Many studies investigating the anterior segment and ICA have utilized modern optical coherence tomography (OCT) methods to obtain cross sectional views of ocular tissue. Anterior segment OCT (AS-OCT) was conceptually demonstrated not long after OCT was invented and has since garnered interest to evolve and become systematically optimized for imaging the anterior eye. Currently AS-OCT is a well-established technique with applications including investigation of cornea and lens pathology and visualizing the risk of angle closure (28). The non-contact nature of AS-OCT is generally more attractive from the patient’s perspective and also eliminates potential problems from inducing unnatural stresses on the cornea and ICA. This can be especially problematic in studies probing TM stress or angle configuration (29). Measuring anterior chamber biometric parameters with AS-OCT has become an imaging standard in terms of both image metric quality and ease of use (30–32).

OCT techniques have been continuously developed and tailored to anterior segment imaging (33). One technique is the use of comparatively longer laser wavelengths than currently used with spectral domain ocular coherence tomography (SD-OCT). This has the advantage of maintaining high contrast and signal strength while imaging directly through the overlying multiply-scattering limbus and sclera. Early commercial AS-OCT devices were time-domain OCT systems that did use long wavelength light, but also had slow scan speeds. Commercial SD-OCT systems optimized for retinal imaging are capable of high imaging speeds and have better axial resolution, but use shorter wavelength light sources (~880nm) with shallow depth range into the anterior segment. New AS-OCT systems utilize swept source OCT (SS-OCT) over SD-OCT, which permits fast imaging speed with longer laser wavelength (~1300nm), optimized to maintain sensitivity for deeper tissue imaging. Differences in technical specifications of these OCT systems are outlined in Table 1 .

In 2018, a study by Crowell et al. sought to address and characterize features and anatomical landmarks that can be visualized with new AS-OCT technology (34). The group reviewed AS-OCT images obtained using a commercial SS-OCT, CASIA SS-1000 (Tomey, Nagoya, Japan). They presented a novel landmark which was coined as the band of extracanalicular limbal lamina (BELL), a feature consists of an avascular layering of collagenous tissues that is posterior to the TM and is adjacent to external SC ( Figure 1D ). Though clinical and pathological significance of the BELL is currently unclear, it has been hypothesized that this tissue provides structural support to the ICA, and may correlate with fibers observed when entering SC intraoperatively.

While successful, trans-tissue SS-OCT imaging can still be limited by light scattering. Crowell et al. reported visualization of TM and BELL in 73% and 93% of studied individuals, respectively, but identified SC in only 40% of people. A further attempt by Ueno et al. utilized a custom polarization-sensitive OCT (PS-OCT) technique to detect and identify TM and BELL at higher rates by removing birefringence artifacts (31). With PS-OCT, BELL was identified in 99.2% of cases and TM identified in 95.1%. Conversely, SC is poorly resolved with PS-OCT due to its luminal nature, with visibility in only one subject (0.3%). Other strategies to better image SC have included a custom high speed 1.66-MHz SS-OCT prototype system, demonstrated to successfully obtain full 3D reconstruction renderings of SC in two eyes but with a relatively coarse lateral resolution of 17.54 μm (35).

AS-OCT output of both cross-sectional 2D images or reconstructed 3D ICA images also allows for easy assessment of structures relative to each other, which has proven useful to aid diagnosis of angle closure glaucoma (36–38). Common parameters derived from AS-OCT images include anterior chamber depth and width, and lens parameters like vault and thickness. Many features describing the angle have been established as biomarkers for angle closure, such as angle opening distance at 500 μm from the scleral spur, trabeculo-iris space, and angle recess area (39–41).

Many studies have also explored the implementation of AI algorithms to reliably detect ICA pathology in AS-OCT images (42–44). Success has been demonstrated in AI capability to screen for angle closure risk and identify features such as iridocorneal apposition and PAS (45). Future directions of deep learning supported algorithms must be able to provide diagnostic aid across multiple ethnicities and classify ICA opening grade to identify risk of angle closure.

