Autofluorescence Imaging Research Articles

Medical femtosecond laser

Medical femtosecond laser devices are used in dermatology for non-linear high-resolution imaging to obtain non-invasive and label-free optical skin biopsies (multiphoton tomography) as well as in ophthalmology for refractive corneal surgery and cataract surgery. Applications of commercial certified multiphoton tomographs include early detection of skin cancer within minutes by two-photon autofluorescence imaging of coenzymes and melanin and second harmonic imaging of collagen as well as by testing the efficacy of pharmaceutical and cosmetical products. Goals are (i) to reduce the number of physically taken human skin biopsies in hospitals and research institutions, (ii) to optimize personalized medicine, and (iii) to reduce animal studies in pharmacy. Current diagnostic tools in dermatology include surface microscopy with a dermatoscope and ultrasound but have poor resolution. Optical coherence tomography and confocal reflectance microscopy have better resolution but provide limited information based on changes of the intratissue refractive index. Multiphoton tomography provides the best resolution of all clinical imaging methods and offer functional imaging such as optical metabolic imaging based on autofluorescence lifetime imaging. Goals of femtosecond laser eye treatment are (i) the replacement of mechanical microkeratomes for corneal flap generation, (ii) the replacement of the UV nanosecond excimer laser for stroma removal, and (iii) to replace, in part, the scalpel in the surgery of cataracts and other eye diseases. So far, millions of eye treatments have been conducted around the world. The major disadvantage of current certified medical femtosecond laser devices is the high price compared with the standard mechanical and optical medical devices.

Early onset of age-related changes in the retina of cystine/glutamate antiporter knockout mice

To determine the role of the cystine/glutamate antiporter on retinal structure and function, retinas of C57Bl/6J wild-type and xCT knockout mice, lacking the xCT subunit of the cystine/glutamate antiporter were examined from 6 weeks to 12 months of age. Fundoscopy, optical coherence tomography (OCT), and whole mount retinal autofluorescence imaging were used to visualise age-related retinal spots. Glial fibrillary acidic protein (GFAP) immunolabelling was used to assess retinal stress. Retinal function was evaluated using full-field and focal electroretinograms. Examinations revealed retinal spots in both wild-type and xCT knockout mice with the number of spots greater at 9 months in the knockout compared to wild-type. OCT confirmed these discrete spots were located at the retinal pigment epithelium (RPE)–photoreceptor junction and did not label with drusen markers. Whole mount lambda scans of the 9 month xCT knockout retinas revealed that the photoreceptor autofluorescence matched the spots, suggesting these spots were retinal debris. GFAP labelling was increased in knockout retinas compared to wild-type indicative of retinal stress, and the discrete spots were associated with migration of microglia/macrophages to the RPE-retina intersection. OCT revealed that the superior retina was thinner at 9 months in knockout compared to wild-type mice due to changes to the outer nuclear and photoreceptor layers. While global retinal function was not affected by loss of xCT, focal changes in retinal function were detected in areas where spots were present. Tother these results suggest that the xCT KO mice exhibit features of accelerated ageing and suggests that this mouse model may be useful for studying the underlying cellular pathways in retinal ageing.

Fluorescence intensity and lifetime imaging of lipofuscin-like autofluorescence for label-free predicting clinical drug response in cancer

Conventional techniques for in vitro cancer drug screening require labor-intensive formalin fixation, paraffin embedding, and dye staining of tumor tissues at fixed endpoints. This way of assessment discards the valuable pharmacodynamic information in live cells over time. Here, we found endogenous lipofuscin-like autofluorescence acutely accumulated in the cell death process. Its unique red autofluorescence could report the apoptosis without labeling and continuously monitor the treatment responses in 3D tumor-culture models. Lifetime imaging of lipofuscin-like red autofluorescence could further distinguish necrosis from apoptosis of cells. Moreover, this endogenous fluorescent marker could visualize the apoptosis in live zebrafish embryos during development. Overall, this study validates that lipofuscin-like autofluorophore is a generic cell death marker. Its characteristic autofluorescence could label-free predict the efficacy of anti-cancer drugs in organoids or animal models.

