Autofluorescence Imaging Research Articles

Two-photon microendoscope for label-free imaging in stereotactic neurosurgery.

We demonstrate a gradient refractive index (GRIN) microendoscope with an outer diameter of ∼1.2 mm and a length of ∼186 mm that can fit into a stereotactic surgical cannula. Two photon imaging at an excitation wavelength of 900 nm showed a field of view of ∼180 microns and a lateral and axial resolution of 0.86 microns and 9.6 microns respectively. The microendoscope was tested by imaging autofluorescence and second harmonic generation (SHG) in label-free human brain tissue. Furthermore, preliminary image analysis indicates that image classification models can predict if an image is from the subthalamic nucleus or the surrounding tissue using conventional, bench-top two-photon autofluorescence.

Fluorescence microscopy imaging of mitochondrial metabolism in cancer cells.

Mitochondrial metabolism is an important contributor to cancer cell survival and proliferation that coexists with enhanced glycolytic activity. Measuring mitochondrial activity is useful to characterize cancer metabolism patterns, to identify metabolic vulnerabilities and to identify new drug targets. Optical imaging, especially fluorescent microscopy, is one of the most valuable tools for studying mitochondrial bioenergetics because it provides semiquantitative and quantitative readouts as well as spatiotemporal resolution of mitochondrial metabolism. This review aims to acquaint the reader with microscopy imaging techniques currently used to determine mitochondrial membrane potential (ΔΨm), nicotinamide adenine dinucleotide (NADH), ATP and reactive oxygen species (ROS) that are major readouts of mitochondrial metabolism. We describe features, advantages, and limitations of the most used fluorescence imaging modalities: widefield, confocal and multiphoton microscopy, and fluorescent lifetime imaging (FLIM). We also discus relevant aspects of image processing. We briefly describe the role and production of NADH, NADHP, flavins and various ROS including superoxide and hydrogen peroxide and discuss how these parameters can be analyzed by fluorescent microscopy. We also explain the importance, value, and limitations of label-free autofluorescence imaging of NAD(P)H and FAD. Practical hints for the use of fluorescent probes and newly developed sensors for imaging ΔΨm, ATP and ROS are described. Overall, we provide updated information about the use of microscopy to study cancer metabolism that will be of interest to all investigators regardless of their level of expertise in the field.

Fundus autofluorescence imaging using red excitation light

Retinal disease accounts significantly for visual impairment and blindness. An important role in the pathophysiology of retinal disease and aging is attributed to lipofuscin, a complex of fluorescent metabolites. Fundus autofluorescence (AF) imaging allows non-invasive mapping of lipofuscin and is a key technology to diagnose and monitor retinal disease. However, currently used short-wavelength (SW) excitation light has several limitations, including glare and discomfort during image acquisition, reduced image quality in case of lens opacities, limited visualization of the central retina, and potential retinal light toxicity. Here, we establish a novel imaging modality which uses red excitation light (R-AF) and overcomes these drawbacks. R-AF images are high-quality, high-contrast fundus images and image interpretation may build on clinical experience due to similar appearance of pathology as on SW-AF images. Additionally, R-AF images may uncover disease features that previously remained undetected. The R-AF signal increases with higher abundance of lipofuscin and does not depend on photopigment bleaching or on the amount of macular pigment. Improved patient comfort, limited effect of cataract on image quality, and lack of safety concerns qualify R-AF for routine clinical monitoring, e.g. for patients with age-related macular degeneration, Stargardt disease, or for quantitative analysis of AF signal intensity.

MORPHOLOGIC CHARACTERISTICS AND PROGRESSION PATTERNS OF MACULAR NEOVASCULARIZATION-RELATED ATROPHY IN EYES WITH PATHOLOGIC MYOPIA.

