Autofluorescence Imaging Research Articles

Fluorescence lifetime imaging with distance and ranging using a miniaturised SPAD system

In this work we demonstrate a miniaturised imaging system based around a time-gated SPAD array operating in a “chip-on-tip” manner. Two versions of the system are demonstrated, each measuring 23 mm × 23 mm × 28 mm with differing fields of view and working distances. Initial tests demonstrate contrast between materials in widefield fluorescence imaging (WFLIm) mode, with frame rates of > 2 Hz achievable. Following this, WFLIm images of autofluorescence in ovine lung tissue are obtained at frame rates of ~ 1 Hz. Finally, the ability of the second system to perform simultaneous WFLIm and time of flight (aka Flourescence Lifetime Imaging Distance and Ranging, FLImDAR) is also tested. This shows that the system is capable of 4 mm resolution of object separation when tested on 3D printed samples. It is further demonstrated as being able to perform scene reconstruction on autofluorescent lung tissue. This system is, to date, the smallest chip on tip WFLIm system published, and is the first demonstration of the FLImDAR technique in a compact, portable system.

Assessing Pediatric Burn Wound Infection Using a Point-of-Care Fluorescence Imaging Device.

Wound infection is the most common complication among pediatric burn patients. When not treated promptly, burn wound infection may lead to delayed healing, failure of skin grafts, or death. Standard burn wound assessment includes inspection for visual signs and symptoms of infection (VSSI) and microbial sampling. To aid in the assessment of burn wound infection, the MolecuLight, a point-of-care autofluorescence imaging device, was introduced at our pediatric burn program in 2020. The MolecuLight uses violet light to illuminate the wound bed, causing clinically relevant quantities of 29 different species of bacteria (>104 CFU/g) to fluoresce in real time. The objectives of this study were to evaluate the role of the MolecuLight in the management of pediatric burn wounds and determine if the findings from the MolecuLight corresponded to VSSI and/or microbial sampling. A retrospective review of patients 0-18 years who had burn wounds assessed with the MolecuLight between November 1, 2020 and June 8, 2023 was conducted. Data were extracted from the medical records of 178 eligible patients with 218 wounds imaged with the device. Fluorescence corresponded with VSSI in 81% of wounds and microbial findings in 82% of wounds. MolecuLight fluorescence, in combination with VSSI, improved sensitivity for detecting wound infections by 39% and decreased specificity by 19% compared to visual signs and symptoms in isolation. Incorporation of the MolecuLight in standard burn wound assessments can improve the detection of infections, which may promote improved wound healing outcomes and antimicrobial stewardship.

Retinal Characteristics of Female Choroideremia Carriers: Multimodal Imaging, Microperimetry, and Genetics

PurposeDescribe visual function and retinal features of female carriers of choroideremia (CHM), using multimodal imaging and microperimetry. DesignCross-sectional cohort study Participants and controlsCHM carriers seen in Australia (Melbourne or Perth) or United Kingdom (Oxford or Cambridge) between 2012 and 2023. Healthy age-matched controls seen in Melbourne, Australia, between 2022 and 2023. MethodsParticipants had visual acuity, fundus-tracked microperimetry, optical coherence tomography (OCT), and fundus autofluorescence (FAF) imaging performed. CHM carriers were either genetically and/or clinically confirmed (i.e., obligate carriers). CHM carriers were grouped according to their retinal phenotype and compared to healthy controls. Statistical analyses were performed on StataBE (v18.0). Main outcome measuresBest-corrected visual acuity (BCVA), low-luminance visual acuity (LLVA), average retinal sensitivity, volume of macular hill of vision (HoV), inner retinal thickness (IRT), and photoreceptor complex (PRC) thickness. ResultsEighty-six eyes of 43 CHM carriers and 60 eyes of 30 healthy controls were examined using multimodal imaging and microperimetry. Median age was 54 and 48.5 years for CHM carriers and controls, respectively (p=0.18). Most CHM carriers (86%) were genetically confirmed. CHM carriers and controls had strong inter-eye correlation between eyes for BCVA and average retinal sensitivity (p<0.001). LLVA and macular HoV tests were sensitive tests to detect changes in CHM carriers with mild phenotypes (i.e., fine and coarse). CHM carriers with geographic and/or male pattern phenotypes had reduced BCVA, LLVA, retinal sensitivity, and retinal thinning, compared to healthy controls. Retinal thickening of the inner retina was observed in the central 1 degree, despite generalised thinning of the PRC in the central 7 degrees, indicating retinal remodelling in CHM carriers, compared to controls. There were no genotype-phenotype correlations observed. ConclusionsFemale carriers of CHM with severe retinal phenotypes (i.e., geographic or male pattern) have significantly decreased visual function and retinal structural changes, when compared to age-matched controls and those carriers with milder phenotypes. LLVA and volumetric measures of the macular HoV were found to be the most sensitive functional tests to detect milder retinal disease (fine and coarse phenotypes) in CHM carriers.

