In Vivo Imaging of Rodent Retina in Retinal Disease.

High-quality imaging of the retina is crucial to the diagnosis and monitoring of disease, as well as for evaluating the success of therapeutics in human patients and in preclinical animal models. Here, we describe the basic principles and methods for in vivo retinal imaging in rodents, including fundus imaging, fluorescein angiography, optical coherence tomography, fundus autofluorescence, and infrared imaging. After providing a concise overview of each method and detailing the retinal diseases and conditions that can be visualized through them, we will proceed to discuss the advantages and disadvantages of each approach. These protocols will facilitate the acquisition of optimal images for subsequent quantification and analysis. Additionally, a brief explanation will be given regarding the potential results and the clinical significance of the detected abnormalities.

Simple design of fluorescein-based probe for rapid and in situ visual monitoring of histamine levels in food spoilage.

With the emergence of numerous food safety problems, rapid and accurate detection of histamine in food spoilage remains a challenge. To this end, we developed a simple design and easy synthesis of fluorescein-based probe FCHO to achieve specific and rapid (<1 s) quantitative detection of histamine through "imine formation" reaction. Significant enhanced fluorescence signal in response to histamine enabled our probe with high sensitivity as low as 51 nM. Utilizing the visualized fluorescence color changes of the probe as histamine increasing, we combined it with paper-based test chip to construct a color-resolved and highly selective recognition system. In addition, our proposed probe has been successfully used to visually imaging histamine changes in fish samples. Finally, for the first time, we have proved it possesses reliable ability to directly in situ imaging the distribution of histamine in whole spoiled fish. Thus, our strategy will provide great potential for monitoring food spoilage.

Reassessment of arterial versus venous perfusion of diabetic retinal neovascularization using ultrawide-field fluorescein angiography.

PURPOSE: The purpose of this study was to assess whether diabetic NV is perfused by the arterial or the venous circulation. METHODS: This is a retrospective, consecutive case series evaluating patients with proliferative diabetic retinopathy (PDR) imaged with ultrawide-field (UWF) fluorescein angiography (FA). Areas of neovascularization elsewhere (NVE) and neovascularization of the disc (NVD) were assessed. Perfusion was defined as arterial, arteriovenous, or venous if the area of diabetic neovascularization (NV) began to hyperfluoresce either prior, during, or after laminar venous flow, respectively. RESULTS: A total of 180 eyes from 176 patients with 928 NV were identified (830 NVE, 98 NVD). Of those, 5.1% of NVE were classified as arterial and 58.2% of NVD were classified as arterial. The remaining NV were classified as arteriovenous except for a small subset (6.1%) which were indeterminate. None of the NV were classified as venous. Noteworthy examples demonstrated NV that nearly fully perfused prior to any detectable fluorescence within nearby veins as well as clear shunting of blood from a feeding artery to a draining vein. CONCLUSIONS: UWF FA images suggest that some NV is perfused by retinal arteries. This may be useful in devising strategies for early detection and treatment of NV precursors. KEY MESSAGES: What is known • Diabetic retinal neovascularization has long been thought to be perfused by the retinal venous circulation. • Vascular endothelial growth factor has been shown to play key roles in both angiogenesis and arteriogenesis. What is new • Ultrawide-field fluorescein angiography demonstrates that at least some diabetic neovascularization is perfused by the retinal arterial circulation. • This supports the hypothesis that diabetic neovascularization may arise from arterially-perfused intraretinal microvascular abnormalities in the capillary bed.

Visualization of the Plasmid DNA Delivery System by Complementary Fluorescence Labeling of Arginine-Rich Peptides.

Cell-penetrating peptides, such as arginine-rich peptides, encapsulate nucleic acid drugs and deliver them to intracellular compartments. Comprehensive tracking of drug delivery systems (DDSs) provides information about the behavior of the drug as well as the fate of the drug carrier after drug release, which is crucial for minimizing side effects. In this study, we labeled peptides designed to carry plasmid DNA with two types of dyes, traditional dye fluorescein and aggregation-induced emission (AIE) dye tetraphenylethylene, and subsequently tracked the DDS through the complementary ON and OFF fluorescence behaviors of the dyes. Traditional fluorescent dyes are susceptible to aggregation-caused quenching during bioimaging, a problem that is mitigated by using AIE dyes. However, by using both of these contrasting fluorescent labels, we were able to clearly visualize the DDS at different stages of its deployment, from drug transport and delivery to carrier dissociation and migration, demonstrating the feasibility of accurate DDS visualization by complementary fluorescence labeling.