To investigate the development and progression patterns of macular neovascularization (MNV)-related atrophies in eyes with pathologic myopia. Twenty-seven eyes of 26 patients with MNV followed from its onset to progression to macular atrophy were studied. A longitudinal series of autofluorescence and optical coherence tomography images were examined for the patterns of MNV-related atrophy. Changes in best-corrected visual acuity were determined for each pattern. The mean age was 67.2 ± 8.7 years. The mean axial length was 29.6 ± 1.5 mm. Three patterns of atrophy were identified: multiple-atrophic pattern, 63% of the eyes had small atrophies occurring at multiple sites around the MNV edge; single-atrophic pattern, 18.5% had atrophies occurring only on one side of the MNV edge; and exudation-related pattern, 18.5% had atrophy occurring within a previous serous exudation or hemorrhagic area and slightly away from the MNV edge. Eyes with atrophies in multiple-atrophic and exudation-related patterns progressed to large macular atrophies involving the central fovea and showed decrease in best-corrected visual acuity during the 3-year follow-up period. Eyes with single-atrophic pattern had a sparing of the fovea and had good recovery of the best-corrected visual acuity. There are three patterns of MNV-related atrophy in eyes with pathologic myopia with different courses of progression.

The value of pre-symptomatic genetic risk assessment for age-related macular degeneration: the Moran AMD Genetic Testing Assessment (MAGENTA) study—a study protocol for a randomized controlled trial

BackgroundAge-related macular degeneration (AMD) is an irreversible blinding eye condition with complex genetic and environmental etiologies. Genetic testing for AMD for previously identified multiple-risk single nucleotide polymorphisms can help determine an individual’s future susceptibility. However, such testing has been discouraged until evidence shows that providing such information to symptomatic or pre-symptomatic individuals will alter their disease course. Therefore, we designed this study to investigate whether knowledge of AMD risk could stimulate the adoption of a healthier lifestyle that could lower the incidence of AMD later in life. We hypothesize that pre-symptomatic individuals informed of a high genetic risk of AMD are more likely to make quantifiable, positive lifestyle changes relative to participants informed of lower genetic risk or randomized to deferred disclosure of genetic testing results.MethodsThe Moran AMD Genetic Testing Assessment (MAGENTA) study is a phase 2, single-center, prospective, double-masked, randomized controlled trial conducted at the John A. Moran Eye Center, University of Utah, Salt Lake City, Utah, USA. Participants are randomized by a 3:1 allocation ratio to immediate and deferred disclosure groups and followed for 12 months. Skin, ocular, and serum carotenoid status, as well as nutritional and social surveys, are assessed at study visits. Skin carotenoid assessment is by resonance Raman spectroscopy and reflectance spectroscopy, ocular carotenoids are measured with Heidelberg Spectralis autofluorescence imaging and fluorescence lifetime imaging ophthalmoscopy (FLIO), and serum carotenoids are quantified using high-performance liquid chromatography. The primary outcome evaluates changes in skin carotenoid status in response to genetic risk disclosure. The secondary outcomes examine changes in ocular and serum carotenoid status in response to genetic risk disclosure. Also, we will correlate AMD genetic risk with baseline ocular and systemic carotenoid status and FLIO.DiscussionMAGENTA will provide much-needed evidence on whether pre-symptomatic testing for AMD risk can lead to quantifiable long-term changes in behavior and lifestyle associated with a lower incidence of AMD later in life. Findings from the MAGENTA trial will facilitate the design of a future larger, longer-term, multicenter phase 3 trial that could feature subgroup analysis, expanded measures of lifestyle modification, and potential active nutritional interventions.Trial registrationClinicalTrials.gov NCT05265624. Registered on March 3, 2022.

Central Serous Chorioretinopathy by Autofluorescence, Enface and SLO-Retromode Imaging.