Multispectral imaging for characterizing autofluorescent tissues

Selective Plane Illumination Microscopy (SPIM) has become an emerging technology since its first application for 3D in-vivo imaging of the development of a living organism. An extensive number of works have been published, improving both the speed of acquisition and the resolution of the systems. Furthermore, multispectral imaging allows the effective separation of overlapping signals associated with different fluorophores from the spectrum over the whole field-of-view of the analyzed sample. To eliminate the need of using fluorescent dyes, this technique can also be applied to autofluorescence imaging. However, the effective separation of the overlapped spectra in autofluorescence imaging necessitates the use of mathematical tools. In this work, we explore the application of a method based on Principal Component Analysis (PCA) that enables tissue characterization upon spectral autofluorescence data without the use of fluorophores. Thus, enabling the separation of different tissue types in fixed and living samples with no need of staining techniques. Two procedures are described for acquiring spectral data, including a single excitation based method and a multi-excitation scanning approach. In both cases, we demonstrate the effective separation of various tissue types based on their unique autofluorescence spectra.

Ageing and Polypharmacy in Mesenchymal Stromal Cells: Metabolic Impact Assessed by Hyperspectral Imaging of Autofluorescence.

The impact of age on mesenchymal stromal cell (MSC) characteristics has been well researched. However, increased age is concomitant with increased prevalence of polypharmacy. This adjustable factor may have further implications for the functionality of MSCs and the effectiveness of autologous MSC procedures. We applied hyperspectral microscopy of cell autofluorescence-a non-invasive imaging technique used to characterise cytometabolic heterogeneity-to identify changes in the autofluorescence signals of MSCs from (1) young mice, (2) old mice, (3) young mice randomised to receive polypharmacy (9-10 weeks of oral therapeutic doses of simvastatin, metoprolol, oxycodone, oxybutynin and citalopram), and (4) old mice randomised to receive polypharmacy. Principal Component Analysis and Logistic Regression Analysis were used to assess alterations in spectral and associated metabolic characteristics. Modelling demonstrated that cells from young mice receiving polypharmacy had less NAD(P)H and increased porphyrin relative to cells from old control mice, allowing for effective separation of the two groups (AUC of ROC curve > 0.94). Similarly, cells from old polypharmacy mice were accurately separated from those from young controls due to lower levels of NAD(P)H (p < 0.001) and higher porphyrin (p < 0.001), allowing for an extremely accurate logistic regression (AUC of ROC curve = 0.99). This polypharmacy regimen may have a more profound impact on MSCs than ageing, and can simultaneously reduce optical redox ratio (ORR) and increase porphyrin levels. This has implications for the use of autologous MSCs for older patients with chronic disease.

Systematic study of ophthalmological findings in 10 patients with PEX1-mediated Zellweger spectrum disorder

ABSTRACT Purpose This cross-sectional study describes the ophthalmological and general phenotype of 10 patients from six different families with a comparatively mild form of Zellweger spectrum disorder (ZSD), a rare peroxisomal disorder. Methods Ophthalmological assessment included best-corrected visual acuity (BCVA), perimetry, microperimetry, ophthalmoscopy, fundus photography, spectral-domain optical coherence tomography (SD-OCT), and fundus autofluorescence (FAF) imaging. Medical records were reviewed for medical history and systemic manifestations of ZSD. Results Nine patients were homozygous for c.2528 G > A (p.Gly843Asp) variants in PEX1 and one patient was compound heterozygous for c.2528 G>A (p.Gly843Asp) and c.2097_2098insT (p.Ile700TyrfsTer42) in PEX1. Median age was 22.6 years (interquartile range (IQR): 15.9 – 29.9 years) at the most recent examination, with a median symptom duration of 22.1 years. Symptom onset was variable with presentations of hearing loss (n = 7) or nyctalopia/reduced visual acuity (n = 3) at a median age of 6 months (IQR: 1.9–8.3 months). BCVA (median of 0.8 logMAR; IQR: 0.6–0.9 logMAR) remained stable over 10.8 years and all patients were hyperopic. Fundus examination revealed a variable retinitis pigmentosa (RP)-like phenotype with rounded hyperpigmentations as most prominent feature in six out of nine patients. Electroretinography, visual field measurements, and microperimetry further established the RP-like phenotype. Multimodal imaging revealed significant intraretinal fluid cavities on SD-OCT and a remarkable pattern of hyperautofluorescent abnormalities on FAF in all patients. Conclusion This study highlights the ophthalmological phenotype resembling RP with moderate to severe visual impairment in patients with mild ZSD. These findings can aid ophthalmologists in diagnosing, counselling, and managing patients with mild ZSD.

