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Cross-sectional OCT images of the healthy human inferior limbus acquired in-vivo, in a tangential direction with the 5x microscope objective, closer to the corneal end of the limbus (A) and closer to the scleral end of the limbus (B). The POV appear as highly reflective, narrow, loop-like structures in Fig. 1(A). Blood and lymph vessels (V) appear as dark shadows on the highly scattering background of the collagen matrix. Capillaries extend from the inside of the fibrous sacks of the POV in vertical direction and connect with a lateral vessels network located ~20 µm to 50 µm below the base of the POV. Volumetric images of the inferior limbus (C, D).
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A research-grade OCT system was used to image in-vivo and without contact with the tissue, the cellular structure and microvasculature of the healthy human corneo-scleral limbus. The OCT system provided 0.95 µm axial and 4 µm (2 µm) lateral resolution in biological tissue depending on the magnification of the imaging objective. Cross-sectional OCT...
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MiR-146a is upregulated in the stem cell-enriched limbal region vs. central human cornea and can mediate corneal epithelial wound healing. The aim of this study was to identify miR-146a targets in human primary limbal epithelial cells (LECs) using genomic and proteomic analyses. RNA-seq combined with quantitative proteomics based on multiplexed iso...
Citations
... However, conventional post-processing to trace, for instance, nerve fibers in 3D is possible with simple techniques. Evaluating magnified regions from the real-time snapshots permits the discrimination of different anatomic structures in vivo, such as the corneal epithelium (EP), the basal cell layer (BCL) of the corneal epithelium, the Bowman layer (BM), the stroma (ST), the endothelial layer (EL), and the Descemet's membrane (Fig. 9) [47]. Because the focus is adjusted to the corneal epithelium the Descemet's membrane may be merged with the endothelial layer ( Fig. 9(c)). ...
A real-time line-field optical coherence tomography (LF-OCT) system is demonstrated with image acquisition rates of up to 5000 B-frames or 2.5 million A-lines per second for 500 A-lines per B-frame. The system uses a high-speed low-cost camera to achieve continuous data transfer rates required for real-time imaging, allowing the evaluation of future applications in clinical or intraoperative environments. The light source is an 840 nm super-luminescent diode. Leveraging parallel computing with GPU and high speed CoaXPress data transfer interface, we were able to acquire, process, and display OCT data with low latency. The studied system uses anamorphic beam shaping in the detector arm, optimizing the field of view and sensitivity for imaging biological tissue at cellular resolution. The lateral and axial resolution measured in air were 1.7 µm and 6.3 µm, respectively. Experimental results demonstrate real-time inspection of the trabecular meshwork and Schlemm’s canal on ex vivo corneoscleral wedges and real-time imaging of endothelial cells of human subjects in vivo.
... Although there has been a concerted effort to characterize the limbal stem cell niche in humans, only a limited number of studies have attempted to identify an apparent limbal niche in other animals and categorize any species-related differences that may exist [4][5][6][7]. Here we identified, as in the human limbal stem cell niche, anatomical features akin to PoV and LCs [2,3,[8][9][10][11] in the mature avian limbus, as well as evidence of direct cellcell interactions between basal epithelial cells and stromal cells [9]. The chicken model has long been used to investigate corneal development [12][13][14][15][16][17] and has proven to be an effective model for understanding corneal repair mechanisms [18,19]. ...
... SBF-SEM allowed for high-resolution imaging and 3D reconstruction of the adult chicken limbal zone, which identified structures that were anatomically similar to human LCs and PoV as described in the literature [8][9][10][11]. In the human limbus, FSPs are distinct from limbal crypts and are described as finger-like projections of the limbal stroma containing an upward-extending central blood vessel. Shortt and colleagues found that FSPs were surrounded by a compact arrangement of basal cells and, when imaged using SEM, the decellularization of the epithelium surrounding them left remnants of protruding blood vessels visible in the limbal zone [3]. ...