The aim of our study was to investigate the clinical features of central serous chorioretinopathy (CSC) with autofluorescence (AF), retromode (RM), and enface imaging. This retrospective study was conducted at Fondazione Policlinico Universitario A. Gemelli, IRCCS, Rome (Italy), between September and December 2022. Each patient underwent a complete ophthalmological examination, which included optical coherence tomography (OCT), enface image analysis, AF, and RM imaging. We further evaluated the presence and area of extension of serous retinal detachment and retinal pigment epithelium (RPE) atrophy through AF, RM, and enface imaging. We included 32 eyes from 27 patients (mean age: 52.7 ± 13.3 years). The median AF area was 19.5 mm2 (IQR 6.1-29.3), while the median RM area was 12.3 mm2 (IQR 8.1-30.8), and the median enface area was 9.3 mm2 (IQR 4.8-18.6). RPE atrophy was diagnosed in 26 cases (81.3%) with RM imaging and in 75% of cases with AF. No difference emerged between AF and RM analysis in the detection of central serous detachment in CSC. However, RM imaging showed a high specificity (91.7%) and negative predictive value (84.6%) to detect RPE changes when compared to the AF standard-of-care technique. Thus, RM imaging could be considered an adjunctive imaging method in CSC.

Prognostication in Stargardt Disease Using Fundus Autofluorescence: Improving Patient Care

To explore fundus autofluorescence (FAF) imaging as an alternative to electroretinography as a noninvasive, quick, and readily interpretable method to predict disease progression in Stargardt disease (STGD). Retrospective case series of patients who attended Moorfields Eye Hospital (London, United Kingdom). Patients with STGD who met the following criteria were included: (1) biallelic disease-causing variants in ABCA4, (2) electroretinography testing performed in house with an unequivocal electroretinography group classification, and (3) ultrawidefield (UWF) FAF imaging performed up to 2 years before or after the electroretinography. Patients were divided into 3 electroretinography groups based on retinal function and 3 FAF groups according to the extent of hypoautofluorescence and retinal background appearance. Fundus autofluorescence images of 30° and 55° were reviewed subsequently. Electroretinography and FAF concordance and its association with baseline visual acuity (VA) and genetics. Two hundred thirty-four patients were included in the cohort. One hundred seventy patients (73%) were in electroretinography and FAF groups of the same severity, 33 (14%) were in a milder FAF than electroretinography group, and 31 (13%) were in a more severe FAF than electroretinography group. Children < 10 years of age (n= 23) showed the lowest electroretinography and FAF concordance at 57% (9 of the 10 with discordant electroretinography and FAF showed milder FAF than electroretinography), and adults with adult onset showed the highest (80%). In 97% and 98% of patients, 30° and 55° FAF imaging, respectively, matched with the group defined by UWF FAF. We demonstrated that FAF imaging is an effective method to determine the extent of retinal involvement and thereby inform prognostication by comparing FAF with the current gold standard of electroretinography. In 80% of patients in our large molecularly proven cohort, we were able to predict if the disease was confined to the macula or also affected the peripheral retina. Children assessed at a young age, with at least 1 null variant, early disease onset, poor initial VA, or a combination thereof may have wider retinal involvement than predicted by FAF alone, may progress to a more severe FAF phenotype over time, or both. Proprietary or commercial disclosure may be found in the Footnotes and Disclosures at the end of this article.

Emerging clinical applications in oncology for non-invasive multi- and hyperspectral imaging of cell and tissue autofluorescence.

Hyperspectral and multispectral imaging of cell and tissue autofluorescence is an emerging technology in which fluorescence imaging is applied to biological materials across multiple spectral channels. This produces a stack of images where each matched pixel contains information about the sample's spectral properties at that location. This allows precise collection of molecularly specific data from a broad range of native fluorophores. Importantly, complex information, directly reflective of biological status, is collected without staining and tissues can be characterised in situ, without biopsy. For oncology, this can spare the collection of biopsies from sensitive regions and enable accurate tumour mapping. For in vivo tumour analysis, the greatest focus has been on oral cancer, whereas for ex vivo assessment head-and-neck cancers along with colon cancer have been the most studied, followed by oral and eye cancer. This review details the scope and progress of research undertaken towards clinical translation in oncology.