Efficacy of Administration Routes in Crayfish: Comparative Analysis of Intracoelomic and Intrapericardial Techniques Using Fluorescein Dye.

Crayfish are emerging as model organisms for various disciplines. Moreover, decapod crustaceans also exhibit pain-like reactions and heightened anxiety when exposed to harmful stimuli, leading to short-term or persistent behavioral shifts. Awareness of decapod crustacean sentience and thus, suffering calls for refinement of current laboratory protocols. This study aims to enhance the standard methodology for injecting substances into crayfish by minimizing stress-inducing manipulation. We examined the impacts of various administration routes on the persistence of injected chemicals in marbled crayfish, its excretion, and animal survival. Fluorescein dye was used as a visual marker. It was administered via three alternative injection routes-intracoelomic (IC), intrapericardial administration through areola (IP-A), and intrapericardial administration through arthrodial membrane (IP-AM). Continuous video observations were made for a 4-h period under UV light, followed by intermittent observations at 12-h intervals over 48 h. The highest mortality (20%) was observed in IP-A administration. The IP-A method also provided the fastest systemic distribution of the dye in the body. Results indicated visibly higher urination frequency in IP-AM compared to IP-A. IC mirrored IP-AM outcomes without any observed mortality. We conclude that IC administration proved superior to intrapericardial methods, offering the least harmful but effective approach for crayfish injections.

Telomere Biology Disorders: Report on Clinical and Angiographic Findings.

PURPOSE: To evaluate the retinal vasculature in pediatric patients with telomere biology disorders (TBD). DESIGN: Retrospective consecutive case series. SUBJECTS: Pediatric patients with a diagnosis of TBD who underwent widefield fluorescein angiography (FA). METHODS: Electronic medical records of pediatric patients with TBD at a tertiary referral eye center were reviewed from January 2019 to July 2023. Vascular phenotype was assessed by reviewing FA images. MAIN OUTCOMES MEASURES: Incomplete peripheral vascularization, aneurysmal dilatation, terminal arborization, anastomotic loops, capillary dropout, neovascularization, tortuosity, leakage from tractional membranes, and blockage from hemorrhage. RESULTS: Fourteen eyes from 7 patients were included. All patients were genetically confirmed for TBD. The most common genetic variants were in CTC1 (5 patients; 71.4%), ACD (1 patient; 14.3%), and RTEL1 (1 patient; 14.3%). On FA, the most common findings were incomplete peripheral vascularization (14 eyes, 100%), aneurysmal dilatation (12 eyes, 85.7%), terminal arborization (12 eyes, 85.7%), anastomotic loops (12 eyes, 85.7%), capillary dropout (10 eyes, 71.4%), and neovascularization (9 eyes, 64.3%). Regarding treatment, laser photocoagulation (14 eyes, 100%), intravitreal bevacizumab injection (13 eyes, 92.6%), and sub-tenon's Kenalog (11 eyes, 78.6%) were utilized. All patients managed with laser photocoagulation and/or bevacizumab required multiple treatments. CONCLUSION: Our study describes a spectrum of vascular changes evidenced by widefield FA in pediatric patients with genetically confirmed TBD. Although further research is warranted to fully understand the etiology of these subtle vascular anomalies, widefield FA should be conducted in patients with genetically confirmed or suspected TBD.

Comparative fluorophotometric evaluation of the ocular surface retention time of cross-linked and linear hyaluronic acid ocular eye drops on healthy dogs.