Clinical predictive factors and imaging biomarkers of treatment response to half dose PDT in patients with chronic central serous chorioretinopathy

PurposeTo determine the clinical and imaging biomarkers of the response to half-dose photodynamic therapy (HD-PDT) in patients with central serous chorioretinopathy (CSC) MethodsClinical records and baseline ophthalmic images of 67 chronic CSC patients who underwent HD-PDT were assessed. In addition to demographic data, optical coherence tomography (OCT), fluorescein angiography (FA) and fundus autofluorescence (FAF) images were analyzed for specific biomarkers. The patients were categorized to early responder and late responder based on the time needed for complete resolution of subretinal fluid after PDT (less than 1 month vs. more than 1 month). The baseline clinical and imaging biomarkers were compared between the two groups. ResultsSeventy-three eyes of 67 patients were included in the study. The mean response time to PDT was 1.63 ± 1.48 months with 82.2% (60/73) of eyes categorized as early responder. The mean response time to PDT in delayed-response group was 4.15±1.51 months. In multivariate analysis, delayed response to PDT was associated with lacking history of systemic corticosteroid consumption, lacking history of pretreatment with eplerenone or acetazolamide before PDT and presence of hyperreflective foci in baseline OCT images (all p values < 0.05). There was no association between final visual outcome and late response to PDT. ConclusionThe presence of inflammatory biomarkers such as hyperreflective foci in baseline OCT images might be indicative of resistance to PDT. Moreover, the effect of pretreatment with mineralocorticoid antagonist on the response to PDT in chronic CSC should be explored in future prospective studies.

#328 Non-invasive assessment of urinary exfoliated proximal tubule cells using multispectral autofluorescence imaging features for early detection of CKD

Abstract Background and Aims Early detection of CKD is important to allow for timely intervention which may reduce adverse clinical outcomes and cost of renal replacement therapies. Histopathological diagnosis of CKD remains the gold standard, but routine kidney biopsy is costly and associated with risks such as pain and bleeding. There is a need to develop accurate non-invasive methods for early diagnosis and prognostication of CKD. Kidney cell exfoliation into urine is an active process, which may generate significant information regarding an individual's kidney status. In particular, proximal tubule cells (PTCs) make up majority of the kidney mass and represents the hallmark of CKD in reflecting the degree of tubulointerstitial fibrosis and atrophy. Multispectral assessment of native cell autofluorescence has been specifically shown to be sensitive to metabolic changes and oxidative stress, which are key factors involved in kidney function decline. Hence, we evaluated whether multispectral autofluorescence signals in urinary exfoliated PTCs reflect kidney function as determined by eGFR. Method Individuals aged 18 or above were included and informed consent was obtained from all participants involved in this study. Spot urine samples were collected and stored at -80°C. On assessment, exfoliated PTCs were extracted from thawed urine using a validated specific immunomagnetic separation method based on anti-CD13 and anti-SGLT2 antibodies. Multispectral autofluorescence imaging was performed using a custom-made autofluorescence microscopy system (standard Olympus iX83™ fluorescence microscope and multispectral excitation lamp from Quantitative™, AU). Images were captured by a Nüvü™ EMCCD camera HNü 1024 cooled to -65°C in our laboratory to reduce sensor-induced noise. For the 34 image channels taken per sample, image acquisition times of up to 5 seconds per channel, with multiple averaging (3 times) was applied to optimize image quality. Image analysis was conducted through a workflow encompassing data preprocessing, data normalization and outlier handling, followed by basic model training and evaluation. An entropy-based image feature selection methodology was initially completed by hand, with results subsequently compared to that generated from the AutoGluon machine-learning framework [1]. The models developed from this process were assessed using ROC (Receiver Operating Characteristic) curves and AUC (Area Under the Curve) values. Two discriminative studies were performed – an overall study between exfoliated PTCs of individuals eGFR≥60 and eGFR &amp;lt; 60 ml/min/1.73 m2 and a subgroup study between that of individuals eGFR≥90 and eGFR 60-90 ml/min/1.73 m2. Results 40 individuals were included in our study. 20 individuals had eGFR≥60 and 20 had eGFR &amp;lt; 60 ml/min/1.73 m2. Multispectral assessment of cell autofluorescence features differentiated between exfoliated PTCs of these 2 groups with AUC value of 0.97 (95% CI 0.94-1.00) (Fig. 1), model accuracy being 82%. Subgroup analysis included 20 individuals of which 10 had eGFR≥90 and 10 had eGFR 60-90 ml/min/1.73 m2. Multispectral assessment of cell autofluorescence features differentiated between exfoliated PTCs of these 2 groups with AUC value of 0.99 (95% CI 0.99-1.00) (Fig. 2), model accuracy being 85%. Conclusion An excellent degree of differentiation between urinary PTCs derived from those with normal or high eGFR and mild degrees of CKD is now reliably demonstrated, complementing conclusions from our group's original small pilot study which evaluated between urinary PTCs from CKD and non-CKD cohorts [2]. Further studies are required to determine whether our novel method can be used to non-invasively predict individuals at risk of progressive kidney disease and to monitor whether intervention is effective.