Autofluorescence Image-Guided Endoscopy in the Management of Upper Aerodigestive Tract Tumors.

At this juncture, autofluorescence and narrow-band imaging have resurfaced in the medicine arena in parallel with current technology advancement. The emergence of newly developed optical instrumentation in addition to the discovery of new fluorescence biomolecules have contributed to a refined management of diseases and tumors, especially in the management of upper aerodigestive tract tumors. The advancement in multispectral imaging and micro-endoscopy has also escalated the trends further in the setting of the management of this tumor, in order to gain not only the best treatment outcomes but also facilitate early tumor diagnosis. This includes the usage of autofluorescence endoscopy for screening, diagnosis and treatment of this tumor. This is crucial, as microtumoral deposit at the periphery of the gross tumor can be only assessed via an enhanced endoscopy and even more precisely with autofluorescence endoscopic techniques. Overall, with this new technique, optimum management can be achieved for these patients. Hence, the treatment outcomes can be improved and patients are able to attain better prognosis and survival.

PRPH2-Associated Retinopathy: Novel Variants and Genotype–Phenotype Correlations

A broad spectrum of autosomal-dominant inherited retinal diseases (IRDs), ranging from mild macular pattern dystrophy to severe cone-rod degeneration, is associated with PRPH2 variants (peripherinopathies). We present detailed clinical and molecular characterization of patients affected by peripherinopathies, aiming to expand the mutational spectrum, and propose novel genotype-phenotype correlations. Observational, retrospective case series. Patients with an IRD related to a molecularly proven PRPH2 variant. Data from ophthalmic examinations and retinal imaging were collected for each follow-up visit. The standard imaging protocol included OCT, blue-light autofluorescence, near-infrared autofluorescence, and ultra-widefield fundus imaging. Genetic analysis was performed with a genomic approach by next-generation sequencing. Results of ophthalmic examination, retinal imaging, and molecular genetic analysis. Overall, a total of 19 patients with an IRD and a (likely) pathogenic PRPH2 variant were identified. Their age at presentation had a median of 48 years, whereas the symptomatic disease onset was in their 30s or 40s in 74% of cases. The median follow-up time was 4 years. Clinically, 6 patients were diagnosed with cone-rod dystrophy and 13 with pattern dystrophy. Among the 13 PRPH2 pathogenic variants identified in our cohort, 7 were missense, 3 nonsense, 2 frame shifting, and 1 splice site. Missense variants in the D2 loop were associated with cone-rod dystrophies and poor visual prognosis, whereas predicted loss-of-function alleles with pattern dystrophies and retention of a good visual function into adulthood. Overall, the following 7 variants were novel and never associated to a clinical phenotype: c.68delT, c.290G>A, c.413T>G, c.642C>G, c.702_706dupCAGTT, c.771_772delinsGA, and c.850C>G. Here, we report the findings of a retrospective case series that provided a detailed clinical and molecular characterization of 19 patients harboring 13 different PRPH2 pathogenic variants, 7 of which were previously unreported, expanding the mutational spectrum of the PRPH2 gene. Loss-of-function variants might be preferentially associated with mild-pattern dystrophies, whereas missense dominant-negative variants might be preferentially associated with severely blinding cone-rod degenerations. Further studies are needed to better define the pathogenetic mechanisms and the functional effects of most variants to allow the development of successful gene therapy. Proprietary or commercial disclosure may be found after the references.