Application of Nanosecond Pulsed Electric Field and Autofluorescence Lifetime Microscopy of FAD in Lung Cells.

Exposure of nanosecond pulsed electric fields (nsPEFs) to live cells is an increasing research interest in biology and medicine. Despite extensive studies, a question still remains as to how effects of application of nsPEF on intracellular functions are different between cancerous cells and normal cells and how the difference can be detected. Herein, we have presented an approach of autofluorescence lifetime (AFL) microscopy of flavin adenine dinucleotide (FAD) to detect effects of application of nsPEF having 50 ns of a pulse width, nsPEF(50), on intracellular function in lung cancerous cells, A549 and H661, which show nsPEF(50)-induced apoptosis, and normal cells, MRC-5, in which the field effect is less or not induced. Then, the application of nsPEF(50) is shown to increase the lifetime of FAD autofluorescence in lung cancerous cells, whereas the electric field effects on the autofluorescence of FAD was not significant in normal healthy cells, which indicates that the lifetime measurements of FAD autofluorescence are applicable to detect the field-induced change in intracellular functions. Lifetime and intensity microscopic images of FAD autofluorescence in these lung cells were also acquired after exposure to the apoptosis-inducer staurosporine (STS). Then, it was found that the AFL of FAD became longer after exposure not only in the cancerous cells but also in the normal cells. These results indicate that nsPEF(50) applied to lung cells induced apoptotic cell death only in lung cancerous cells (H661 and A549) but not in lung normal cells (MRC-5), whereas STS induced apoptotic cell death both in lung cancerous cells and in lung normal cells. The lifetime microscopy of FAD autofluorescence is suggested to be very useful as a sensitive detection method of nsPEF-induced apoptotic cell death.

Short-wavelength excitation two-photon intravital microscopy of endogenous fluorophores.

The noninvasive two-photon excitation autofluorescence imaging of cellular and subcellular structure and dynamics in live tissue could provide critical in vivo information for biomedical studies. However, the two-photon microscopy of short-wavelength endogenous fluorophores, such as tryptophan and hemoglobin, is extremely limited due to the lack of suitable imaging techniques. In this study, we developed a short-wavelength excitation time- and spectrum-resolved two-photon microscopy system. A 520-nm femtosecond fiber laser was used as the excitation source, and a time-correlated single-photon counting module connected with a spectrograph was used to provide time- and spectrum-resolved detection capability. The system was specially designed for measuring ultraviolet and violet-blue fluorescence signals and thus was very suitable for imaging short-wavelength endogenous fluorophores. Using the system, we systematically compared the fluorescence spectra and fluorescence lifetimes of short-wavelength endogenous fluorophores, including the fluorescent molecules tyrosine, tryptophan, serotonin (5-HT), niacin (vitamin B3), pyridoxine (vitamin B6), and NADH and the protein group (keratin, elastin, and hemoglobin). Then, high-resolution three-dimensional (3D) label-free imaging of different biological tissues, including rat esophageal tissue, rat oral cheek tissue, and mouse ear skin, was performed in vivo or ex vivo. Finally, we conducted time-lapse imaging of leukocyte migration in the lipopolysaccharide injection immunization model and a mechanical trauma immunization model. The results indicate that the system can specifically characterize short-wavelength endogenous fluorophores and provide noninvasive label-free 3D visualization of fine structures and dynamics in biological systems. The microscopy system developed here can empower more flexible imaging of endogenous fluorophores and provide a novel method for the 3D monitoring of biological events in their native environment.

Advanced diagnostic endoscopy in the upper gastrointestinal tract: Review of the Japan Gastroenterological Endoscopy Society core sessions.

