming a network that enter the cornea close to the inferior, superior, nasal, and temporal positions of the eye. The size of these lamellae in the central cornea range from as low as 10µm to up to 320µm in width and between 1.5 and 2.5µm in height [46].
Less is known about the structure at the limbus. Only one study has in vivo data in humans focused on the limbus where the corneal and scleral tissues intersect [53] and require large, highly customized systems. In this region, collagen lamellae run limbus to limbus and become thicker creating bundles at the junction of the cornea and the sclera. Peripheral layers receive insertions from the scleral bundles, mostly in line with the rectus muscles. In the sclera, the collagen bundles range in sizes of about 10-16µm thick and 100-140µm wide, running mostly parallel to the scleral surface but cross each other in all directions. At the limbus, deeper bundles take a circular course running coronally, parallel to the limbus [44], and which may also correspond to the orientation of collagen in BELL. This would explain why the polarization orientation of the OCT beam changes the brightness of BELL, allowing to enhance the contrast of SC against it.
The collagen bundle diameters decrease markedly from the sclera to the cornea. Meek and Knupp [54] call these regions of larger diameter fibrils “anchoring lamellae”, and they seem to be of scleral origin. The anchoring lamellae are directed towards the ocular rectus muscles, crossing the peripheral cornea on a curved path but do not reach the central optical zone of the cornea. It has been proposed that this complex structure of the collagen increases corneal rigidity and helps maintain the anterior corneal curvature [37,46]. The anchoring lamellae may coincide with the CSB imaged in this study and the measured diameters of the CSB are in good agreement to the values given in the literature for scleral collagen bundles [40]. As collagen, the CSB are polarization sensitive, such that their visibility is dependent on the orientation of s-polarization with the highest visibility of the CSB, seen at 0° and 90° from the vertical. Further analysis of their visibility as a function of polarization orientation at different locations around the eye may allow an approach to characterizing the anatomical orientation of collagen bundles in the limbal area. To our knowledge, this is the first time the corneal-scleral collagen bundles have been imaged in vivo and studied further by altering the orientation of s-polarization.
Our study has several limitations. First, the resolution is currently insufficient to characterize the fine structures of the TM. For the gonioscopic imaging, this could be improved by optimizing the design of the commercial goniolens currently being used. A shorter goniolens body would increase the flexibility of imaging by increasing the working distance and allowing higher tip and tilt angles. The optical design of the lens can also be improved by optimizing the focusing lens placed on the anterior surface of the goniolens, reducing the spot size at the image plane. To resolve the cellular level structure of the TM, an adaptive optics OCT would be needed [22]. Secondly, the goniolens was fixed within the mount and could not be rotated while placed on the eye. A freely rotating 360° mount would allow imaging different sectors of the ICA without removing the goniolens from the eye between locations, reducing the imaging session time and improving patient comfort. Thirdly, there was no fixation target for the subject, which reduced ocular stability and limited the acquisition of OCT volumes. While external fixation was available, the need to move the instrument often blocked fixation targets. Finally, the current software is optimized for retinal imaging which limited the acquisition of the OCT volume scans. Improved software would allow increased acquired volume size, reduced acquisition times, and in turn, increase the number of regions being imaged in a single session.
5. Conclusions
A modified goniolens and the use of polarization rotation is able to improve non-invasive, in vivo gonioscopic OCT in humans. These improvements allowed 3D imaging of the TM, including the anterior uveal meshwork structure, band of extracanalicular limbal lamina (BELL), and the corneoscleral bands (CSB). Transverse enface images constructed from gonioscopic OCT volume scans allowed imaging Schlemm’s canal (SC) over space in some subjects, including visualization and measurements of SC narrowing at a specific location. Varying the orientation of the linear s-polarization of the OCT device allowed modifying the contrast of BELL, improving the visibility of SC, and enhancing the ability to make measurements of its dimensions. Gonioscopic OCT imaging and anterior segment OCT was used to image the CSB. The brightness of these bands was dependent on polarization orientation, presumably due to the birefringent nature of the corneoscleral collagen bundles.
Funding
National Institutes of Health NEI (1R0EY024315); Alcon Research Institute; National Center for Advancing Translational Sciences (TL1TR002531).
Disclosures
The authors declare no conflicts of interest.
Data availability
Data underlying the results presented in this paper are not publicly available at this time but may be obtained from the authors upon reasonable request.
Supplemental document
See Supplement 1 for supporting content.
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