A novel compound heterozygous BEST1 gene mutation in two siblings causing autosomal recessive bestrophinopathy

PurposeTo describe the clinical features, imaging characteristics, and genetic test results associated with a novel compound heterozygous mutation of the BEST1 gene in two siblings with autosomal recessive bestrophinopathy.MethodsTwo siblings underwent a complete ophthalmic examination, including dilated fundus examination, fundus photography, fundus autofluorescence imaging, spectral-domain optical coherence tomography, fluorescein angiography, electroretinography, and electrooculography. A clinical diagnosis of autosomal recessive bestrophinopathy was established based on ocular examination and multimodal retinal imaging. Subsequently, clinical exome sequencing consisting of a panel of 6670 genes was carried out to confirm the diagnosis and assess genetic alterations in the protein-coding region of the genome of the patients. The identified mutations were tested in the two affected siblings and one of their parents.ResultsTwo siblings (a 17-year-old female and a 15-year-old male) presented with reduced visual acuity and bilaterally symmetrical subretinal deposits of hyperautofluorescent materials in the posterior pole, which showed staining in the late phase of fluorescein angiogram. Spectral-domain optical coherence tomography demonstrated hyperreflective subretinal deposits and subretinal fluid accumulation. Both patients shared two mutations in the protein-coding region of the BEST1 gene, c.103G > A, p.(Glu35Lys) and c.313C > A, p.(Arg105Ser) (a novel disease-causing mutation). Sanger sequencing confirmed that the unaffected mother of the proband was carrying p.(Glu35Lys) variant in a heterozygous state.ConclusionsWe have identified and described the phenotype of a novel disease-causing mutation NM_004183.4:c.313C > A, p.(Arg105Ser) in a heterozygous state along with a previously reported mutation NM_004183.4:c.103G > A, p.(Glu35Lys) of the BEST1 gene in two related patients with autosomal recessive bestrophinopathy.

Autofluorescence Imaging to Evaluate Retinal Disease Progression in Rodent.

Fundus autofluorescence (FAF) imaging is a noninvasive retinal imaging methodology that allows mapping of lipofuscin distribution in the retinal pigment epithelium cell (RPE). Excessive accumulation of lipofuscin granules in the lysosomal compartment of RPE cells represents a common downstream pathogenetic pathway in various hereditary and complex retinal diseases, including age-related macular degeneration. The clinical applications of FAF coupled with its ease of use, and the noninvasive nature of characterizing retinal diseases, are increasingly valuable to the field of ophthalmology and in assessing the progression of retinitis pigmentosa (RP). Quantitative AF (qAF) enhances the understanding of retinal disease processes, serves as a diagnostic aid, and allows for the monitoring of the effects of therapeutic interventions. This chapter introduces basic principles of FAF and general protocols of FAF evaluating retinal disease progression in rodents.

Reticular Pseudodrusen Status, ARMS2/HTRA1 Genotype, and Geographic Atrophy Enlargement: Age-Related Eye Disease Study 2 Report 32

To determine whether reticular pseudodrusen (RPD) status, ARMS2/HTRA1 genotype, or both are associated with altered geographic atrophy (GA) enlargement rate and to analyze potential mediation of genetic effects by RPD status. Post hoc analysis of an Age-Related Eye Disease Study 2 cohort. Eyes with GA: n = 771 from 563 participants. Geographic atrophy area was measured from fundus photographs at annual visits. Reticular pseudodrusen presence was graded from fundus autofluorescence images. Mixed-model regression of square root of GA area was performed by RPD status, ARMS2 genotype, or both. Change in square root of GA area. Geographic atrophy enlargement was significantly faster in eyes with RPD (P < 0.0001): 0.379 mm/year (95% confidence interval [CI], 0.329-0.430 mm/year) versus 0.273 mm/year (95% CI, 0.256-0.289 mm/year). Enlargement was also significantly faster in individuals carrying ARMS2 risk alleles (P < 0.0001): 0.224 mm/year (95% CI, 0.198-0.250 mm/year), 0.287 mm/year (95% CI, 0.263-0.310 mm/year), and 0.307 mm/year (95% CI, 0.273-0.341 mm/year) for 0, 1, and 2, respectively. In mediation analysis, the direct effect of ARMS2 genotype was 0.074 mm/year (95% CI, 0.009-0.139 mm/year), whereas the indirect effect of ARMS2 genotype via RPD status was 0.002 mm/year (95% CI, -0.006 to 0.009 mm/year). In eyes with incident GA, RPD presence was not associated with an altered likelihood of central involvement (P= 0.29) or multifocality (P= 0.16) at incidence. In eyes with incident noncentral GA, RPD presence was associated with faster GA progression to the central macula (P= 0.009): 157 μm/year (95% CI, 126-188 μm/year) versus 111 μm/year (95% CI, 97-125 μm/year). Similar findings were observed in the Age-Related Eye Disease Study. Geographic atrophy enlargement is faster in eyes with RPD and in individuals carrying ARMS2/HTRA1 risk alleles. However, RPD status does not mediate the association between ARMS2/HTRA1 genotype and faster enlargement. Reticular pseudodrusen presence and ARMS2/HTRA1 genotype are relatively independent risk factors, operating by distinct mechanisms. Reticular pseudodrusen presence does not predict central involvement or multifocality at GA incidence but is associated with faster progression toward the central macula. Reticular pseudodrusen status should be considered for improved predictions of enlargement rate. Proprietary or commercial disclosure may be found after the references.