PURPOSE: Evaluate and compare the retention time on the canine ocular surface of crosslinked hyaluronic acid (X-HA), linear hyaluronic acid (L-HA) and saline solution using fluorescent compounds (fluorescein sodium salt, Alexa Fluor 488 cadaverine and Alexa Fluor 488 maleimide). METHODS: 0.9% saline solution (SAL) was combined with fluorescein sodium salt. X-HA and L-HA were covalently modified using Alexa Fluor 488 reactive moieties. Eye drops were applied to 70 eyes of 35 dogs that were previously assessed and determined to have a normal ocular surface. Employing a blue light filter (450-490 nm), digital images were captured from instillation to 180 min. Images were analyzed to assess the percent of the total ocular area covered with green fluorescence at various time points. RESULTS: X-HA exhibited a dual phase behavior: A broad microgel coverage first, followed by accumulation in tear film meniscus and medial canthus in the second phase, remaining in contact with the ocular surface up to 180 min. Coverage with L-HA and SAL eye drops quickly migrated to the tear meniscus. No traces of the fluorescent compounds were observed by 45 min in eyes treated with SAL solution compound and, by 120 min, eyes treated with L-HA. CONCLUSIONS: X-HA exhibited a significantly increased ocular surface contact time with the ocular surface compared with L-HA and SAL. Not only could this indicate extended lubrication time but also supports the potential use of this compound as a method for topical sustained-release drug application.

Fluorescein Angiography for Monitoring Neural Blood Flow in Chronic Nerve Compression Neuropathy: Experimental Animal Models and Preliminary Clinical Observations.

Pathologies associated with neural blood disturbance have been reported in patients with chronic nerve compression (CNC) neuropathy. Fluorescein angiography (FAG) and laser Doppler flowmetry (LDF) are effective for real-time peripheral nerve blood flow assessment. However, their reliability in severe neuropathy models in large animals or clinical conditions remains unclear. Initially, we aim to apply FAG to two different CNC animal models and evaluate their characteristics in comparison with those of LDF. In FAG, we quantified the peak luminance at the compression site following fluorescein injection. Then, we positioned the LDF probe at the center of the compression site and recorded the blood flow. Subsequently, we analyzed whether the FAG characteristics obtained in this animal experiment were consistent with those of clinical studies in patients with severe carpal tunnel syndrome (CTS). In the CNC rat model, FAG and LDF effectively monitored reduced neural blood flow over time. We observed significant blood flow reduction using both techniques in a newly developed severe CNC rabbit model. Notably, FAG correlated strongly with the compound muscle action potential (CMAP) amplitude in electrodiagnostic findings, unlike LDF. As a next step, we performed FAG after open carpal tunnel release in clinical cases of CTS. FAG correlated significantly with preoperative CMAP amplitude. This indicates FAG's importance for assessing nerve blood flow during surgery, potentially improving diagnostic accuracy and surgical outcomes.

Imaging-Based Drug Penetration Profiling in an Excised Sheep Cornea Model.

Formulations designed to address ocular conditions and diseases are predominantly administered topically. While in vitro test systems have been developed to assess corneal permeation under extended contact conditions, methods focusing on determining the penetration depth and kinetics of a substance within the cornea itself rather than through it, are scarce. This study introduces a method for time-dependent penetration depth analysis (10 and 60 min) by means of a semiquantitative imaging method in comparison with a quantitative corneal depth-cut technique, employing fluorescein sodium at concentrations of 0.2 and 0.4 mg/mL as a small molecule model substance and sheep cornea as a human surrogate. Excised tissues exhibited sustained viability in modified artificial aqueous humor and maintained thickness (746 ± 43 µm) and integrity (electrical resistance 488 ± 218 Ω∙cm2) under the experimental conditions. Both methods effectively demonstrated the expected concentration- and time-dependent depth of penetration of fluorescein sodium, displaying a significantly strong correlation. The traceability of the kinetic processes was validated with polysorbate 80, which acted as a penetration enhancer. Furthermore, the imaging-based method enabled detecting the retention of larger structures, such as hyaluronic acid and nanoemulsions from the commercial eyedrop formulation NEOVIS® TOTAL multi, inside the lacrimal layer.

Fluorescence guidance in skull base surgery: Applications and limitations - A systematic review.

