PURPOSE:Fuchs endothelial corneal dystrophy (FECD) results in the death of the nonproliferative endothelial cells of the posterior corneal surface, leading to corneal swelling, clouding, and potential blindness. Few studies have suggested the potential role of transcription factors in the endothelial to mesenchymal transition (EnMT) during disease progression. This study aimed to evaluate the expression of selected transcription factors in the corneal endothelium of FECD patients to understand their role in disease pathogenesis. DESIGN:This is a prospective, pilot, case-control study for studying the gene expression in patients with FECD. Transcription factors ZEB1, TCF4, smad proteins (SMAD3, SMAD4), zinc finger proteins SNAI1 and SNAI2, lymphoid enhancer binding factor 1 (LEF1), N-cadherin (CDH2), claudin 10 (CLDN10), and nuclear factor (erythroid-derived 2)-like 2 (NFE2L2/NRF2), which are involved in the EnMT, were selected for the study. Fourteen FECD endothelia were compared with 15 control endothelia for the gene expression analyses using quantitative real-time PCR. RESULTS:Significant differential expressions were seen in the levels of SMAD3 (P = 0.0251) in moderate cases compared to control tissues. Further, TCF4 and NFE2L2 showed significantly different expressions among the moderate and severe cases (P = 0.0262 and P = 0.0350, respectively), with expressions decreasing with severity, indicating their possible role in disease progression. CONCLUSIONS:Our pilot study on transcription factor gene expressions in FECD patients' tissue samples suggests NRF2 and TCF4 to play an important role in the disease pathogenesis and progression.

PURPOSE:To investigate dysregulated ion channels and epithelial-to-mesenchymal transition (EMT) genes in Fuchs endothelial corneal dystrophy (FECD) and to identify potential drugs interacting with dysregulated ion channels. METHODS:Five RNA-sequencing datasets from patients with FECD were analyzed with DESeq2 to identify differentially expressed genes. Dysregulated ion channels and EMT-related genes underwent enrichment analysis and EMT scoring via the KS method. Coexpression patterns were identified using weighted gene coexpression network analysis. A pathway map highlighted altered cellular processes in FECD, and potential drugs were identified using the Drug-Gene Interaction Database. Molecular docking assessed binding of drugs with TRPV4 and TRPM8 . Selected gene expression changes were validated by quantitative real-time polymerase chain reaction. RESULTS:Analysis identified 12,550 differentially expressed genes, including 229 ion channels (157 up-regulated, 72 down-regulated) and 502 EMT-related genes (304 up-regulated, 198 down-regulated). Functional enrichment revealed disrupted biological processes including interleukin-1 production and extracellular matrix organization organization. Coexpression analysis uncovered 2 modules linked to apoptosis and senescence. Up-regulated transient receptor potential cation (TRP) cation channels correlated with elevated senescence; altered NFKBIA , NFKB1 , and TNFSF10 expression with endothelial apoptosis. We identified 1052 drugs targeting 133 dysregulated ion channels and performed docking analysis for TRP channels. Experimental validation confirmed altered expression of TRPV1 , CDKN2A , and KRT7 . CONCLUSIONS:Dysregulated TRP cation channels contribute to cellular senescence and peripheral sensitization in FECD, whereas altered apoptotic regulators drive corneal endothelial cell death. Most likely, these pathways represent potential targets for treatment of patients with FECD.

Abstract The aims of this study were to use an isogenic cell model system to investigate the proteomic consequences of TCF4 trinucleotide repeat expansion in Fuchs endothelial corneal dystrophy (FECD) and to identify potential molecular pathways contributing to disease pathogenesis. We used our previously established immortalized FECD cells (iFECD) that harbor CTG repeat expansion in TCF4 and CRISPR/Cas9 genome editing to generate an isogenic counterpart (iFECD TCF4ΔCTG) in which the expansion was deleted. Comprehensive proteomic analysis was then performed using tandem mass tag (TMT)-labeled quantitative LC-MS/MS. The resulting data were subjected to differential expression analysis, functional enrichment analysis, and protein-protein interaction network construction to elucidate the molecular impact of the CTG repeat expansion. Deletion of the CTG repeat expansion significantly altered the corneal endothelial proteome, with 90 upregulated and 111 downregulated proteins (|log2 fold change| ≥ 0.5, P-value < 0.05). Functional enrichment analysis revealed that the downregulated proteins were predominantly associated with extracellular matrix organization and cell–substrate adhesion pathways, while the upregulated proteins were enriched in interferon signaling and antigen-processing pathways. The most significantly upregulated proteins included neuropilin-1, Cip1-interacting zinc finger protein, and protein-glutamine gamma-glutamyltransferase 2, while protein phosphatase 1 regulatory subunit 14 C, alpha-crystallin B chain, and 14-3-3 protein sigma showed the greatest downregulation. Our findings demonstrate that TCF4 CTG repeat expansion significantly impacts the corneal endothelial proteome, particularly affecting the extracellular matrix and cell adhesion proteins that likely contribute to guttae formation. These proteomic alterations provide mechanistic insights connecting trinucleotide repeat expansion to endothelial dysfunction and suggest potential therapeutic targets for FECD.