Improvement of hydroxychloroquine retinopathy monitoring by informatic systems

AbstractPurpose: To study the proportion of patients treated with hydroxychloroquine in Health Area VII of Murcia following the screening recommended by the international guidelines and to implement a system to improve the screening efficacy.Methods: We studied the patients in Health Area VII of Murcia, Spain, treated with hydroxychloroquine between January and November 2019 to find out whether patients were correctly screened for retinopathy. Demographic data, medical specialty prescribing hydroxychloroquine, disease, year of onset and examinations performed in our Ophthalmology Service were collected.Results: A total of 202 medical records were reviewed. Baseline ophthalmologic exams were performed in 63.9% of cases: fundus examination (96.9%) and optical coherence tomography (OCT) (33.3%). Follow‐up examination was performed in 61.8% of cases. Follow‐up was complete, with OCT, 10‐2 visual field and autofluorescence image in only 2.4% of patients. The time between revisions was 1.6 years on average. These results strongly deviated from the guidelines. We planned a strategy to improve the screening of these patients. We developed a computer database that allows the voluntary and automatic registration of patients treated with hydroxychloroquine, including contact and demographic data, start date of treatment, the daily dose and retinopathy risk factors. With this database, the patient's appointment in ophthalmology will be provided with the corresponding tests. The examination will be assessed by telemedicine and the time of the next check‐up will be determined according to the results.Conclusions: This plan aims to capture most patients starting the treatment with hydroxychloroquine and to carry out the appropriate tests at each visit with strict follow‐up, thus reducing the care load.