Intraoperative spectroscopic evaluation of sentinel lymph nodes in breast cancer surgery

Background and objectivesSentinel lymph node (SLN) biopsy is a standard procedure for patients with breast cancer and normal axilla on imaging. Positive SLNs on histological examination can lead to a subsequent surgery for axillary lymph node clearance (ALNC). Here we report a non-destructive technique based on autofluorescence (AF) imaging and Raman spectroscopy for intra-operative assessment of SLNs excised in breast cancer surgery.MethodsA microscope integrating AF imaging and Raman spectroscopy modules was built to allow scanning of lymph node biopsy samples. During AF-Raman measurements, AF imaging determined optimal sampling locations for Raman spectroscopy measurements. After optimisation of the AF image analysis and training of classification models based on data from 85 samples, the AF-Raman technique was tested on an independent set of 81 lymph nodes comprising 58 fixed and 23 fresh specimens. The sensitivity and specificity of AF-Raman were calculated using post-operative histology as a standard of reference.ResultsThe independent test set contained 66 negative lymph nodes and 15 positive lymph nodes according to the reference standard, collected from 78 patients. For this set of specimens, the area under the receiver operating characteristic (ROC) curve for the AF-Raman technique was 0.93 [0.83–0.98]. AF-Raman was then operated in a regime that maximised detection specificity, producing a 94% detection accuracy: 80% sensitivity and 97% specificity. The main confounders for SLN metastasis were areas rich in histiocytes clusters, for which only few Raman spectra had been included in the training dataset.DiscussionThis preliminary study indicates that with further development and extension of the training dataset by inclusion of additional Raman spectra of histiocytes clusters and capsule, the AF-Raman may become a promising technique for intra-operative assessment of SLNs. Intra-operative detection of positive biopsies could avoid second surgery for axillary clearance.

Genotype-phenotype severity correlation in a multicentric portuguese cohort of ABCA4-associated retinopathy

PurposeTo investigate genotype-phenotype correlations in ABCA4-associated retinopathy and further validate a recently proposed genotype-phenotype correlation model. DesignMulticentric, cross-sectional cohort study. MethodsConsecutive patients with genetically confirmed ABCA4-associated retinopathy from three Portuguese centres were included. Patients were categorized into distinct phenotype groups according to the degree of hypoautofluorescence and retinal background appearance in ultra-widefield fundus autofluorescence (UW-FAF) imaging. Genotype classification was performed using two criteria: one according to the presence of the p.Gly1961Glu variant, a hypomorphic variant, at least one moderate variant, or two biallelic severe/PVS1 variants (genotype classification A, which corresponds to the newly described criteria); and another one based on the number of null variants identified (genotype classification B). Associations between clinical data and phenotype and genotype groups were analysed. ResultsA total of 50 patients were included. Significant correlations between age of onset, best-corrected visual acuity (BCVA), and both phenotype and genotype groups were found, with patients in more severe phenotype and genotype categories exhibiting earlier disease onset and poorer visual function (p < 0.001; p < 0.001; p < 0.001; p < 0.001, p < 0.001; and p = 0.004, respectively). Genotype classification A better predicted phenotype severity on UW-AF imaging, demonstrating milder genotypes in patients with less severe phenotypes and more severe genotypes in those with advanced disease (p < 0.001). A genotype-phenotype correlation matrix was constructed based on the classification of the two disease-causing variants and their corresponding phenotypic staging. ConclusionOur findings support the utility of the newly described genotype classification in evaluating ABCA4-associated retinopathy phenotype severity, with possible implications in future understanding of the disease genetics and assessment of individual prognosis for patients.

When Two Eyes Don't Suffice-Learning Difficult Hyperfluorescence Segmentations in Retinal Fundus Autofluorescence Images via Ensemble Learning.