Introduction: Intraoperative fluorescence guidance is a well-established surgical adjunct in high-grade glioma surgery. In contrast, the clinical use of such dyes and technology has been scarcely reported in skull base surgery. Research question: We aimed to systematically review the clinical applications of different fluorophores in both open and endonasal skull base surgery. Material and methods: We performed a systematic review and discussed the current literature on fluorescence guidance in skull base surgery. Results: After a comprehensive literature search, 77 articles on skull base fluorescence guidance were evaluated. A qualitative analysis of the articles is presented, discussing clinical indications and current controversies. The use of intrathecal fluorescein was the most frequently reported in the literature. Beyond that, 5-ALA and ICG were two other fluorescent dyes most extensively discussed, with some experimental fluorophore applications in skull base surgery. Discussion and conclusion: Intraoperative fluorescence imaging can serve as an adjunct technology in skull base surgery. The scope of initial indications of these fluorophores has expanded beyond malignant glioma resection alone. We discuss current use and controversies and present an extensive overview of additional indications for fluorescence imaging in skull base pathologies. Further quantitative studies will be needed in the future, focusing on tissue selectivity and time-dependency of the different fluorophores currently commercially available, as well as the development of new compounds to expand applications and facilitate skull base surgeries.

Modern Approach to Diabetic Retinopathy Diagnostics.

This article reviews innovative diagnostic approaches for diabetic retinopathy as the prevalence of diabetes mellitus and its complications continue to escalate. Novel techniques focus on early disease detection. Technological innovations, such as teleophthalmology, smartphone-based photography, artificial intelligence with deep learning, or widefield photography, can enhance diagnostic accuracy and accelerate the treatment. The review highlights teleophthalmology and handheld photography as promising solutions for remote eye care. These methods revolutionize diabetic retinopathy screening, offering cost-effective and accessible solutions. However, the use of these techniques may be limited by insurance coverage in certain world regions. Ultra-widefield photography offers a comprehensive view of up to 80.0% of the retina in a single image, compared to the 34.0% coverage of the traditional seven-field imaging protocol. It allows retinal imaging without pupil dilation, especially for individuals with compromised mydriasis. However, they also have drawbacks, including high costs, artifacts from eyelashes, eyelid margins, and peripheral distortion. Recent advances in artificial intelligence and machine learning, particularly through convolutional neural networks, are revolutionizing diabetic retinopathy diagnostics, enhancing screening efficiency and accuracy. FDA-approved Artificial Intelligence-powered devices such as LumineticsCore™, EyeArt, and AEYE Diagnostic Screening demonstrate high sensitivity and specificity in diabetic retinopathy detection. While Artificial Intelligence offers the potential to improve patient outcomes and reduce treatment costs, challenges such as dataset biases, high initial costs, and cybersecurity risks must be considered to ensure safety and efficiency. Nanotechnology advancements further enhance diagnosis, offering highly branched polyethyleneimine particles with fluorescein sodium (PEI-NHAc-FS) for better fluorescein angiography or vanadium oxide-based metabolic fingerprinting for early detection.

Grad-CAM-Based Investigation into Acute-Stage Fluorescein Angiography Images to Predict Long-Term Visual Prognosis of Branch Retinal Vein Occlusion.

Background/Objectives: The purpose of this study was to analyze relevant areas in acute-stage fluorescein angiography (FA) images, predicting the long-term visual prognosis of branch retinal vein occlusion (BRVO) based on gradient-weighted class activation mapping (Grad-CAM). Methods: This retrospective observational study included 136 eyes with BRVO that were followed up for more than a year post-FA. Cropped grayscale images centered on the fovea (200 × 200 pixels) were manually pre-processed from early-phase FA at the acute phase. Pairs of the cropped FA images and the best-corrected visual acuity (BCVA) in remission at least one year post-FA were used to train a 38-layer ResNet with five-fold cross-validation. Correlations between the ResNet-predicted and true (actually measured) logMAR BCVAs in remission, and between the foveal avascular zone (FAZ) area measured by ImageJ (version 1.52r) from FA images and true logMAR BCVA in remission were evaluated. The heat maps generated by Grad-CAM were evaluated to determine which areas were consumed as computational resources for BCVA prediction. Results: The correlation coefficient between the predicted and true logMAR BCVAs in remission was 0.47, and that between the acute-stage FAZ area and true logMAR BCVA in remission was 0.42 (p < 0.0001 for both). The Grad-CAM-generated heat maps showed that retinal vessels adjacent to the FAZ and the FAZ per se had high selectivity (95.7% and 62.2%, respectively). Conclusions: The Grad-CAM-based analysis demonstrated FAZ-neighboring vessels as the most relevant predictor for the long-term visual prognosis of BRVO.

