Autofluorescence Imaging Research Articles

Non-syndromic OTX2-associated pattern dystrophy: a 10-year multimodal imaging study

PurposeTo report novel multimodal imaging features and long-term follow-up of Orthodenticle Homeobox 2 (OTX2)-associated pattern Gdystrophy.MethodsA 14-year-old boy referred with glaucoma suspect and macular pigmentation underwent fundus autofluorescence imaging, optical coherence tomography, fluorescein and indocyanine green angiography, visual field test, microperimetry and electrophysiology over a ten-year period. Next-generation sequencing panel identified a de novo heterozygous likely pathogenic OTX2 variant, c.259G>A, [p.(Glu87Lys)].ResultsVisual acuity was 20/40 OD and 20/30 OS. Examination showed bilateral enlarged optic nerve heads and increased disc cupping, multiple cilioretinal arteries, a pigmentary maculopathy with stellate-shaped region of hypoautofluorescence, shallow serous macular detachment, subretinal deposits and temporal avascular retina. Angiography showed no source of leakage and absence of retinal neovascularisation despite extensive peripheral non perfusion. Electrophysiological assessments demonstrated mild progressive rod and cone pathway abnormalities, reduced light-adapted b:a ratio, and reduced Arden ratio on electro-oculogram. Ten-year follow-up confirmed a stable disease course despite persistent submacular fluid. There was no associated pituitary structural abnormality or dysfunction.ConclusionsThis case study contributes to further understanding of OTX2-associated pattern dystrophy, highlighting its stability over 10 years. Further investigation into inter-individual and intrafamilial variability is warranted.

Advanced optical imaging techniques for bladder cancer detection and diagnosis: a systematic review.

To systematically assess the current available literature concerning advanced optical imaging methods for the detection and diagnosis of bladder cancer (BCa), focusing particularly on the sensitivity and specificity of these techniques. First a scoping search was performed to identify all available optical techniques for BCa detection and diagnosis. The optical imaging techniques used for detecting BCa are: the Storz professional image enhancement system (IMAGE1 S), narrow-band imaging (NBI), photoacoustic imaging (PAI), autofluorescence imaging (AFI), photodynamic diagnosis (PDD), and scanning fibre endoscopy (SFE). The staging and grading techniques for BCa are: optical coherence tomography (OCT), confocal laser endomicroscopy (CLE), Raman spectroscopy, endocytoscopy, and non-linear optical microscopy (NLO). Then a systematic literature search was conducted using MEDLINE, EMBASE and Web of Science from inception to 21 November 2023. Articles were screened and selected by two independent reviewers. Inclusion criteria were: reporting on both the sensitivity and specificity of a particular technique and comparison to histopathology, and in the case of a detection technique comparison to white light cystoscopy (WLC). Out of 6707 articles, 189 underwent full-text review, resulting in 52 inclusions. No articles met criteria for IMAGE1 S, PAI, SFE, Raman spectroscopy, and endocytoscopy. All detection techniques showed higher sensitivity than WLC, with NBI leading (87.8-100%). Overall, detection technique specificity was comparable to WLC, with PDD being most specific (23.3-100%). CLE and OCT varied in sensitivity and specificity, with OCT showing higher specificity for BCa diagnosis, notably for carcinoma in situ (97-99%) compared to CLE (62.5-81.3%). NLO demonstrated high sensitivity and specificity (90-97% and 77-100%, respectively) based on limited data from two small ex vivo studies. Optical techniques with the most potential are PDD for detecting and OCT for staging and grading BCa. Further research is crucial to validate their integration into routine practice and explore the value of other imaging techniques.

In vivo and in situ robust quantitative optical biopsy of hepatocellular carcinoma and metastasis based on two-photon autofluorescence imaging.

Two-photon autofluorescence (TPAF) imaging is able to offer precise cellular metabolic information with high spatiotemporal resolution, making it a promising biopsy tool. The technique is greatly hampered by the complexity of either the optical system or data processing. Here, the excitation wavelength was optimized to simultaneously excite both flavin adenine dinucleotide and nicotinamide adenine dinucleotide and eliminate the unexpected TPAF. The optical redox ratio (ORR) images were robustly achieved without additional calibration under the optimized single-wavelength excitation. The in vitro, ex vivo, and in vivo biopsy by the TPAF method were systematically studied and compared using hepato-cellular carcinoma and metastasis as examples. It was demonstrated that the proposed TPAF method simplified the optical system, improved the robustness of ORR, and enabled early-stage cancer diagnosis, showing distinguished advantages as compared with previous methods.