While the clinical value of current AS-OCT is clear, image resolution is still a limitation of this modality, and tissue resolution is not sufficient for investigation of trabecular biomechanics at the cellular level and therefore provides no clinically useful information with regard to open angle glaucoma. These images also struggle to identify angle neovascularization and level of pigment, which means gonioscopy remains a necessity for comprehensive evaluation of the ICA (46).

In addition to using OCT to image TM and ICA structures externally through the limbus, OCT based gonioscopy approaches have also been successfully reported. This concept utilizes a contact gonioscopy lens to image the ICA from inside the eye, circumventing total internal reflection. McNabb et al. described a customized OCT gonioscopy system they designed to obtain 360° ICA images (47). They constructed an SS-OCT system to be coupled with a curved 360° paraboloidal mirror gonio lens to image the entire ICA circumference with a single volume. While this method proved effective and could construct entire 360° ICA 3D volume renderings with relatively short acquisition time, the lateral resolution of this system was 24 μm, far too large for resolving the smaller trabecular structures.

Carmichael et al. reported a similar OCT gonioscopy technique, but with the use of a commercially available SD-OCT (Heidelberg Spectralis, Heidelberg Engineering, Heidelberg, Germany) and modified clinical goniolens (48). With this strategy, they improved system ease of use and reduced cost of specialization, and were able to obtain gonio OCT images of the ICA with an axial resolution calculated at 8.35 μm ( Figures 1E–I ). They displayed reconstructed enface images and were able to obtain representations of uveal meshwork in higher detail. This is an efficient strategy that can potentially permit cost-effective ICA imaging at an easier uptake rate.

While the previously mentioned OCT-based designs offer many benefits like ease of use and low-cost, the reported resolution of the CASIA SS-1000 is not suited for resolving cellular features at axial 10 μm and lateral 30 μm (33). Although gonioscopic OCT methods successfully obtained images of ICA with improved resolution, neither design was able to provide cellular level structural information of the ICA region. These methods also both employ the use of contact imaging which can limit patient comfort.

To address this, Mazlin et al. recently developed a novel multimodal anterior OCT system that combined time-domain full-field OCT (TD-FF-OCT) and SD-OCT. Full field OCT obtains high resolution en face images via simultaneous illumination of the entire scanning field. The use of TD-FF-OCT provides the benefit of local, cell-resolution images, which this group first reported for in vivo human cornea imaging in 2018 (49). Recent upgrades to this system incorporated benefits of an SD-OCT arm. This strategy yields cellular-resolution en face TD-FF-OCT images in the cornea, while simultaneously acquiring cross-sectional SD-OCT images with large field of view, so that real-time imaging position can be determined (50).

Recently, they were able to acquire in vivo human ICA images with a reported lateral resolution of 1.7 μm (51). Using this method to image a single subject, Mazlin et al. produced images of TM fibers and corneoscleral and uveal meshwork were able to be differentiated. Notably, their OCT based system corneal images can be acquired quickly, with alignment and acquisition reported to take only minutes. Alignment time for trabecular meshwork imaging, however, was said to be substantial.

Two-photon(2P) microscopy is a popular imaging technique utilized to investigate biological tissue in many different scientific domains. Its use to image ex vivo and in situ human and animal ICA tissue has also been established to differentiate and characterize structure details (52–54). Photon emission signals from fluorophores are collected to form an image upon stimulation with excitation light. 2P microscopy provides many beneficial attributes considering its application to obtaining ICA images. In particular, tissue photo-absorption is restricted to the imaging plane, which permits deep laser penetration and affords intrinsic optical sectioning.