Purpose:Fuchs endothelial corneal dystrophy (FECD) is a progressive corneal disorder characterized by excessive extracellular matrix (ECM) accumulation and corneal endothelial cell death. CTG trinucleotide repeat expansion in the transcription factor 4 (TCF4) gene represents the most significant genetic risk factor. This study aimed to elucidate the role of TCF4 in FECD pathogenesis through comprehensive proteomic analysis. Methods:Corneal endothelial cells isolated from patients with FECD harboring TCF4 trinucleotide repeat expansion were immortalized to establish an FECD cell model (iFECD). CRISPR/Cas9-mediated genome editing was employed to generate TCF4-knockout iFECD cells. Whole-cell proteome analysis was performed using liquid chromatography-mass spectrometry, followed by pathway enrichment analysis of differentially expressed proteins (DEPs). The effects of TCF4 deletion on TGF-β-mediated protein aggregation and cell death were evaluated using Western blot analysis, flow cytometry, and aggresome detection assays. Results:Proteomic analysis identified 88 DEPs among 6510 detected proteins. Pathway analysis revealed significant enrichment in ECM-associated pathways, oxidative stress responses, and cellular motility. TCF4 deletion attenuated TGF-β-induced cell death in iFECD cells. Concordantly, Western blot analysis demonstrated that TCF4 deletion suppressed TGF-β2-mediated cleavage of caspase-3 and poly (ADP-ribose) polymerase. Flow cytometric analysis of Annexin V-positive cells confirmed reduced apoptosis in TCF4-deleted cells following TGF-β2 treatment. Additionally, aggresome detection assays revealed that TCF4 deletion diminished TGF-β2-induced protein aggregation. Conclusions:This study demonstrates a crucial role for TCF4 in FECD pathogenesis, particularly in ECM regulation and protein aggregation-induced cell death.

Anatomically inflammation in the anterior chamber of the eye, specifically in the iris and choroid is named as anterior uveitis. For the effective management of the disease it is essential for regular monitoring. Quantifying aqueous flare as a continuous measure of the intensity of light scatter (ILS) assists in accurately evaluating inflammation. Nevertheless, there is a potential for the subject’s blinking to disrupt the ILS data. This leads to increased and misleading levels of aqueous flare when assessing the extent of inflammation. Thus, our objective was to use an EOG-based spot fluorometer to examine the influence of eyeblink artifacts on ILS outcomes. This approach was founded on empirical data collected by quantifying the blink-induced and blink-artifact-free ILS in individuals with good health. A dataset of synthetic uveitis was generated using the LSTM deep learning technique. In addition, unsupervised machine learning techniques including k-means clustering, agglomerative hierarchical clustering, and Gaussian mixture clustering were used to identify blink artifacts in both the healthy and synthetic uveitis data. The optimal choice for minimizing artifacts was found to be the model that demonstrated superior performance. Our study findings indicate that the Gaussian mixture model outperformed other models in predicting blink-induced ILS, resulting in the most substantial decrease in blink artifacts. Furthermore, we successfully resolved the ILS by using our artifact removal technique, resulting in an impressive accuracy rate of 89%. The experiment verifies that our methodology successfully mitigates the occurrence of blinking errors in ILS measurements, thereby allowing a spot fluorometer to precisely grade uveitis.