We held four upper gastrointestinal tract advanced diagnostic endoscopy sessions from the 89th to the 92nd Congress of the Japan Gastroenterological Endoscopy Society. The most common region addressed was the stomach in 25 presentations, followed by the esophagus in 23, duodenum in five, and other in one. Looking at techniques discussed, the most common image enhancement method discussed was narrowband imaging in 29 presentations, blue laser imaging, and linked color imaging (LCI) in 10 each, dual red imaging in three, and autofluorescence imaging in one. Furthermore, there were presentations of new techniques such as M-Chromo-LCI and acetic acid-indigo carmine mixture LCI. There were also six presentations regarding probe-based confocal laser endomicroscopy, and one of endocytoscopy techniques. We also saw presentations of images of gastric subepithelial tumors within the submucosa, 3D endoscopy, the development of computer-aided detection systems for early cancers, and fluorescent imaging.

Scale-adaptive model for detection and grading of age-related macular degeneration from color retinal fundus images

Age-related Macular Degeneration (AMD), a retinal disease that affects the macula, can be caused by aging abnormalities in number of different cells and tissues in the retina, retinal pigment epithelium, and choroid, leading to vision loss. An advanced form of AMD, called exudative or wet AMD, is characterized by the ingrowth of abnormal blood vessels beneath or into the macula itself. The diagnosis is confirmed by either fundus auto-fluorescence imaging or optical coherence tomography (OCT) supplemented by fluorescein angiography or OCT angiography without dye. Fluorescein angiography, the gold standard diagnostic procedure for AMD, involves invasive injections of fluorescent dye to highlight retinal vasculature. Meanwhile, patients can be exposed to life-threatening allergic reactions and other risks. This study proposes a scale-adaptive auto-encoder-based model integrated with a deep learning model that can detect AMD early by automatically analyzing the texture patterns in color fundus imaging and correlating them to the vasculature activity in the retina. Moreover, the proposed model can automatically distinguish between AMD grades assisting in early diagnosis and thus allowing for earlier treatment of the patient’s condition, slowing the disease and minimizing its severity. Our model features two main blocks, the first is an auto-encoder-based network for scale adaption, and the second is a convolutional neural network (CNN) classification network. Based on a conducted set of experiments, the proposed model achieves higher diagnostic accuracy compared to other models with accuracy, sensitivity, and specificity that reach 96.2%, 96.2%, and 99%, respectively.

Clinical Characteristics and Progression of Geographic Atrophy in a Japanese Population

To elucidate the clinical characteristics and progression rate of geographic atrophy (GA) associated with age-related macular degeneration (AMD) in a Japanese population. Retrospective, multicenter, observational study. A total of 173 eyes from 173 patients from six university hospitals in Japan were included. Of 173 study eyes, 101 eyes from 101 patients were included in the follow-up group. All patients were Japanese, aged ≥ 50 years, and had definite GA associated with AMD in at least one eye. The GA area was measured semi-automatically using fundus autofluorescence (FAF) images. In the follow-up group followed for more than six months with FAF images, the GA progression rate was calculated by two methods: mm2/year and mm/year using the square-root transformation (SQRT) strategy. Simple and multiple linear regression analyses were used to identify the baseline factors associated with the GA progression rate. Clinical characteristics of GA and the GA progression rate. The mean age was 76.8 ± 8.8 years and 109 (63.0%) were males. Sixty-two (35.8%) patients had bilateral GA. The mean GA area was 3.06 ± 4.00 mm2 (1.44 ± 1.00 mm [SQRT]). Thirty-eight eyes (22.0%) were classified as having pachychoroid GA. Drusen and reticular pseudodrusen were detected in 115 (66.5%) and 73 eyes (42.2%), respectively. The mean subfoveal choroidal thickness was 194.7 ± 105.5 μm. In the follow-up group (follow-up period: 46.2 ± 28.9 months), the mean GA progression rate was 1.01 ± 1.09 mm2/year (0.23 ± 0.18 mm/year [SQRT]). In the multivariable analysis, baseline GA area [SQRT] (P=0.002) and the presence of reticular pseudodrusen (P<0.001) were significantly associated with a greater GA progression rate [SQRT]. Certain clinical characteristics of GA in Asian populations may differ from those in Caucasians. Asian patients with GA showed male-dominance and relatively thicker choroid than Caucasian patients. There was a group with GA without drusen but with features of pachychoroid. The GA progression rate in this Asian population was relatively lower than that in Caucasians. Large GA and reticular pseudodrusen were associated with a greater GA progression rate.