(1) Background: Owing to its ready availability and ease of acquisition, developing chick corneal tissue has long been used for research purposes. Here, we seek to ascertain the three-dimensional microanatomy and spatiotemporal interrelationships of the cells (epithelial and stromal), extracellular matrix, and vasculature at the corneo-scleral limbus as the site of the corneal stem cell niche of the chicken eye. (2) Methods: The limbus of developing (i.e., embryonic days (E) 16 and 18, just prior to hatch) and mature chicken eyes was imaged using scanning electron microscopy (SEM), transmission electron microscopy (TEM), and the volume electron microscopy technique, serial-block face SEM (SBF-SEM), the latter technique allowing us to generate three-dimensional reconstructions from data sets of up to 1000 serial images; (3) Results: Data revealed that miniature limbal undulations of the embryonic basement membrane, akin to Palisades of Vogt (PoV), matured into distinct invaginations of epithelial cells that extended proximally into a vascularized limbal stroma. Basal limbal epithelial cells, moreover, occasionally exhibited a high nuclear:cytoplasmic ratio, which is a characteristic feature of stem cells. SBF-SEM identified direct cell–cell associations between corneal epithelial and stromal cells at the base of structures akin to limbal crypts (LCs), with cord-like projections of extracellular matrix extending from the basal epithelial lamina into the subjacent stroma, where they made direct contact with stomal cells in the immature limbus. (4) Conclusion: Similarities with human tissue suggest that the corneal limbus of the mature chicken eye is likely the site of a corneal stem cell niche. The ability to study embryonic corneas pre-hatch, where we see characteristic niche-like features emerge, thus provides an opportunity to chart the development of the limbal stem cell niche of the cornea.
... The anterior segment possesses tissues with potential clinical interest but its relatively long axial extension and surface topography preclude the use of medium-NA, thus imaging is typically limited to a relatively coarse lateral resolution of around 20 µm [11][12][13]. Therefore, anterior segment imaging is an attractive application that could benefit from CR to achieve highresolution polarimetric parameters of tissue micro-structures such as the arrangement of collagen fibrils in the stroma [4] and the palisades of Vogt [14]. We imaged the limbus, targeting the trabecular meshwork, and acquired an out-of-focus (OOF) tomogram with the focal plane located deep into the tissue. ...
... Indeed, a relationship between birefringence and IOP has been suggested in the sclera [12]. PS-OCT in combination with CR could also benefit future explorations of polarimetric properties of the palisades of Vogt, which requires a medium-NA system to resolve its ridgelike structure with ∼ 50 µm periodicity [14]. These organized fibrovascular structures contain stem cells responsible for the maintenance and regeneration of corneal tissue. ...
... These organized fibrovascular structures contain stem cells responsible for the maintenance and regeneration of corneal tissue. The potential use of CR in OCT imaging of the palisades of Vogt has been suggested [14]. CR could also be used in tissue polarimetry for understanding scarring processes in the limbus, such as blebs scarring in glaucoma patients treated with trabeculectomy [1]. ...
We present computational refocusing in polarization-sensitive optical coherence tomography (PS-OCT) to improve spatial resolution in the calculated polarimetric parameters and extend the depth-of-field in phase-unstable, fiber-based PS-OCT systems. To achieve this, we successfully adapted short A-line range phase-stability adaptive optics (SHARP), a computational aberration correction technique compatible with phase-unstable systems, into a Stokes-based PS-OCT system with inter-A-line polarization modulation. Together with the spectral binning technique to mitigate system-induced chromatic polarization effects, we show that computational refocusing improves image quality in tissue polarimetry of swine eye anterior segment ex vivo with PS-OCT. The benefits, drawbacks, and potential applications of computational refocusing in anterior segment imaging are discussed.
... This seems counterintuitive as the cornea is completely avascular, and the limbus is considered the vessel barrier safeguarding the cornea's vascular and immune privilege. However, the limbus itself is indeed heavily vascularized and features a delicate network of arcade-forming blood and lymphatic vessels which are thought to provide nutrients and facilitate the passage of growth factors essential for stem cell maintenance [41]. Contrarily, the differentiated epithelium of the peripheral and central cornea expresses antiangiogenic factors that tip the balance towards avascularity, including soluble and membrane-bound VEGFR-3 [9,42,43]. ...