We present a case of scleral lens surface crazing in a patient with keratoconus (KC) and chronic giant papillary conjunctivitis (GPC). A 45-year-old Asian male with known keratoconus (KC) and allergic eye disease in both eyes, who is an experienced scleral lens user, presented with a 3-month history of lens intolerance in the right eye. On examination, the right eye scleral lens showed extensive lens surface crazing with poor surface wettability. The upper tarsal conjunctiva showed conjunctival hypertrophy – active GPC. After allergy treatment and symptomatic improvement, the scleral lens was refitted after intensive cleaning and polishing with aluminum oxide. The lens surface showed significant improvement and good fitting characteristics, and the patient resumed lens wear. At 6 months of follow-up, the patient was asymptomatic, and the ocular condition was stable.

Keratoconus is an etiologically complex, degenerative corneal disease that eventually leads to loss of corneal integrity. Cells in corneal epithelium and endothelium express various types of ion channels that play important roles in ocular pathology. This emphasizes the need of understanding alterations of ion channels in keratoconus. Differential gene expression analysis was performed to identify deregulated ion channels in keratoconus patients using transcriptomic data. Thereafter correlation analysis of ion channel expression was performed to obtain the changed correlation between ion channels’ expression in keratoconus patients versus control samples. Moreover, Protein-protein interaction networks and a pathway map was constructed to identify cellular processes altered due to the deregulation of ion channels. Furthermore, drugs interacting with deregulated ion channels were identified. Total 75 ion channels were found to be deregulated in keratoconus, of which 12 were upregulated and 63 were downregulated. Correlations between ion channel expressions found to be different in control and keratoconus samples. Thereafter, protein-protein interactions network was generated to identify hub ion channels in network. Furthermore, the pathway map was constructed to depict calcium signalling, MAPK signalling, synthesis and secretion of cortisol, and cAMP signalling. The 19 FDA- approved drugs that interact with the 5 deregulated ion channels were identified. Down-regulation of voltage-gated calcium channels can be attributed to reduced cell proliferation and differentiation. Additionally, deregulated ion channels in 3’,5’- cyclic adenosine monophosphate signalling may be responsible for elevated cortisol level in progressive keratoconus patients.

Purpose The relevance of corneal biomechanics and the importance of including it in the clinical assessment of corneal ectasias are being increasingly recognized. The connection between corneal ultrastructure, biomechanical properties, and optical function is exemplified by a condition like keratoconus. Biomechanical instability is seen as the underlying basis for the secondary morphological changes in the cornea. Asymmetric biomechanical weakening is believed to drive progressive corneal steepening and thinning. Biomechanical strengthening is the principle of collagen crosslinking that has been shown to effectively arrest progression of the keratoconus. Corneal biomechanics has therefore ignited the interest of researchers and clinicians alike and has given us new insights into the cause and course of the disease. This article is an overview of the extensive work published, predominantly in the last two decades, on the biomechanical aspect of keratoconus. Methods Published articles on corneal biomechanics in the specific context of keratoconus were reviewed, based on an electronic search using PubMed, Elsevier, and Science Direct. The search terms used included "Corneal Biomechanics," "Mechanical properties of the cornea," "Corneal ultrastructure," "Corneal Collagen," and "Keratoconus". Articles pertaining to refractive surgery, keratoplasty, collagen crosslinking, or intrastromal rings were excluded. Results The electronic search revealed more than 500 articles, from which 80 were chosen for this article Conclusions The structural and organizational pattern of the corneal stroma determines its mechanical properties and are responsible for the maintenance of the normal shape and function of the cornea. Changes in the ultrastructure are responsible for the biomechanical instability that leads to corneal ectasia. As non-invasive methods for evaluating corneal biomechanics in vivo evolve, our ability to diagnose subclinical keratoconus will improve, allowing identification of patients at risk to develop ectasia and to allow early treatment to arrest progression of the disease.