A molecular rotor FLIM probe reveals dynamic coupling between mitochondrial inner membrane fluidity and cellular respiration

The inner mitochondrial membrane (IMM), housing components of the electron transport chain (ETC), is the site for respiration. The ETC relies on mobile carriers; therefore, it has long been argued that the fluidity of the densely packed IMM can potentially influence ETC flux and cell physiology. However, it is unclear if cells temporally modulate IMM fluidity upon metabolic or other stimulation. Using a photostable, red-shifted, cell-permeable molecular-rotor, Mitorotor-1, we present a multiplexed approach for quantitatively mapping IMM fluidity in living cells. This reveals IMM fluidity to be linked to cellular-respiration and responsive to stimuli. Multiple approaches combining invitro experiments and live-cell fluorescence (FLIM) lifetime imaging microscopy (FLIM) show Mitorotor-1 to robustly report IMM 'microviscosity'/fluidity through changes in molecular free volume. Interestingly, external osmotic stimuli cause controlled swelling/compaction of mitochondria, thereby revealing a graded Mitorotor-1 response to IMM microviscosity. Lateral diffusion measurements of IMM correlate with microviscosity reported via Mitorotor-1 FLIM-lifetime, showing convergence of independent approaches for measuring IMM local-order. Mitorotor-1 FLIM reveals mitochondrial heterogeneity in IMM fluidity; between-and-within cells and across single mitochondrion. Multiplexed FLIM lifetime imaging of Mitorotor-1 and NADH autofluorescence reveals that IMM fluidity positively correlates with respiration, across individual cells. Remarkably, we find that stimulating respiration, through nutrient deprivation or chemically, also leads to increase in IMM fluidity. These data suggest that modulating IMM fluidity supports enhanced respiratory flux. Our study presents a robust method for measuring IMM fluidity and suggests a dynamic regulatory paradigm of modulating IMM local order on changing metabolic demand.

Macular Telangiectasia Type 2: A Classification System Using MultiModal Imaging MacTel Project Report Number 10.

To develop a severity classification for macular telangiectasia type 2 (MacTel) disease using multimodal imaging. An algorithm was used on data from a prospective natural history study of MacTel for classification development. A total of 1733 participants enrolled in an international natural history study of MacTel. The Classification and Regression Trees (CART), a predictive nonparametric algorithm used in machine learning, analyzed the features of the multimodal imaging important for the development of a classification, including reading center gradings of the following digital images: stereoscopic color and red-free fundus photographs, fluorescein angiographic images, fundus autofluorescence images, and spectral-domain (SD)-OCT images. Regression models that used least square method created a decision tree using features of the ocular images into different categories of disease severity. The primary target of interest for the algorithm development by CART was the change in best-corrected visual acuity (BCVA) at baseline for the right and left eyes. These analyses using the algorithm were repeated for the BCVA obtained at the last study visit of the natural history study for the right and left eyes. The CART analyses demonstrated 3 important features from the multimodal imaging for the classification: OCT hyper-reflectivity, pigment, and ellipsoid zone loss. By combining these 3 features (as absent, present, noncentral involvement, and central involvement of the macula), a 7-step scale was created, ranging from excellent to poor visual acuity. At grade 0, 3 features are not present. At the most severe grade, pigment and exudative neovascularization are present. To further validate the classification, using the Generalized Estimating Equation regression models, analyses for the annual relative risk of progression over a period of 5 years for vision loss and for progression along the scale were performed. This analysis using the data from current imaging modalities in participants followed in the MacTel natural history study informed a classification for MacTel disease severity featuring variables from SD-OCT. This classification is designed to provide better communications to other clinicians, researchers, and patients. Proprietary or commercial disclosure may be found after the references.