Hyperfluorescence (HF) and reduced autofluorescence (RA) are important biomarkers in fundus autofluorescence images (FAF) for the assessment of health of the retinal pigment epithelium (RPE), an important indicator of disease progression in geographic atrophy (GA) or central serous chorioretinopathy (CSCR). Autofluorescence images have been annotated by human raters, but distinguishing biomarkers (whether signals are increased or decreased) from the normal background proves challenging, with borders being particularly open to interpretation. Consequently, significant variations emerge among different graders, and even within the same grader during repeated annotations. Tests on in-house FAF data show that even highly skilled medical experts, despite previously discussing and settling on precise annotation guidelines, reach a pair-wise agreement measured in a Dice score of no more than 63-80% for HF segmentations and only 14-52% for RA. The data further show that the agreement of our primary annotation expert with herself is a 72% Dice score for HF and 51% for RA. Given these numbers, the task of automated HF and RA segmentation cannot simply be refined to the improvement in a segmentation score. Instead, we propose the use of a segmentation ensemble. Learning from images with a single annotation, the ensemble reaches expert-like performance with an agreement of a 64-81% Dice score for HF and 21-41% for RA with all our experts. In addition, utilizing the mean predictions of the ensemble networks and their variance, we devise ternary segmentations where FAF image areas are labeled either as confident background, confident HF, or potential HF, ensuring that predictions are reliable where they are confident (97% Precision), while detecting all instances of HF (99% Recall) annotated by all experts.

Pegcetacoplan Treatment and Consensus Features of Geographic Atrophy Over 24 Months

Despite widespread availability and consensus on its advantages for detailed imaging of geographic atrophy (GA), spectral-domain optical coherence tomography (SD-OCT) might benefit from automated quantitative OCT analyses in GA diagnosis, monitoring, and reporting of its landmark clinical trials. To analyze the association between pegcetacoplan and consensus GA SD-OCT end points. This was a post hoc analysis of 11 614 SD-OCT volumes from 936 of the 1258 participants in 2 parallel phase 3 studies, the Study to Compare the Efficacy and Safety of Intravitreal APL-2 Therapy With Sham Injections in Patients With Geographic Atrophy (GA) Secondary to Age-Related Macular Degeneration (OAKS) and Study to Compare the Efficacy and Safety of Intravitreal APL-2 Therapy With Sham Injections in Patients With Geographic Atrophy (GA) Secondary to Age-Related Macular Degeneration (DERBY). OAKS and DERBY were 24-month, multicenter, randomized, double-masked, sham-controlled studies conducted from August 2018 to July 2020 among adults with GA with total area 2.5 to 17.5 mm2 on fundus autofluorescence imaging (if multifocal, at least 1 lesion ≥1.25 mm2). This analysis was conducted from September to December 2023. Study participants received pegcetacoplan, 15 mg per 0.1-mL intravitreal injection, monthly or every other month, or sham injection monthly or every other month. The primary end point was the least squares mean change from baseline in area of retinal pigment epithelium and outer retinal atrophy in each of the 3 treatment arms (pegcetacoplan monthly, pegcetacoplan every other month, and pooled sham [sham monthly and sham every other month]) at 24 months. Feature-specific area analysis was conducted by Early Treatment Diabetic Retinopathy Study (ETDRS) regions of interest (ie, foveal, parafoveal, and perifoveal). Among 936 participants, the mean (SD) age was 78.5 (7.22) years, and 570 participants (60.9%) were female. Pegcetacoplan, but not sham treatment, was associated with reduced growth rates of SD-OCT biomarkers for GA for up to 24 months. Reductions vs sham in least squares mean (SE) change from baseline of retinal pigment epithelium and outer retinal atrophy area were detectable at every time point from 3 through 24 months (least squares mean difference vs pooled sham at month 24, pegcetacoplan monthly: -0.86 mm2; 95% CI, -1.15 to -0.57; P < .001; pegcetacoplan every other month: -0.69 mm2; 95% CI, -0.98 to -0.39; P < .001). This association was more pronounced with more frequent dosing (pegcetacoplan monthly vs pegcetacoplan every other month at month 24: -0.17 mm2; 95% CI, -0.43 to 0.08; P = .17). Stronger associations were observed in the parafoveal and perifoveal regions for both pegcetacoplan monthly and pegcetacoplan every other month. These findings offer additional insight into the potential effects of pegcetacoplan on the development of GA, including potential effects on the retinal pigment epithelium and photoreceptors. ClinicalTrials.gov Identifiers: NCT03525600 and NCT03525613.