Changes in visual function associated with hydroxychloroquine retinal toxicity

AbstractIntroduction: Non‐infectious inflammatory diseases, such as systemic lupus erythematosus, rheumatoid arthritis, dermatomyositis, antiphospholipid antibody syndrome and others cause significant morbidity and are treated with immunosuppression. Hydroxychloroquine (HCQ) sulfate is used as a second line immunosuppressant and has many advantages over other agents. These include increased life expectancy, reduced risk of neoplasia, reduced risk of diabetes and hypercholesterolaemia. HCQ is safe in pregnancy. After more than 5 years of treatment, HCQ can cause a degenerative maculopathy characterized by loss of parafoveal photoreceptors and RPE cells, leading to a ring‐like visual field defect. Current detection of early retinal toxicity relies on central visual field testing, optical coherence tomography (OCT) scan and fundus autofluorescence. The reason for the parafoveal predilection for retinopathy is not understood. HCQ is concentrated in melanin‐containing cells, and there is increased melanin concentration in the macular RPE cells. A hypothesis is that HCQ concentrated in the macular RPE cells leads to toxicity, with macular pigment protecting the fovea but leaving a ring of potentially vulnerable tissue in the parafoveal region. We were interested to investigate parafoveal function in patients with mild HCQ maculopathy and report the results of detailed macular function assessment of red/green (RG) and yellow / blue (YB) colour vision and rod‐ and cone‐mediated rapid flicker sensitivity.Methods: Two patients with confirmed early hydroxychloroquine maculopathy participated in the study. RG and YB colour thresholds were measured with the Colour Assessment and Diagnosis (CAD) test. Cone and rod‐mediated rapid flicker sensitivity was also measured in both central and paracentral vision using the Flicker‐Plus test. Both patients had taken HCQ for more than 15 years and had paracentral scotomas on 10–2 field test and loss of the parafoveal outer nuclear layer and ellipsoid layer, confirming the presence of maculopathy. To test the hypothesis of parafoveal toxicity risk, 20 short wavelength (BAF) and infrared wavelength (IRAF) autofluorescence images were reviewed. The BAF images are surrogate markers of the macular pigment and the IRAF the melanin concentration. Normalized maps of the difference between BAF and IRAF were calculated to investigate the possibility of a ring of vulnerable tissue.Results: Both patients had normal RG, but abnormal YB colour thresholds. One subject had normal cone flicker thresholds, but the rod flicker thresholds were raised in all quadrants in both eyes. The second subject had significantly elevated rod thresholds and elevated cone flicker thresholds in both eyes. Of note is that the degree of retinopathy in the second patient was greater than in the first. The areas of greatest threshold elevation correlated with the areas of greatest field loss and anatomical disruption on OCT.Conclusion: These new findings show that hydroxychloroquine retinopathy affects preferentially both YB colour vision and rod‐mediated flicker sensitivity. The cone‐mediated flicker sensitivity is also affected in more advanced disease. A model for the anatomical location of the toxicity will be presented, based on the hypothesis outlined above.

Scanning laser ophthalmoscopy – basic optical principles

AbstractScanning Laser Ophthalmoscopy (SLO) produces an ocular fundus image by moving a focused laser beam via scanning mirrors over the retina in a grid pattern and registering the reflected light from each scanned point. In confocal SLO, a pinhole is placed in front of the detector to cut off scattered or defocused light coming from outside the point of interest, which otherwise can blur the image. This results in a focused, high‐contrast image of a single tissue layer located at the focal plane. Tomographic information can be extracted by moving the plane of the pinhole. The use of various wavelengths allows for different applications, such as fluorescein angiography, indocyanine green angiography, and autofluorescence imaging. This lecture will present the basic principles of SLO, as well as discuss notable applications and variants of the technology.

Optical coherence tomography angiography in Bietti crystalline dystrophy.

Bietti crystalline dystrophy (BCD) is a rare autosomal recessive retinal dystrophy caused by pathogenic variants of CYP4V2 gene and characterized by shiny yellow deposits in the retina and progressive atrophy of the retinal pigment epithelium (RPE) and choriocapillaris. The main aim of the present study is to describe the optical coherence tomography angiography (OCTA) characteristics of a patient affected by BCD. A 59-years-old female with genetically confirmed BCD underwent an ophthalmological examination complete of OCTA performed in the atrophic retina, the junctional zone and the apparently normal retina. The area of choriocapillaris (CC) atrophy was compared to the area of RPE atrophy on fundus autofluorescence (FAF) imaging. A severe vessel density (VD) deficit at the level of superficial and deep capillary plexa as well as CC was registered in atrophic areas, which resulted deeper with respect to the junctional area, whereas the apparently preserved retina revealed VD values similar to that of control eyes. The area of RPE atrophy on FAF was larger (55.90 mm2 in right eye and 48.76 mm2 in left eye) than the area of CC atrophy on OCTA imaging (51.86 mm2 and 42.44 mm2 respectively in right and left eye). Our findings suggest that VD impairment of retinal plexa and CC follows the degeneration of RPE as demonstrated by the greater size of the area of RPE atrophy compared to CC atrophy. Further investigations based on OCTA imaging are necessary to enhance our knowledge of this rare disease.