The limbus, the vascularized junction between the cornea and conjunctiva, is thought to function as a barrier against corneal neovascularization. However, the exact mechanisms regulating this remain unknown. In this study, the limbal epithelial stem cell (LESC) marker ABCB5 was used to investigate the role of LESCs in corneal neovascularization. In an ABCB5KO model, a mild but significant increase of limbal lymphatic and blood vascular network complexity was observed in developing mice (4 weeks) but not in adult mice. Conversely, when using a cornea suture model, the WT animals exhibited a mild but significant increase in the number of lymphatic vessel sprouts compared to the ABCB5KO, suggesting a contextual anti-lymphangiogenic effect of ABCB5 on the limbal vasculature during development, but a pro-lymphangiogenic effect under inflammatory challenge in adulthood. In addition, conditioned media from ABCB5-positive cultured human limbal epithelial cells (ABCB5+) stimulated human blood and lymphatic endothelial cell proliferation and migration. Finally, a proteomic analysis demonstrated ABCB5+ cells have a pro(lymph)angiogenic as well as an anti-inflammatory profile. These data suggest a novel dual, context-dependent role of ABCB5+ LESCs, inhibiting developmental but promoting inflammatory (lymph)angiogenesis in adulthood and exerting anti-inflammatory effects. These findings are of high clinical relevance in relation to LESC therapy against blindness.
... Anterior segment optical coherence tomography (AS-OCT) is a highly precise, noninvasive imaging modality that can be successfully used in patients with LSCD [25][26][27][28]. In our practice, we have experience with various machines but currently prefer to utilize the Avanti xR for AS-OCT examination and Angio-OCT modes using the AngioVue program and the SSADA algorithm. ...
Background:
Limbal stem cell deficiency (LCSD) presents several challenges. Currently, there is no clearly defined systematic approach to LSCD diagnosis that may guide surgical tactics.
Methods:
The medical records of 34 patients with LSCD were analyzed. Diagnostic modalities included standard (visometry, tonometry, visual field testing, slit-lamp biomicroscopy with corneal fluorescein staining, Schirmer test 1, ultrasonography) and advanced ophthalmic examination methods such as anterior segment optical coherence tomography, in vivo confocal microscopy, impression cytology, and enzyme-linked immunoassay.
Results:
Standard ophthalmological examination was sufficient to establish the diagnosis of LSCD in 20 (58.8%) cases, whereas advanced evaluation was needed in 14 (41.2%) cases. Depending on the results, patients with unilateral LSCD were scheduled to undergo glueless simple limbal epithelial transplantation (G-SLET) or simultaneous G-SLET and lamellar keratoplasty. Patients with bilateral LSCD with normal or increased corneal thickness were enrolled in the paralimbal oral mucosa epithelium transplantation (pLOMET) clinical trial.
Conclusions:
Based on the diagnostic and surgical data analyzed, the key points in LSCD diagnosis were identified, helping to guide the surgeon in selecting the appropriate surgical procedure. Finally, we proposed a novel step-by-step diagnostic algorithm and original surgical guidelines for the treatment of patients with LSCD.
... Оптическая когерентная томография (ОКТ) переднего сегмента (AS-OCT) появилась в качестве альтернативного, неинвазивного метода визуализации пациентов с СЛН [58][59][60][61][62]. ...