Ex vivo 2P microscopy TM imaging of the last decade was highly valuable for establishing cellular level information regarding TM structure (55–57). More recently, Avila et al. reported a safe in vivo technique to non-invasively image the human eye, and showed 2P images of living human TM, sclera, and cornea for the first time ( Figures 2A, B ) (58). Two subjects were imaged with the 2P prototype system that uses a trans-limbal incident beam perpendicular to the TM. Acquired TM images revealed structural and individual cell detail within the juxta-canalicular tissue at a pixel resolution of 1.5 μm. Though the apparent center of interest in their report was on cornea imaging, Avila et al. discuss the potential value of using 2P imaging to track effects of high IOP and characterize TM morphology to aid in diagnosing glaucoma. Non-OCT based imaging techniques discussed in this manuscript are summarized in Table 2 .

Utilizing Bessel beams generated with axicon lenses in ICA imaging provides many advantages, and has been explored to attain high-resolution images in ex vivo and in vivo animal models. The ability of Bessel beams to “self-heal” around light scattering obstacles improves tissue penetration while maintaining enhanced contrast to provide intrinsic optical sectional capability. An axicon-assisted gonioscopy approach was described by Perinchey et al, who designed a prototype setup featuring a handheld probe with a central imaging sensor optimized to attain ICA images at different depths (59). Although they demonstrated a 3 μm lateral resolution with this concept, many system difficulties were described in its operation. They only reported images in ex vivo porcine eyes and reported many user difficulties. Hong et al. reported a prototype system also implementing an axicon lens to generate Bessel beam illumination in a light sheet fluorescence microscopy (LSFM) scheme (60, 61). This resulted in a more accessible system with a long working-distance objective, to impart a noncontact nature to image acquisition. This LSFM modality required the application of fluorescein injection to ex vivo porcine tissue and topical drops to the in vivo rabbit eye, then imaging from the opposite angle at a 20mm working distance. With this improved imaging platform, TM network arrangement could be visualized with a lateral spatial resolution of 2.19 μm. Although utilization of this imaging technique has not yet been performed in humans, the authors suggest can complement existing imaging methods to help clinicians with glaucoma diagnosis.

High resolution imaging of the trabecular meshwork was also recently achieved with a first-of-kind, proof-of-concept paradigm that utilized an adaptive-optics scanning laser ophthalmoscope (AOSLO) to image in vivo human trabecular meshwork through a custom modified gonioscopy lens (62). This system employed a button lens centered on the surface above the mirror on a clinical gonio lens. This allowed the AOSLO to focus onto the TM in a gonioscopy approach.

This adaptive optics gonioscopy (AOG) approach proved successful and provided in vivo cellular scale (2.5μm) resolution of human ICA structures including Schwalbe’s line, corneal collagen, and posterior and anterior TM structures. ( Figures 2C–E ) While system optimization is required, this setup generated a valuable closer look into meshwork features, as TM endothelial cells and macrophages could be visualized with morphology consistent with findings based on histology. As with any gonioscopy approach, physical contact is also required between the cornea and gonio lens, which may be uncomfortable during the longer imaging sessions described, and the AOG method also prohibits optical sectioning.

Assessment of aqueous humor outflow pathways has also been an attractive area to scientists and researchers interested in exploring aqueous outflow mechanics. This information can be used to develop and optimize interventions to lower IOP, most notably in the form of MIGS. Visualizing conventional aqueous outflow has been established by different aqueous angiography (AA) techniques ( Figures 2F, G ) (63, 64). These strategies, including fluorescein AA and AS-OCT angiography, are very much akin to their respective posterior segment strategies that are already clinically utilized.

Huang et al. were the first to perform fluorescein AA in live human subjects, and have demonstrated segmental, pulsatile, and dynamic aqueous outflow patterns (65). Human AA imaging is performed by tracer addition into the eye, followed by fluorescence capture by an angiographer on a customized arm. An experimental method was developed that sequentially delivers different tracers, which can be used to assess the conventional outflow system before and after intervention. This information can potentially inform future decision making about MIGS placement in the eye. In a recent case study, putative post-surgical recruitment of previously unvisualized aqueous outflow channels was demonstrated in a patient who underwent bent needle ab-interno goniectomy (66, 67).