Metabolic changes in Toxoplasma gondii-infected host cells measured by autofluorescence imaging.

Toxoplasma gondii, the causative agent of toxoplasmosis, is an obligate intracellular parasite that infects warm-blooded vertebrates across the world. In humans, seropositivity rates of T. gondii range from 10% to 90% across communities. Despite its prevalence, few studies address how T. gondii infection changes the metabolism of host cells. In this study, we investigate how T. gondii manipulates the host cell metabolic environment by monitoring the metabolic response over time using noninvasive autofluorescence lifetime imaging of single cells, metabolite analysis, extracellular flux analysis, and reactive oxygen species (ROS) production. Autofluorescence lifetime imaging indicates that infected host cells become more oxidized and have an increased proportion of bound NAD(P)H compared to uninfected controls. Over time, infected cells also show decreases in levels of intracellular glucose and lactate, increases in oxygen consumption, and variability in ROS production. We further examined changes associated with the pre-invasion "kiss and spit" process using autofluorescence lifetime imaging, which also showed a more oxidized host cell with an increased proportion of bound NAD(P)H over 48 hours compared to uninfected controls, suggesting that metabolic changes in host cells are induced by T. gondii kiss and spit even without invasion.IMPORTANCEThis study sheds light on previously unexplored changes in host cell metabolism induced by T. gondii infection using noninvasive, label-free autofluorescence imaging. In this study, we use optical metabolic imaging (OMI) to measure the optical redox ratio (ORR) in conjunction with fluorescence lifetime imaging microscopy (FLIM) to noninvasively monitor single host cell response to T. gondii infection over 48 hours. Collectively, our results affirm the value of using autofluorescence lifetime imaging to noninvasively monitor metabolic changes in host cells over the time course of a microbial infection. Understanding this metabolic relationship between the host cell and the parasite could uncover new treatment and prevention options for T. gondii infections worldwide.

Computer-assisted discrimination of cancerous and pre-cancerous from benign oral lesions based on multispectral autofluorescence imaging endoscopy.

Diagnosis of cancerous and pre-cancerous oral lesions at early stages is critical for the improvement of patient care, to increase survival rates and minimize the invasiveness of tumor resection surgery. Unfortunately, oral precancerous and early-stage cancerous lesions are often difficult to distinguish from oral benign lesions with the existing diagnostic tools used during standard clinical oral examination. In consequence, early diagnosis of oral cancer can be achieved in only about 30% of patients. Therefore, clinical diagnostic technologies for fast, minimally invasive, and accurate oral cancer screening are urgently needed. This study investigated the use of multispectral autofluorescence imaging endoscopy for the automated and noninvasive discrimination of cancerous and precancerous from benign oral epithelial lesions. In vivo multispectral autofluorescence endoscopic images of clinically suspicious oral lesions were acquired from 67 patients undergoing tissue biopsy examination. The imaged lesions were classified as precancerous (n = 4), cancerous (n = 29), and benign (n = 34) lesions based on histopathology diagnosis. Multispectral autofluorescence intensity feature maps were generated for each oral lesion and used to train and optimize support vector machine (SVM) models for automated discrimination of cancerous and precancerous from benign oral lesions. After a leave-one-patient-out cross-validation strategy, an optimized SVM model developed with four multispectral autofluorescence features yielded levels of sensitivity and specificity of 85% and 71%, respectively and overall accuracy of 78% in the discrimination of cancerous/precancerous versus benign oral lesions. This study demonstrates the potentials of a computer-assisted detection system based on multispectral autofluorescence imaging endoscopy for the early detection of cancerous and precancerous oral lesions.

Machine Learning for Extraction of Image Features Associated with Progression of Geographic Atrophy

Background: Several studies have investigated various features and models in order to understand the growth and progression of the ocular disease geographic atrophy (GA). Commonly assessed features include age, sex, smoking, alcohol consumption, sedentary lifestyle, hypertension, and diabetes. There have been inconsistencies regarding which features correlate with GA progression. Chief amongst these inconsistencies is whether the investigated features are readily available for analysis across various ophthalmic institutions. Methods:In this study, we focused our attention on the association of fundus autofluorescence (FAF) imaging features and GA progression. Our method included feature extraction using radiomic processes and feature ranking by machine learning incorporating the algorithm XGBoost to determine the best-ranked features. This led to the development of an image-based linear mixed-effects model, which was designed to account for slope change based on within-subject variability and inter-eye correlation. Metrics used to assess the linear mixed-effects model included marginal and conditional R2, Pearson’s correlation coefficient (r), root mean square error (RMSE), mean error (ME), mean absolute error (MAE), mean absolute deviation (MAD), the Akaike Information Criterion (AIC), the Bayesian Information Criterion (BIC), and loglikelihood. Results: We developed a linear mixed-effects model with 15 image-based features. The model results were as follows: R2 = 0.96, r = 0.981, RMSE = 1.32, ME = −7.3 × 10−15, MAE = 0.94, MAD = 0.999, AIC = 2084.93, BIC = 2169.97, and log likelihood = −1022.46. Conclusions: The advantage of our method is that it relies on the inherent properties of the image itself, rather than the availability of clinical or demographic data. Thus, the image features discovered in this study are universally and readily available across the board.