A Workflow to Quantitatively Determine Age-Related Macular Degeneration Lesion-Specific Variations in Fundus Autofluorescence.

Fundus autofluorescence (FAF) imaging allows the noninvasive mapping of intrinsic fluorophores of the ocular fundus, particularly the retinal pigment epithelium (RPE), now quantifiable with the advent of confocal scanning laser ophthalmoscopy-based quantitative autofluorescence (QAF). QAF has been shown to be generally decreased at the posterior pole in age-related macular degeneration (AMD). The relationship between QAF and various AMD lesions (drusen, subretinal drusenoid deposits) is still unclear. This paper describes a workflow to determine lesion-specific QAF in AMD. A multimodal in vivo imaging approach is used, including but not limited to spectral domain optical coherence tomography (SD-OCT) macular volume scanning and QAF. Using customized FIJI plug-ins, the corresponding QAF image is aligned with the near-infrared image from the SD-OCT scan (characteristic landmarks; i.e., vessel bifurcations). The foveola and the edge of the optic nerve head are marked in the OCT images (and transferred to the registered QAF image) for accurate positioning of the analysis grids. AMD-specific lesions can then be marked on individual OCT BScans or the QAF image itself. Normative QAF maps are created to account for the varying mean and standard deviation of QAF values throughout the fundus (QAF images from a representative AMD group were averaged to build normative standard retinal QAF AMD maps). The plug-ins record the X and Y coordinates, z-score (a numerical measurement that describes the QAF value in relation to the mean of AF maps in terms of standard deviation from the mean), mean intensity value, standard deviation, and number of pixels marked. The tools also determine z-scores from the border zone of marked lesions.This workflow and the analysis tools will improve the understanding of the pathophysiology and clinical AF image interpretation in AMD.

Exudative age-related macular degeneration lesion components predicting microperimetric retinal sensitivity during anti-vascular endothelial growth factor treatment.

To analyse the effect of exudative age-related macular degeneration (eAMD) lesion components on retinal sensitivity during anti-vascular endothelial growth factor treatment. Visual acuity, fluorescein and indocyanine green (ICG) angiographies, autofluorescence images, microperimetries and optical coherence tomographies (OCTs) of 24 eyes of 24 patients were prospectively analysed in a 2-year study of pro-re-nata bevacizumab treatment for eAMD. Microperimetries were aligned with the OCTs, angiographies and autofluorescence images. Thicknesses of the neuroretina, pigment epithelial (RPE) elevation, neuroepithelial detachment (NED), subretinal tissue (SRT) and cystic intraretinal fluid were measured under each stimulus site, and areas of type 1 and type 2 macular neovascularizations (MNVs), ICG plaque, haemorrhage and RPE atrophy were identified. The effects and predictive values of lesion components on retinal sensitivity were analysed with multivariate mixed linear models for repeated measurements. The overall microperimetric retinal sensitivity increased during the first year (from 10.1 dB at baseline to 11.9 dB at 1 year; p = 0.021, Wilcoxon signed ranks), but remained the same during the second year (11.5 dB, p = 0.301). The baseline lesion components most strongly predicting deteriorated sensitivity at 1 year were RPE atrophy, the area of Type 2 MNV, intraretinal cysts, haemorrhage, Type 1 MNV and retinal thickening >350 μm. NED and RPE elevation had only small effects. At 2 years, the predictive values of the baseline lesion components remained quite unchanged. The most powerful predictors of retinal sensitivity loss during 2 years of treatment were RPE atrophy, areas of haemorrhage, the area of MNVs, intraretinal cysts and SRT. RPE elevation and NED had lesser effects.

Retromode Scanning Laser Ophthalmoscopy for Choroidal Nevi: A Preliminary Study.