Relevance. Limbal stem cell deficiency (LSCD) is a pathology that leads to substantial vision decrease, corneal neovascularization, corneal epithelial persistent erosions and have a negative impact on patient's the quality of life. It is known that the efficiency of corneal transplantation in LSCD patients is low and requires repeated interventions. One of the important problems is the lack of a clear diagnostic algorithm and a standardized set of diagnostic techniques for this condition. Purpose. Systematically analyze domestic and foreign literature on the currently existing methods for diagnosing LSCD. Material and methods. A systematic analysis of scientific publications from 1992 to 2021 was carried out. Its included domestic and foreign authors on the resources of PubMed, Medline, eLibrary, as well as dissertations and textbooks devoted to currently existing methods for diagnosing LSCD. Results. In addition to biomicroscopy, the main in vivomethods for diagnosing LSCD are staining of the ocular surface, impression cytology with the detection of specific cytokeratins, confocal microscopy, optical coherence tomography of the anterior segment (OST) and OST-angiography. Conclusion. Physician awareness and vigilance are required for the diagnosis of LSCD, especially in patients with conjunctivalization and corneal neovascularization. It is impossible to use only biomicroscopy and the patient's complaints, since LSCD is not always obvious. That is why diagnostics should include an integrated approach, including all available research methods, which are briefly described in Table No. 1 with their disadvantages and advantages. Key words: limbal stem cell deficiency, impression cytology, laser confocal scanning microscopy, goblet cells, cytokeratin, penetrating keratoplasty, cytokines
... Visualization of goblet cells on IVCM is indicative of late LSCD. 44,47,48 AS-OCT allows quasi-histological non-contact in vivo imaging of the cornea with 3-dimensional quantification of layer thickness. Central corneal epithelial thickness and limbal epithelial thickness on AS-OCT appear to correlate with LCSD disease, making these measurements a potentially useful diagnostic aid. ...
The corneal epithelium serves to protect the underlying cornea from the external environment and is essential for corneal transparency and optimal visual function. Regeneration of this epithelium is dependent on a population of stem cells residing in the basal layer of the limbus, the junction between the cornea and the sclera. The limbus provides the limbal epithelial stem cells (LESCs) with an optimal microenvironment, the limbal niche, which strictly regulates their proliferation and differentiation. Disturbances to the LESCs and/or their niche can lead to the pathologic condition known as limbal stem cell deficiency (LSCD) whereby the corneal epithelium is not generated effectively. This has deleterious effects on the corneal and visual function, due to impaired healing and secondary corneal opacification. In this concise review, we summarize the characteristics of LESCs and their niche, and present the current and future perspectives in the management of LSCD with an emphasis on restoring the function of the limbal niche.
... Axial resolution is defined as the distinguish-ability of two nearest objects along the direction of the incident light, which mainly depends on the wavelength. Lateral resolution is defined as the distinguishability along the direction perpendicular to incident light, which primarily depends on the magnification of the imaging objective (Bizheva et al 2017). To improve the spatial resolution of OCT, many methods have been proposed. ...
Optical Coherence Tomography (OCT) is a promising non-invasive imaging technique that owns many biomedical applications. In this paper, a deep neural network is proposed for enhancing the spatial resolution of OCTen faceimages. Different from the previous reports, the proposed can recover high resolutionen faceimages from low resolutionen faceimages at arbitrary imaging depth. This kind of imaging depth adaptive resolution enhancement is achieved through an external attention mechanism, which takes advantage of morphological similarity between the arbitrary-depth and full-depthen faceimages. Firstly, the deep feature maps are extracted by a feature extraction network from the arbitrary- depth and full-depthen faceimages. Secondly, the morphological similarity between the deep feature maps is extracted and utilized to emphasize the features strongly correlated to the vessel structures by using the external attention network. Finally, the SR image is recovered from the enhanced feature map through an up-sampling network. The proposed network is tested on a clinical skin OCT data set and an open-access retinal OCT dataset. The results show that the proposed external attention mechanism can suppress invalid features and enhance significant features in our tasks. For all tests, the proposed SR network outperformed the traditional image interpolation method, e.g., bi-cubic method, and the state-of-the-art image super-resolution networks, e.g., enhanced deep super-resolution network, residual channel attention network, and second-order attention network. The proposed method may increase the quantitative clinical assessment of micro-vascular diseases which is limited by OCT imaging device resolution.
... Recently, studies have used AS-OCT to visualize the POV in healthy 9,13 and pathological conditions [11][12][13] . Although SD-OCT has not yet been able to identify the structure of individual limbal stem cells, research-grade OCT with a high resolution (0.95-μm axial resolution and 2-μm lateral resolution) could visualize individual pigmented cells along fibrous folds and red blood cells inside the capillaries of the POV 22 . In addition, Chen et al. reported that full-field OCT could provide both crosssectional and en face images of the POV at a submicron resolution, and even the corneal nerve could be clearly observed 23 www.nature.com/scientificreports/ ...