Comprehensive analysis of two hotspot codons in the TUBB4B gene and associated phenotypes

Our purpose was to elucidate the genotype and ophthalmological and audiological phenotype in TUBB4B-associated inherited retinal dystrophy (IRD) and sensorineural hearing loss (SNHL), and to model the effects of all possible amino acid substitutions at the hotspot codons Arg390 and Arg391. Six patients from five families with heterozygous missense variants in TUBB4B were included in this observational study. Ophthalmological testing included best-corrected visual acuity, fundus examination, optical coherence tomography, fundus autofluorescence imaging, and full-field electroretinography (ERG). Audiological examination included pure-tone and speech audiometry in adult patients and auditory brainstem response testing in a child. Genetic testing was performed by disease gene panel analysis based on genome sequencing. The molecular consequences of the substitutions of residues 390 and 391 on TUBB4B and its interaction with α-tubulin were predicted in silico on its three-dimensional structure obtained by homology modelling. Two independent patients had amino acid exchanges at position 391 (p.(Arg391His) or p.(Arg391Cys)) of the TUBB4B protein. Both had a distinct IRD phenotype with peripheral round yellowish lesions with pigmented spots and mild or moderate SNHL, respectively. Yet the phenotype was milder with a sectorial pattern of bone spicules in one patient, likely due to a genetically confirmed mosaicism for p.(Arg391His). Three patients were heterozygous for an amino acid exchange at position 390 (p.(Arg390Gln) or p.(Arg390Trp)) and presented with another distinct retinal phenotype with well demarcated pericentral retinitis pigmentosa. All showed SNHL ranging from mild to severe. One additional patient showed a variant distinct from codon 390 or 391 (p.(Tyr310His)), and presented with congenital profound hearing loss and reduced responses in ERG. Variants at codon positions 390 and 391 were predicted to decrease the structural stability of TUBB4B and its complex with α-tubulin, as well as the complex affinity. In conclusion, the twofold larger reduction in heterodimer affinity exhibited by Arg391 substitutions suggested an association with the more severe retinal phenotype, compared to the substitution at Arg390.

Genotypic and Phenotypic Characterization of a Cohort of Patients Affected by Rod Cyclic Nucleotide Channel-Associated Retinitis Pigmentosa.

Retinitis pigmentosa (RP), a heterogeneous inherited retinal disorder causing gradual vision loss, affects over 1 million people worldwide. Pathogenic variants in CNGA1 and CNGB1 genes, respectively, accounting for 1% and 4% of cases, impact the cyclic nucleotide-gated channel in rod photoreceptor cells. The aim of this study was to describe and compare genotypic and clinical characteristics of a cohort of patients with CNGA1- or CNGB1-related RP and to explore potential genotype-phenotype correlations. The following data from patients with CNGA1- or CNGB1-related RP, followed in five Italian inherited retinal degenerations services, were retrospectively collected: genetic variants in CNGA1 and CNGB1, best-corrected visual acuity (BCVA), ellipsoid zone (EZ) width, fundus photographs, and short-wavelength fundus autofluorescence (SW-AF) images. Comparisons and correlation analyses were performed by first dividing the cohort in two groups according to the gene responsible for the disease (CNGA1 and CNGB1 groups). In parallel, the whole cohort of RP patients was divided into two other groups, according to the expected impact of the variants at protein level (low and high group). In total, 29 patients were recruited, 11 with CNGA1- and 18 with CNGB1-related RP. In both CNGA1 and CNGB1, 5 novel variants in CNGA1 and 5 in CNGB1 were found. BCVA was comparable between CNGA1 and CNGB1 groups, as well as between low and high groups. CNGA1 group had a larger mean EZ width compared to CNGB1 group, albeit not statistically significant, while EZ width did not differ between low and high groups A statistically significant correlation between EZ width and BCVA as well as between EZ width and age were observed in the whole cohort of RP patients. Fundus photographs of all patients in the cohort showed classic RP pattern, and in SW-AF images an hyperautofluorescent ring was observed in 14/21 patients. Rod CNG channel-associated RP was demonstrated to be a slowly progressive disease in both CNGA1- and CNGB1-related forms, making it an ideal candidate for gene augmentation therapies.

Past, Present, and Future Diagnostic Methods for the Early Noninvasive Detection of Oral Premalignant Lesions: A State of the Art and Systematic Review.

Objectives: To provide an in-depth analysis of noninvasive methods for the early diagnosis of oral premalignant lesions, focusing on novel biomarkers and optical technologies, and to discuss their potential in improving the prognosis of patients with oral oncological diseases. Methods: This state-of-the-art review examines various noninvasive diagnostic techniques, including the utilization of salivary microRNAs and optical technologies such as Raman spectroscopy, elastic scattering spectroscopy, diffuse reflectance spectroscopy, narrow-band imaging, autofluorescence imaging, toluidine blue staining, and microendoscopy. Results: Several noninvasive techniques have shown varying degrees of effectiveness in detecting oral cancer. Autofluorescence imaging exhibited sensitivities up to 100% but had variable specificity. toluidine blue staining reported sensitivity between 77% and 100% for high-risk lesions or cancer, with specificity around 45% to 67%. Spectroscopy techniques achieved 72% to 100% sensitivities and specificities of 75% to 98%. Microendoscopy presented a sensitivity of 84% to 95% and a specificity of 91% to 95%. Conclusion: The review highlights the strengths and limitations of each noninvasive diagnostic method and their recent advancements. Although promising results have been demonstrated, there is a need for further development of reliable strategies for early detection and intervention in oral oncology.