PURPOSE:To develop a new virtual surgery simulation platform to predict postoperative corneal stiffness (Kc mean ) after laser vision correction (LVC) surgery. SETTING:Narayana Nethralaya Eye Hospital and Sankara Nethralaya, India; Humanitas Clinical and Research Center, Italy. DESIGN:Retrospective observational case series. METHODS:529 eyes from 529 patients from 3 eye centers and 10 post-small-incision lenticule extraction (SMILE) ectasia eyes were included. The software (called AcuSimX) derived the anisotropic, fibril, and extracellular matrix biomechanical properties (using finite element calculation) of the cornea using the preoperative Corvis-ST, Pentacam measurement, and inverse finite element method assuming published healthy collagen fibril orientations. Then, the software-computed postoperative Kc mean was adjusted with an artificial intelligence (AI) model (Orange AI) for measurement uncertainties. A decision tree was developed to classify ectasia from normal eyes using the software-computed and preoperative parameters. RESULTS:In the training cohort (n = 371 eyes from 371 patients), the mean absolute error and intraclass correlation coefficient were 6.24 N/m and 0.84 (95% CI, 0.80-0.87), respectively. Similarly, in the test cohort (n = 158 eyes from 158 patients), these were 6.47 N/m and 0.84 (0.78-0.89), respectively. In the 10 ectasia eyes, the measured in vivo (74.01 [70.01-78.01]) and software-computed (74.1 [69.03-79.17]) Kc mean were not statistically different ( P = .96). Although no statistically significant differences in these values were observed between the stable and ectasia groups ( P ≥ .14), the decision tree classification had an area under the receiver operating characteristic curve of 1.0. CONCLUSIONS:The new software provided an easy-to-use virtual surgery simulation platform for post-LVC corneal stiffness prediction by clinicians and was assessed in post-SMILE ectasia eyes. Further assessments with ectasia after surgeries are required.

Abstract: Corneal collagen cross-linking has established itself as the only known treatment with a proven ability to arrest the progression of keratoconus. The efficacy and safety of the procedure have been entirely due to meticulous preparation by the innovators of this technique, before clinical application was confidently advocated. The article by Spoerl et al, entitled “Safety of UVA riboflavin crosslinking of the cornea,” one of the most frequently cited articles in the journal “Cornea,” explains the logic behind the formulation of the “Dresdon protocol” that remains the gold standard in the treatment of keratoconus by collagen cross-linking.

Purpose: To assess real-world clinical outcomes and safety of the Clareon ® intraocular lens (IOL) and AutonoMe ® automated preloaded delivery system in an Indian population. Patients and methods: This was a prospective, single-arm, multicenter, 12-month clinical study in patients aged ≥20 years with unilateral or bilateral cataracts. Surgery was performed by phacoemulsification followed by implantation of the Clareon monofocal IOL (CNA0T0). Monocular best-corrected distance visual acuity (BCDVA) and uncorrected distance visual acuity (UCDVA) were assessed at 1 week and 1, 6, and 12 months after implantation. Posterior capsular opacification (PCO), surface haze, and glistenings were evaluated at all visits. Surgeons’ satisfaction with automated injector system was also evaluated using a questionnaire. Safety was assessed by monitoring adverse events (AEs). Results: A total of 151 eyes received the CNA0T0 IOL. Mean ± SD monocular BCDVA improved from 0.53±0.44 logMAR preoperatively to 0.00±0.08 logMAR at week 1 and −0.03±0.08 logMAR at 12 months after implantation. At 12 months, 137/137 (100%) of eyes achieved BCDVA of 0.3 logMAR or better. Mean ± SD monocular UCDVA was 0.78±0.40 logMAR preoperatively, 0.11±0.15 logMAR at week 1, and 0.08±0.13 logMAR at 12 months after implantation. At 12 months, 132/137 (96%) eyes achieved UCDVA of 0.3 logMAR or better. Serious intraoperative AEs were posterior capsule rupture (n=1) and ciliary zonular dehiscence (n=1). Surgeons reported that the automated preloaded device was more intuitive compared with other push- or screw-style preloaded injector systems. None of the eyes in this study presented surface haze; all were graded as 0 glistenings at all visits. No clinically significant PCO or neodymium-doped yttrium aluminum garnet (Nd:YAG) capsulotomies were reported. Conclusion: The hydrophobic IOL preloaded in an automated injector system provided good visual and refractive outcomes, as well as no surface haze and grade 0 glistenings. None of the patients required Nd:YAG capsulotomy.

PURPOSE:The purpose of this article was to study the clinical, optical, and morphological correlates of visual function in patients with Fuchs endothelial corneal dystrophy (FECD). METHODS:The case records were analyzed for patients diagnosed with FECD between September 2019 and March 2020. The best-corrected visual acuity (BCVA) was recorded as decimal visual acuity and converted to the logarithm of the minimum angle of resolution units. Contrast sensitivity was measured with the Pelli-Robson contrast sensitivity test. Corneal alterations, including central corneal thickness, depression of the posterior cornea, and corneal densitometry values, were evaluated using Scheimpflug images. Corneal epithelial thickness was measured by spectral-domain optical coherence tomography. RESULTS:A total of 107 eyes of 61 patients (18 male and 43 female) with FECD were retrospectively investigated. The Spearman rank correlation coefficient showed moderate correlation between BCVA and contrast sensitivity (ρ = -0.66, P < 0.001), with some patients maintaining relatively good BCVA but having reduced contrast sensitivity. Logistic regression analysis demonstrated that age, central corneal thickness, depression of the posterior cornea, and epithelial thickening were negatively associated with contrast sensitivity but not with BCVA. CONCLUSIONS:Contrast sensitivity is a useful tool for assessing visual dysfunction and should be incorporated into the assessment protocol of patients with FECD. Alterations in the cornea, including central corneal thickness, depression of the posterior cornea, and epithelial thickening, might be objective parameters that can help the clinician in grading the severity of the disease and tracking its progression.