Review for Disease of the Year Clinical Features and Ocular Imaging in Intermediate Uveitis and Pars Planitis.

Intermediate uveitis is a relatively common form of intraocular inflammation that can be seen at any age. The vitreous is the primary site of inflammation; and the presence of snowballs or snowbank defines its subtype, pars planitis. Since it is a clinical diagnosis, it is important to recognize the typical clinical features and associated ocular complications. Recent advances in ocular imaging have made significant contributions to the clinical appraisal and studies of intermediate uveitis. This narrative review includes clinical signs, ocular complications, and ocular imaging of intermediate uveitis and pars planitis.

Recent trends and future perspectives of photoresponsive-based mercury (II) sensors and their biomaterial applications.

Recent advancements in the field of photoresponsive-based mercury (II) sensors have witnessed a surge in research focused on enhancing detection capabilities. Leveraging innovations in materials science, particularly with quantum dots, nanomaterials, and organic semiconductors, these sensors exhibit improved selectivity and sensitivity. Beyond traditional applications, such as environmental monitoring, the integration of photoresponsive principles with emerging technologies like the internet of things (IoT) and wearable promises real-time and remote mercury (II) ion detection. The on-going efforts also explore multifunctional sensors and miniaturization for on-site applications, addressing current challenges and paving the way for broader commercialization. This dynamic landscape underscores the potential for these sensors to play a crucial role in ensuring the effective monitoring and management of mercury (II) levels in diverse settings.

Multiple-ResNet GAN: An enhanced high-resolution image generation method for translation from fundus structure image to fluorescein angiography.

Fluorescein angiography (FA) is a diagnostic method for observing the vascular circulation in the eye. However, it poses a risk to patients. Therefore, generative adversarial networks have been used to convert retinal fundus structure images into FA images. Existing high-resolution image generation methods employ complex deep network models that are challenging to optimize, which leads to issues such as blurred lesion boundaries and poor capture of microleakage and microvessels. In this study, we propose a multiple-ResNet generative adversarial network (GAN) to improve model training, thereby enhancing the ability to generate high-resolution FA images. First, the structure of the multiple-ResNet generator is designed to enhance detail generation in high-resolution images. Second, the Gaussian error linear unit (GELU) activation function is used to help the model converge rapidly. The effectiveness of the multiple-ResNet is verified using the publicly available Isfahan MISP dataset. Experimental results show that our method outperforms other methods, achieving better quantitative results with a mean structural similarity of 0.641, peak signal-to-noise ratio of 18.25, and learned perceptual image patch similarity of 0.272. Compared with state-of-the-art methods, the results showed that using the multiple-ResNet framework and GELU activation function can improve the generation of detailed regions in high-resolution FA images.

Ultra-Wide-Field Fluorescein Angiographic Features Influencing Visual Prognosis in Coats' Disease.