The purpose of the present study was to document pathological findings on retromode imaging in choroidal nevi and evaluate its diagnostic validity, using the confocal scanning laser ophthalmoscope Nidek Mirante (cSLO). A total of 41 choroidal nevi from 41 patients were included. All patients underwent multicolor fundus (mCF), infrared reflectance (IR), green fundus autofluorescence (FAF), dark-field (DF) and retromode (RM) imaging and optical coherence tomography (OCT) scans. We investigated retromode images to evaluate choroidal nevus features by comparing the results with those of mCF, IR, FAF, DF and OCT. In 100% of available images, retromode scanning laser ophthalmoscopy was able to detect choroidal nevi with a characteristic "hypo-retro-reflective" pattern, even the cases not visible on mCF, IR and FAF images. It also made it possible to delineate the margins of lesions with the highest rate of sharpness and accuracy among the imaging modalities examined. These findings seem to demonstrate how RM-SLO is an innovative diagnostic tool to detect and follow up choroidal nevi in a fast, reliable and non-invasive way.

Deep phenotypic characterization of the retinal dystrophy in patients with RNU4ATAC-associated Roifman syndrome.

To characterize the retinal phenotype in RNU4ATAC-associated Roifman syndrome. Ten patients (including 8 males) with molecularly confirmed Roifman syndrome underwent detailed ophthalmologic evaluation including fundus imaging, fundus autofluorescence (FAF) imaging, spectral-domain optical coherence tomography (SD-OCT), and electroretinography (ERG). Six patients had follow-up eye exams. All patients also underwent comprehensive examination for features of extra-retinal Roifman syndrome. All patients had biallelic RNU4ATAC variants. Nyctalopia was common (7/10). Visual acuity at presentation ranged from 20/20 to 20/200 (Age Range: 5-41 years). Retinal exam revealed features of generalized retinopathy with mid-peripheral pigment epithelial changes. A para or peri-foveal ring of hyper-autofluorescence was the commonest FAF abnormality noted (6/8). The SD-OCT demonstrated relative preservation of the foveal ellipsoid zone in six cases; associated features included cystoid changes (5/10) and posterior staphyloma (3/10). The ERG was abnormal in all patients; nine showed generalized rod-cone dystrophy, whilst one patient with sectoral retinal involvement only had isolated rod dystrophy (20 years old). On follow-up examination (Mean duration: 8.16 years), progressive loss of visual acuity (2/6), mid-peripheral retinal atrophy (3/6) or shortening of ellipsoid zone width (1/6) were observed. This study has characterized the retinal phenotype in RNU4ATAC-associated Roifman syndrome. Retinal involvement is universal, early-onset, and overall, the retinal and FAF features are consistent with rod-cone degeneration that is slowly progressive over time. The sub-foveal retinal ultrastructure is relatively preserved in majority of patients. Phenotypic variability independent of age exists, and more study of allelic- and sex-based determinants of disease severity are necessary.

Multimodal microscopic imaging with deep learning for highly effective diagnosis of breast cancer

Accurate and rapid diagnosis of breast cancer is particularly important since it becomes the primary malignant tumor threatening women's health. Although pathological diagnosis with H&E staining section is the gold standard in tumor diagnosis, it is hard to meet quick intraoperative inspection because of its complex procedures and hysteresis. This study adopts multimodal microscopic imaging technology (bright-field imaging, auto-fluorescence imaging and orthogonal polarization imaging) combined with deep learning to achieve rapid intelligent diagnosis of breast cancer by getting the rich information of tissue morphology, content and structure of collagen in tissue slices. By using both fusion classification models of multimodal images at pixel level and decision level, the AUC score and accuracy is 0.9366 and 89.01% for the pixel fusion classification, as well as 0.9421 and 87.53% for the decision fusion classification, respectively. The results suggest that the multimodal microscopic imaging technique proposed in this study has unique advantages in accuracy, speed and feasibility in clinic for breast cancer diagnosis, and has strong clinical potential and application prospects when combined with deep learning.