Deep Learning for prediction of late recurrence of retinal detachment using preoperative and postoperative ultra-wide field imaging.

To elaborate a deep learning (DL) model for automatic prediction of late recurrence (LR) of rhegmatogenous retinal detachment (RRD) using pseudocolor and fundus autofluorescence (AF) ultra-wide field (UWF) images obtained preoperatively and postoperatively. We retrospectively included patients >18 years who underwent either scleral buckling (SB) or pars plana vitrectomy (PPV) for primary or recurrent RRD with a post-operative follow-up >2 years. Records of RRD recurrence between 6 weeks and 2 years after surgery served as a ground truth for the training of the deep learning (DL) models. Four separate DL models were trained to predict LR within the 2 postoperative years (binary outputs) using, respectively, UWF preoperative and postoperative pseudocolor images and UWF preoperative and postoperative AF images. A total of 412 eyes were included in the study (332 eyes treated with PPV and 80 eyes with SB). The mean follow-up was 4.0 ± 2.1 years. The DL models based on preoperative and postoperative pseudocolor UWF imaging predicted recurrence with 85.6% (sensitivity 86.7%, specificity 85.4%) and 90.2% accuracy (sensitivity 87.0%, specificity 90.8%) in PPV-treated eyes, and 87.0% (sensitivity 86.7%, specificity 87.0%) and 91.1% (sensitivity 88.2%, specificity 91.9%) in SB-treated eyes, respectively. The DL models using preoperative and postoperative AF-UWF imaging predicted recurrence with 87.6% (sensitivity 84.0% and specificity 88.3%) and 91.0% (sensitivity 88.9%, specificity 91.5%) accuracy in PPV eyes, and 86.5% (sensitivity 87.5%; specificity 86.2%) and 90.6% (sensitivity 90.0%, specificity 90.7%) in SB eyes, respectively. Among the risk factors detected with visualisation methods, potential novel ones were extensive laser retinopexy and asymmetric staphyloma. DL can accurately predict the LR of RRD based on UWF images (especially postoperative ones), which can help refine follow-up strategies. Saliency maps might provide further insight into the dynamics of RRD recurrence.

Comparison of diagnostic accuracy between linked color imaging and autofluorescence imaging in patients with ulcerative colitis: A prospective observational study.

Image enhancement endoscopy techniques, such as linked color imaging (LCI) and autofluorescence imaging (AFI), have shown promise in diagnosing mucosal inflammation in ulcerative colitis (UC). However, no studies have directly compared the diagnostic efficacy of LCI and AFI. This prospective observational study aimed to compare their diagnostic accuracy for histological healing in UC. This study included 81 UC patients, resulting in a total of 204 endoscopic images captured using LCI and AFI, respectively. Spearman's rank correlation coefficients assessed the correlation between LCI and AFI coloration and Geboes histopathology score (GHS). Six endoscopists, who were blinded to clinicopathological features, evaluated these images, and subsequently, the diagnostic accuracy was evaluated. Spearman's rank correlation coefficients between LCI index, AFI index (reverse gamma value), and GHS were 0.324 and -0.428, respectively (P<0.001), indicating a significant correlation between LCI and AFI coloration and histological healing. In LCI and AFI classifications, mean values for diagnostic accuracy, sensitivity, specificity, positive predictive value, and negative predictive value were 76.3±2.2 versus 77.8±2.7, 91.8±4.0 versus 83.2±7.6, 53.4±10.0 versus 70.0±5.3, 74.0±3.5 versus 80.0±1.6, and 82.9±5.2 versus 75.5±7.5, respectively. No significant difference in diagnostic accuracy existed between LCI and AFI classifications. However, LCI displayed higher sensitivity than AFI while AFI showed higher specificity compared with LCI (P<0.05). LCI and AFI offer comparable diagnostic accuracy for histological healing. Clinically, it is necessary to recognize diagnostic features characterized by higher sensitivity in LCI and greater specificity in AFI.

Developing a Prototype Device for Assessing Meat Quality Using Autofluorescence Imaging and Machine Learning Techniques

Meat quality determination is now more vital than ever, with an ever-increasing demand for meat, especially with a greater desire for high-quality beef. Many existing qualitative methods currently used for meat quality assessment are strenuous, time-consuming, and subjective. The quantitative techniques employed are time-consuming, destructive, and expensive. In the search for a quantitative, rapid, and non-destructive method of determining meat quality, the use of autofluorescence has been employed and has demonstrated its capabilities to characterise meat grades by identifying biochemical features such as the intramuscular fat and tryptophan content through the excitation of meat samples and the collection and analysis of the emission data. Despite its success, the method remains expensive and inaccessible, thus preventing it from being translated into small-scale industry applications. This study will detail the process taken to design and construct a low-cost, miniature prototype device that could successfully distinguish between varying meat grades using autofluorescence imaging and machine learning techniques.