Purpose: To investigate the retinal vascular abnormalities in both affected and fellow eyes of presumed unilateral Coats' disease patients using ultra-wide-field fluorescein angiography (UWF-FA) and their association with visual prognosis. Methods: A retrospective review of 30 patients diagnosed with presumed unilateral Coats' disease, evaluated with UWF-FA from March 2003 to May 2024 in a tertiary referral hospital. Clinical features and multimodal imaging findings were evaluated and factors related to final visual outcomes were analyzed. Results: All 30 patients were diagnosed with presumed unilateral Coats' disease at presentation, comprising 36.7% childhood-onset and 63.3% adult-onset patients. Retinal vascular telangiectasia was observed in 51.7% of fellow eyes. The extent of telangiectasia and exudate in the affected eyes did not significantly correlate with extent of telangiectasia in the fellow eyes. In the more affected eyes, the childhood-onset group had a significantly greater extent of capillary drop-out compared to the adult-onset group (5.0 vs 2.8 clock hours, p = 0.023). In the fellow eyes, telangiectasia tended to be more frequent in the childhood-onset group (63.6% vs. 44.4%), without significance (p = 0.160). In the multivariable regression analysis, the final best-corrected visual acuity (BCVA) in the more affected eye was significantly associated with initial BCVA. The mean extent of telangiectasia in the temporal and nasal quadrants and the initial BCVA of the more affected eyes were identified as prognostic factors for final moderate to severe visual loss (Snellen BCVA < 20/66) (OR = 12.8, p = 0.043; OR = 11.8, p = 0.024, respectively). Conclusions: About half of presumed unilateral Coats' disease cases exhibited features of bilateral disease. Visual prognosis is associated with the peripheral retinal telangiectasia in the temporal and nasal quadrants as well as initial BCVA in the affected eyes while it is not associated with retinal vascular abnormalities in the fellow eyes.

Predictive Role of Fluorescein Angiography in Retinopathy of Prematurity.

BACKGROUND: Fluorescein angiography (FA) has been a pivotal tool for studying the pathophysiology of retinopathy of prematurity (ROP) in vivo. We examined the course of ROP using FA to assess the predictive value of angiographic features. METHODS: This is an observational retrospective cohort study of eyes screened for ROP with a binocular indirect ophthalmoscope and FA. RetCam fundus imaging and video digital fluorescein angiography were performed in the neonatal intensive care unit of Santa Maria Hospital of Perugia. The masked grading of the FA images was retrospectively conducted by two ROP expert ophthalmologists. RESULTS: A total of 80 eyes of 40 patients were included for this study. Among the angiographic features evaluated, leakage, shunts, and tangles were predictive of the development of treatment-requiring ROP (p < 0.05). CONCLUSIONS: FA can add to our understanding of the evolution of vascular abnormalities in the course of ROP and can help predict which eyes will go on to treatment.

Fluorescein Angiography Parameters in Premature Neonates.

Purpose: To describe fluorescein angiography (FA) parameters observed in premature neonates with retinopathy of prematurity (ROP). Design: Retrospective case series. Subjects: Patients with ROP who underwent FA imaging using Retcam at Holtz Children's Hospital from November 2014 to October 2022. Methods: Fluorescein angiography images of the included patients were analyzed with a focus on the timing of angiography phases, including choroidal flush, retinal, and recirculation phases. Gestational age, birth weight (BW), age at imaging, treatment choice, and any FA complications were documented. Main Outcome Measures: Dose of fluorescein administered, onset and duration of each angiography phase, and FA findings in ROP-treated patients. Results: A total of 72 images of 72 eyes were reviewed. Image quality was deemed suitable for inclusion in 64 eyes (88.9%) of 43 patients. The mean gestational age and BW at birth were 24.4 ± 1.9 weeks and 607.8 ± 141.3 g, respectively. The mean postmenstrual age at FA imaging was 50.5 ± 40.8 weeks. All eyes (100%) received treatment with intravitreal injection of anti-VEGF at a mean age of 35.5 ± 2.4 weeks. The onset and duration of angiography phases were relatively variable within the cohort. Choroidal flush occurred at a mean time of 12.2 seconds (range: 6-22 seconds). A subsequent retinal phase was documented at a mean time of 11.96 seconds (range: 3-22 seconds). Recirculation phase was complete at an average time of 2.15 minutes (range: 1-5.45 minutes) postfluorescein injection. None of patients developed allergic reactions to fluorescein injection, such as rash, respiratory distress, tachycardia, fever, or local injection site reactions. Conclusions: Angiographic phases on FA in preterm infants with ROP are variable and may occur earlier than the established references for adults. Financial Disclosures: Proprietary or commercial disclosure may be found in the Footnotes and Disclosures at the end of this article.