PURPOSE:The purpose of this article was to study the impact of instillation of 1% fluorescein and mydriatics on measurements made by Scheimpflug imaging.METHODS:This was a cross-sectional study of patients who had measurements of corneal shape (maximum keratometry and best fit sphere of anterior and posterior corneal surfaces), pachymetry (at the pupil center, corneal apex, and thinnest location), and anterior chamber parameters (depth, volume, and angle of anterior chamber and corneal volume) using the Pentacam HR before and after the instillation of fluorescein and mydriatics. Group A had baseline measurements that were repeated 1.5 hours after the instillation of 1% fluorescein and 0.8% tropicamide+ 5% phenylephrine eye drops. Group B had baseline measurements that were repeated 10 minutes after the instillation of 1% fluorescein, and group C had baseline measurements that were repeated 1.5 hours after the instillation of mydriatics.RESULTS:Overall, 131 eyes of 131 patients were studied: 87 in group A, 28 in group B, and 16 in group C. Significant differences and wide variations were noted in corneal pachymetry and anterior chamber parameters in all 3 groups. The highest magnitude and range of difference was observed in pachymetry at the pupil center (17 ± 53.5 μm) and in anterior chamber volume (26.7 ± 69.8 mm 3 ). Corneal shape measurements of anterior and posterior corneal surfaces were not significantly affected by either fluorescein or mydriatics. Pupillary dilation also affected the ability of the Pentacam to accurately trace the pupil margin in 19.5% of cases.CONCLUSIONS:For consistent and accurate measurements, it is important that Scheimpflug imaging be performed before other tests which may need the instillation of fluorescein or mydriatics.

Purpose: To evaluate the regional corneal biomechanical deterioration with keratoconus (KC) progression as measured by the Stress-Strain Index (SSI) maps.Methods: The preoperative examinations of 29 progressive KC cases that were submitted to corneal cross-linking (CXL) were evaluated. The examinations included the tomography and the SSI measured by the Pentacam HR and the Corvis ST (Oculus, Wetzlar, Germany), respectively. The results were recorded twice, the latter of which was at the last visit before the CXL procedure. The patient-specific SSI maps were built, using data at each examination, based on finite element modelling and employing inverse analysis to represent the regional variation of biomechanical stiffness across the cornea.Results: All cases presented significant shape progression (above the 95% CI of repeatability) in anterior and posterior curvatures and minimum thickness. The overall corneal stiffness as measured by the SSI within the central 8 mm-diameter area underwent slight but significant reductions from the first to the last examination (−0.02 ± 0.02, range: −0.09 to 0, p < 0.001). In all 29 cases, the reduction in stiffness was localised and concentred in the area inside the keratoconus cone. The SSI values inside the cone were significantly lower in the last examination (by 0.15 ± 0.09, range: −0.42 to −0.01, p < 0.001), while the SSI outside the cone presented minimal, non-significant variations (0 ± 0.01, range: −0.04 to 0.01, p = 0.999).Conclusion: It has been observed through the SSI maps that the regional deterioration in stiffness was concerted inside the area of pathology, while only mild non-significant alterations were observed outside the area of pathology.