Retinal Displacement Following Rhegmatogenous Retinal Detachment Repair.

This study aimed to report retinal displacement incidence following rhegmatogenous retinal detachment (RRD) repair with pars plana vitrectomy (PPV), scleral buckle (SB), or combined SB/PPV. A single-center, case series using Optos fundus autofluorescence (FAF) images was performed after RRD repair between April 2020 and February 2022. Retinal displacement was identified by imprinted retinal vessels on FAF imaging. One hundred ninety-four eyes were included. Ninety-seven (50.0%) eyes underwent PPV, 46 (23.7%) underwent SB, and 51 (26.3%) underwent SB/PPV. A total of 25/194 eyes (12.9%) had retinal vessel imprinting on FAF imaging consistent with retinal displacement. The SB/PPV group (9/51, 17.6%) and the PPV group (15/97, 15.5%) had significantly higher displacement rates when compared to the SB group (1/46, 2.2%; P = 0.017, P = 0.021, respectively). Retinal displacement after RRD repair is more prevalent after PPV. There was a low rate of displacement with SB. Retinal displacement was not associated with a visual acuity difference. [Ophthalmic Surg Lasers Imaging Retina 2024;55:XX-XX.].

FLUID-FLUID EXCHANGE AND EXTERNAL NEEDLE DRAINAGE WITH MINIMAL GAS VITRECTOMY ASSOCIATED WITH RETINAL DISPLACEMENT.

To determine whether fluid-fluid exchange (endodrainage) or external needle drainage can result in retinal displacement following minimal gas vitrectomy (MGV) with no fluid-air exchange for rhegmatogenous retinal detachment repair. Two patients with macula-off rhegmatogenous retinal detachment underwent MGV with and without segmental buckle. First case had MGV with segmental buckle (MGV-SB), along with endodrainage, whereas the second case had MGV only with external fluid drainage. At the completion of surgery, the patient was immediately log rolled to face down for 6 hours followed by positioning to the break. Both patients achieved retinal reattachment, and postoperative widefield fundus autofluorescence imaging demonstrated a low-integrity retinal attachment with retinal displacement. Iatrogenic fluid drainage techniques, such as fluid-fluid exchange or external needle drainage during MGV (without fluid-air exchange), may result in retinal displacement. Allowing the retinal pigment epithelial pump to reabsorb the fluid naturally may reduce the risk of retinal displacement.

Artificial Intelligence to Facilitate Clinical Trial Recruitment in Age-Related Macular Degeneration

ObjectiveRecent developments in artificial intelligence (AI) have positioned it to transform several stages of the clinical trial process. In this study, we explore the role of AI in clinical trial recruitment of individuals with geographic atrophy (GA), an advanced stage of age-related macular degeneration, amidst numerous ongoing clinical trials for this condition. DesignCross-sectional study.Subjects, Participants, and/or Controls: Retrospective dataset from the INSIGHT Health Data Research Hub at Moorfields Eye Hospital in London, United Kingdom, including 306,651 patients (602,826 eyes) with suspected retinal disease who underwent optical coherence tomography (OCT) imaging between 1 January 2008 and 10 April 2023. Methods, Intervention, or TestingA deep learning model was trained on OCT scans to identify patients potentially eligible for GA trials, using AI-generated segmentations of retinal tissue. This method's efficacy was compared against a traditional keyword-based electronic health record (EHR) search. A clinical validation with fundus autofluorescence (FAF) images was performed to calculate the positive predictive value (PPV) of this approach, by comparing AI predictions to expert assessments. Main Outcome MeasuresThe primary outcomes included the PPV of AI in identifying trial-eligible patients, and the secondary outcome was the intraclass correlation between GA areas segmented on FAF by experts and AI-segmented OCT scans. ResultsThe AI system shortlisted a larger number of eligible patients with greater precision (1,139, PPV: 63%; 95% CI: 54–71%) compared to the EHR search (693, PPV: 40%; 95% CI: 39–42%). A combined AI-EHR approach identified 604 eligible patients with a PPV of 86% (95% CI: 79–92%). Intraclass correlation of GA area segmented on FAF versus AI-segmented area on OCT was 0.77 (95% CI: 0.68–0.84) for cases meeting trial criteria. The AI also adjusts to the distinct imaging criteria from several clinical trials, generating tailored shortlists ranging from 438 to 1,817 patients. ConclusionsThis study demonstrates the potential for AI in facilitating automated pre-screening for clinical trials in GA, enabling site feasibility assessments, data-driven protocol design, and cost reduction. Once treatments are available, similar AI systems could also be used to identify individuals who may benefit from treatment.