Ocular Effects of Prenatal Carotenoid Supplementation in the Mother and Her Child: The Lutein and Zeaxanthin in Pregnancy (L-ZIP) Randomized Trial - Report Number 2

PurposeLutein and zeaxanthin are xanthophyll carotenoids that have been promoted to enhance maternal health and infant visual and neurodevelopment. In this study, we determined the effects of prenatal lutein and zeaxanthin supplementation on systemic and ocular carotenoid status in the mother and her newborn infant (NCT03750968). This report focuses on the ocular effects of prenatal carotenoid supplementation. DesignA prospective randomized clinical trial with 47 subjects randomly assigned by 1:1 allocation to receive standard-of-care prenatal vitamins along with 10 mg lutein and 2 mg zeaxanthin softgel (Carotenoid Group) or standard-of-care prenatal vitamins with a placebo softgel (Control Group) starting in the first trimester. SubjectsWe enrolled low-risk pregnancy subjects aged 18 years and above from the obstetrics and gynecology clinic of the University of Utah Hospital. MethodsMaternal macular, skin, and serum carotenoid concentrations were measured using autofluorescence imaging, resonance Raman spectroscopy, and high-performance liquid chromatography, respectively. Infants’ ocular carotenoids and retinal architecture were measured by blue light reflectance imaging and spectral domain optical coherence tomography, respectively. Main Outcome MeasuresChanges in maternal and infant macular pigment, skin, and serum carotenoid status over the study period. Differences in infants’ retinal maturity indicators between the two study groups. ResultsFollowing supplementation, there was a statistically significant increase in maternal macular pigment optical volume (p < 0.001) in the Carotenoid Group relative to the Control Group at all study time points, and there was no detectable maternal ocular carotenoid depletion. Infant skin and serum carotenoids increased significantly in the Carotenoid Group compared to the Control Group. As exploratory endpoints, infants in the Carotenoid Group had a 20% increase in macular pigment optical density (p = 0.242) and more mature foveal parameters compared to those in the Control Group. ConclusionPrenatal carotenoid supplementation significantly increased maternal and infant systemic carotenoids and caused a pattern of increased infant ocular carotenoid status, which may benefit both mothers and their infants’ ocular development and function. This study provides important data to design and power a future multicenter study of prenatal carotenoid supplementation in higher-risk pregnancies.

Rediscovering Age-Related Macular Degeneration with Swept-Source OCT Imaging: The 2022 Charles L. Schepens, MD, Lecture

Swept-source OCT angiography (SS-OCTA) scans of eyes with age-related macular degeneration (AMD) were used to replace color, autofluorescence, infrared reflectance, and dye-based fundus angiographic imaging for the diagnosis and staging of AMD. Through the use of different algorithms with the SS-OCTA scans, both structural and angiographic information can be viewed and assessed using both cross sectional and en face imaging strategies. Presented at the 2022 Charles L. Schepens, MD, Lecture at the American Academy of Ophthalmology Retina Subspecialty Day, Chicago, Illinois, on September 30,2022. Patients with AMD. Review of published literature and ongoing clinical research using SS-OCTA imaging in AMD. Swept-source OCT angiography imaging of AMD at different stages of disease progression. Volumetric SS-OCTA dense raster scans were used to diagnose and stage both exudative and nonexudative AMD. In eyes with nonexudative AMD, a single SS-OCTA scan was used to detect and measure structural features in the macula such as the area and volume of both typical soft drusen and calcified drusen, the presence and location of hyperreflective foci, the presence of reticular pseudodrusen, also known as subretinal drusenoid deposits, the thickness of the outer retinal layer, the presence and thickness of basal laminar deposits, the presence and area of persistent choroidal hypertransmission defects, and the presence of treatment-naïve nonexudative macular neovascularization. In eyes with exudative AMD, the same SS-OCTA scan pattern was used to detect and measure the presence of macular fluid, the presence and type of macular neovascularization, and the response of exudation to treatment with vascular endothelial growth factor inhibitors. In addition, the same scan pattern was used to quantitate choriocapillaris (CC) perfusion, CC thickness, choroidal thickness, and the vascularity of the choroid. Compared with using several different instruments to perform multimodal imaging, a single SS-OCTA scan provides a convenient, comfortable, and comprehensive approach for obtaining qualitative and quantitative anatomic and angiographic information to monitor the onset, progression, and response to therapies in both nonexudative and exudative AMD. Proprietary or commercial disclosure may be found in the Footnotes and Disclosures at the end of this article.