Purpose To assess the biomechanical deterioration arising from keratoconus progression in-vivo. Methods The preoperative examinations of 32 progressive KC cases that were submitted to corneal cross-linking were evaluated. The examinations included the corneal tomography using the Pentacam HR and biomechanical parameters assessed by the Corvis ST (Oculus, Wetzlar, Germany). The results were recorded at two time points, the latter of which was at the last visit before the CXL procedure. Keratoconus progression was characterised by a significant change in the ABCD system. Results At the last follow-up visit (41.4 +/- 40.9 months) all morphological parameters of the ABCD grading system showed significant deterioration (p < 0.001). The comparative analyses revealed a significant reduction in corneal stiffness expressed by a significant reduction in the stress-strain index (SSI: -0.10 +/- 0.06, p < 0.001), the Stiffness parameter A1 (SP-A1: -6.1 +/- 12.0 mmHg/mm, p = 0.011), by a significant increase in the integrated Inverse Radius (IIR: 0.95 +/- 1.04 mm-1, p < 0.001) and in the deflection amplitude (DA) ratio (0.23 +/- 0.58, p = 0.034). A barely significant increase in the DA also pointed towards corneal stiffness reduction. (0.04 +/- 0.13 mm, p = 0.056). The SSI and the IIR were the indices with the smallest overlaps between the two examinations. Conclusions It has been demonstrated in-vivo that corneal biomechanical deterioration occurs with keratoconus progression. The larger changes observed in the SSI and the IIR when compared to the remaining biomechanical parameters suggests that these parameters could be suitable to assess the corneal stiffness reduction in keratoconus natural progression.

Purpose: To evaluate the characteristics of corneal material properties in healthy individuals and keratoconic patients using the stress-strain index (SSI). Setting: Vincieye Clinic in Milan, Italy, and Instituto de Olhos Renato Ambrosio in Rio de Janeiro, Brazil. Design: Retrospective observational cross-sectional study. Methods: Records of 1221 patients were divided into 3 groups: healthy corneas (n = 728), bilateral keratoconus (KC, n = 388), and very asymmetric ectasia (VAE, n = 105) when patients presented with clinical ectasia in 1 eye and normal topography (VAE-NT) in the fellow eye. All patients were examined with Pentacam HR and Corvis ST. Severity of KC cases was stratified according to the Pentacam topographic KC classification. The SSI distribution across the different groups and its correlation with age, biomechanically corrected intraocular pressure (bIOP), and central corneal thickness (CCT) were assessed. Results: A statistically significant difference between healthy individuals and each of the keratoconic groups (P < .001) was observed, and a progressive reduction in the SSI was observed across the groups. A significant correlation was observed between the SSI and age in all groups (P < .010) but KC severe subgroup (P = .361). No correlation between the SSI and bIOP and CCT was observed in all KC subgroups and VAE-NT groups (P >.050). Among healthy eyes, there was only a mild correlation between the SSI and bIOP (R = 0.12, P = .002) and CCT (R = 0.13, P = .001). Conclusions: This study estimates the in vivo corneal material properties in healthy individuals and patients with KC using a new method. The SSI showed a progressive deterioration within the advance in disease stages while being relatively independent of bIOP and CCT but positively correlated with age. Copyright (c) 2022 Published by Wolters Kluwer on behalf of ASCRS and ESCRS

Purpose: To evaluate the reliability of intensity of light scatter (ILS) for diagnosing mild-to-moderate uveitis, which is difficult to score by the subjective standardized uveitis nomenclature (SUN) grading. Methods: ILS (in millivolts) was measured in healthy subjects and uveitis patients by a custom-made Spot fluorometer. The reliability was assessed by noise analysis of the instrument using turbid samples and intra- and interobserver variabilities. Results: ILS increased with the severity of inflammation: healthy (0.1498 ± 0.0564) and SUN0 (0.1590 ± 0.0564) < SUN1+ (0.2050 ± 0.0887) < SUN2+ (0.4283 ± 0.1895). The increase in ILS was more significant between healthy and SUN1+ (P < 0.0001) compared to the difference between SUN0 and SUN1+ (P = 0.0006). The instrument noise was small (σ = 0.002). Intraobserver measurements revealed no variability (P > 0.98) between measurements and predicted repeatability coefficients of 0.0754, 0.0522, and 0.0532 in healthy, SUN0, and SUN1+, respectively. Interobserver measurements also showed no variability (P > 0.88) and predicted intraclass correlation coefficient > 0.63 in healthy and uveitis subjects. Conclusions: The fluorometer enables precise measurement of ILS with negligible instrument noise, and the measurements are unaffected by intra- and interobserver variabilities. However, the difference in mean ILS between SUN0 and SUN1+ is greater than the instrument noise and comparable to intra- and interobserver variabilities. Thus, ILS measurements are likely impacted by blinking, eye movements, and focusing jitter. The impact of these factors must be reduced before using the fluorometer to distinguish mild-to-moderate uveitis.