Cellpose as a reliable method for single-cell segmentation of autofluorescence microscopy images.

Autofluorescence microscopy uses intrinsic sources of molecular contrast to provide cellular-level information without extrinsic labels. However, traditional cell segmentation tools are often optimized for high signal-to-noise ratio (SNR) images, such as fluorescently labeled cells, and unsurprisingly perform poorly on low SNR autofluorescence images. Therefore, new cell segmentation tools are needed for autofluorescence microscopy. Cellpose is a deep learning network that is generalizable across diverse cell microscopy images and automatically segments single cells to improve throughput and reduce inter-human biases. This study aims to validate Cellpose for autofluorescence imaging, specifically from multiphoton intensity images of NAD(P)H. Manually segmented nuclear masks of NAD(P)H images were used to train new Cellpose models. These models were applied to PANC-1 cells treated with metabolic inhibitors and patient-derived cancer organoids (across 9 patients) treated with chemotherapies. These datasets include co-registered fluorescence lifetime imaging microscopy (FLIM) of NAD(P)H and FAD, so fluorescence decay parameters and the optical redox ratio (ORR) were compared between masks generated by the new Cellpose model and manual segmentation. The Dice score between repeated manually segmented masks was significantly lower than that of repeated Cellpose masks (p<0.0001) indicating greater reproducibility between Cellpose masks. There was also a high correlation (R2>0.9) between Cellpose and manually segmented masks for the ORR, mean NAD(P)H lifetime, and mean FAD lifetime across 2D and 3D cell culture treatment conditions. Masks generated from Cellpose and manual segmentation also maintain similar means, variances, and effect sizes between treatments for the ORR and FLIM parameters. Overall, Cellpose provides a fast, reliable, reproducible, and accurate method to segment single cells in autofluorescence microscopy images such that functional changes in cells are accurately captured in both 2D and 3D culture.

Optic Disc Drusen in Pseudoxanthoma Elasticum Are Associated with the Extent of Bruch's Membrane Calcification.

Background/Objectives: To assess the frequency, extent, localization and potential progression of optic disc drusen (ODD) and the correlation with the angioid streak (AS) length and retinal atrophy in patients with pseudoxanthoma elasticum (PXE). Methods: This retrospective study included patient data from a dedicated PXE clinic at the Department of Ophthalmology, University of Bonn, Germany (observation period from February 2008 to July 2023). Two readers evaluated the presence, localization, and the extent of the ODD on fundus autofluorescence (FAF) imaging at baseline and the follow-up assessments. Additionally, we measured the length of the longest AS visible at baseline and follow-up and the area of atrophy at baseline, both on FAF. Results: A total of 150 eyes of 75 PXE patients (median age at baseline 51.8 years, IRQ 46.3; 57.5 years, 49 female) underwent retrospective analysis. At baseline, 23 of 75 patients exhibited ODD in a minimum of one eye, resulting in an ODD prevalence of 30.7% in our cohort of PXE patients. Among these, 14 patients showed monocular and 9 binocular ODD that were localized predominantly nasally (46.9%). During the observational period (mean 97.5 ± 44.7 months), only one patient developed de novo ODD in one eye and one other patient showed a progression in the size of the existing ODD. The group of patients with ODD had significantly longer ASs (median 7020 µm, IQR 4604; 9183, vs. AS length without ODD: median 4404 µm, IQR 3512; 5965, p < 0.001). No association with the size of the atrophy was found at baseline (p = 0.27). Conclusions: This study demonstrates a prevalence of ODD of 30.7%. ODD presence is associated with longer ASs (an indicator of the severity and extent of ocular Bruch's membrane calcification), suggesting that ODD formation is tightly related to ectopic calcification-possibly secondary to calcification of the lamina cribrosa. Prospective studies investigating the impact of ODD (in conjunction with intraocular pressure) on visual function in PXE warrant consideration.

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.