... SD-OCT provided information regarding epithelial thickness, epithelial-stromal interface, subepithelial stromal morphology, and stromal vessel diameters in the POV. SD-OCT could also detect the density of POV with high reliability and repeatability 10,22 ; compared with slit-lamp biomicroscopy, SD-OCT was more sensitive and useful in examining the health of limbal microstructure. To the best of our knowledge, information provided by our study has not been reported thus far. ...
... However, despite the aforementioned limitations, we found some novel results of the structural change of the POV in patients with AWE and phlyctenular keratitis/ocular rosacea by using SD-OCT. With new sub-micrometer axial resolution optical coherence tomography, the high spatial OCT resolution allowed for precise identification of POV by visualization of the capillaries that extend from the inside of the palisade' ridges to a lateral micro-vascular network located underneath the POV 22,23 . Extension of the study method to other ocular surface disorders can be a safe and valuable future clinical application. ...
Spectral-domain optical coherence tomography (SD-OCT) has been used to observe the morphology of the palisades of Vogt (POV) with satisfactory resolutions. In this study, we used SD-OCT to examine the microstructure of the POV in ocular surface disorders with limbal involvement. We detect subclinical limbal pathologies based on five parameters, including (1) decreased epithelial thickness, (2) loss of the sharp stromal tip, (3) loss of the smooth epithelial-stromal interface, (4) dilated stromal vessels, and (5) decreased POV density. Eighteen eyes of 10 patients with advancing wavelike epitheliopathy (AWE) and 15 eyes of 9 patients with phlyctenular keratitis/ocular rosacea were recruited. SD-OCT could detect abnormal changes in the POV in 100% of the lesion sites. In presumed-healthy areas of the diseased eyes diagnosed by slit-lamp biomicroscopy, SD-OCT detected abnormal changes in the POV in 100% of the eyes in both groups. In patients with unilateral disease, abnormal changes in the POV were detected by SD-OCT in 50% and 100% of presumed-healthy eyes diagnosed by slit-lamp biomicroscopy in the AWE group and phlyctenular keratitis/ocular rosacea group, respectively. SD-OCT is powerful in detecting POV changes in ocular surface disorders and can provide useful information that cannot be provided by slit-lamp biomicroscopy.
... Support for this hypothesis may be found in the undulations of the epithelium, similar to the columnar organization of the ophidian VSE, found in epidermis, oral epithelium, and limbal epithelium of mammals. These structures, known as rete ridges, correspond to the interdigitations of the vascular lamina propria, which are called dermal papillae in epidermis and oral epithelium (Winning & Townsend, 2000;Wu et al., 2013) or palisade of Vogt in limbal tissue (Bizheva et al., 2017;Goldberg & Bron, 1982). The bottom of the rete ridges harbors the stem cells providing them a suitable microenvironment (Webb et al., 2004;West, 2015). ...
The sensory olfactory epithelium and the vomeronasal sensory epithelium (VSE) are characterized by continuous turnover of the receptor cells during postnatal life and are capable of regeneration after injury. The VSE, like the entire vomeronasal organ, is generally well developed in squamates and is crucial for detection of pheromones and prey odors. Despite the numerous studies on embryonic development of the VSE in squamates, especially in snakes, an ultrastructural analysis, as far as we know, has never been performed. Therefore, we investigated the embryology of the VSE of the grass snake (Natrix natrix) using electron microscopy (SEM and TEM) and light microscopy. As was shown for adult snakes, the hypertrophied ophidian VSE may provide great resolution of changes in neuron morphology located at various epithelial levels. The results of this study suggest that different populations of stem/progenitor cells occur at the base of the ophidian VSE during embryonic development. One of them may be radial glia‐like cells, described previously in mouse. The various structure and ultrastructure of neurons located at different parts of the VSE provide evidence for neuronal maturation and aging. Based on these results, a few non‐mutually exclusive hypotheses explaining the formation of the peculiar columnar organization of the VSE in snakes were proposed.