Geographic atrophy in AMD ‐ prognostic factors based on long‐term follow‐up

AbstractPurpose: So far, the natural course of geographic atrophy (GA) progression in age‐related macular degeneration (AMD) has primarily been investigated by colour fundus photography (CF) and fundus autofluorescence (FAF) imaging. The aim of this long‐term retrospective, observational clinical study was to show the enlargement rate (ER) of GA, defined as complete retinal pigment epithelium and outer retinal atrophy (cRORA) and to find predictors of progression.Methods: All patients available in the database of Vista Augenklinik Binningen, Switzerland, with follow‐up of at least 24 months and existing GA in at least one eye, regardless of neovascular AMD (nAMD) being present initially or in the course of disease, were included. SD‐OCT and FAF evaluations were performed according to a standardized protocol. The cRORA area ER (mm2/year), the cRORA square root area ER (mm/yr), the FAF GA area and the condition of the outer retina (inner−/outer segment (IS/OS) and external limiting membrane (ELM) disruption scores) were determined.Results: 204 eyes of 129 patients (64% females) were included. Mean follow‐up time was 4.2 ± 2.2 (range 2–10) years. 109 of 204 (53.4%) eyes were classified as wet AMD (neovascular aspect being present initially or in the course of disease), 95 of 204 (46.6%) eyes were classified as exclusively dry AMD. The primary lesion was unifocal in 146 (72%) eyes and multifocal in 58 (28%) eyes. A strong correlation was observed between the area of cRORA (SD‐OCT) and the FAF GA area (r = 0.924; p &lt; 0.001). Mean ER was 1.44 ± 1.2 mm2/year and mean square root ER 0.29 ± 0.19 mm/year. There was no significant difference in mean ER between eyes without (dry AMD) and with intravitreal anti‐VEGF injections (nAMD) (0.30 ± 0.19 mm/year vs. 0.28 ± 0.20 mm/year; p = 0.466). Eyes with a multifocal atrophy pattern at baseline (n = 58) had a significantly higher mean ER compared to eyes with a unifocal pattern (0.34 ± 0.19 mm/year vs. 0.27 ± 1.19 mm/year; p = 0.008). There were moderate significant correlations between ELM and IS/OS disruption scores and visual acuity at baseline, 5 and 7 years (all r values ca. −0.5; p &lt; 0.001).Conclusions: SD‐OCT evaluated cRORA area might serve as a GA parameter comparable to traditional FAF measurement. The dispersion pattern might be a predictor of ER, whereas anti‐VEGF treatment seems not to be associated with ER.

Interactive effect of 24-epibrassinolide and plant growth promoting rhizobacteria inoculation restores photosynthetic attributes in Brassica juncea L. under chlorpyrifos toxicity

Chlorpyrifos (CP) is a commonly used organophosphorous pesticide that is frequently utilised in the agricultural industry because of its great efficiency and inexpensive cost. The focus of the present study was to assess the impact of CP toxicity on Brassica juncea L. and to unravel the ameliorative potential of phytohormone, 24-epibrassinolide (EBL) mediated plant-microbe (Pseudomonas aeruginosa (B1), Burkholderia gladioli (B2)) interaction in B. juncea L. The maximum significant increment in the total chlorophyll, carotenoids, xanthophyll, anthocyanin and flavonoid content with EBL and B2 treatment in CP stressed B. juncea seedlings on spectrophotometric analysis were observed. Autofluorescence imaging of photosynthetic pigments i.e. chlorophyll, carotenoids, and total phenols with confocal microscopy showed maximum fluorescence with EBL and B2. Furthermore, when compared to CP stressed seedlings, scanning electron microscopy (SEM) study of the abaxial surface of leaves revealed a recovery in stomatal opening. The supplementation of EBL and PGPR (plant growth promoting rhizobacteria) improved the level of psb A (D1 subunit PSII) and psb B (CP 47 subunit of PSII) genes expression. The expression analysis of chalcone synthase (CHS), Phenylalanine ammonialyase (PAL), Phyotene synthase (PSY) with RT-PCR system showed up-regulation in the expression when supplemented with EBL and PGPR. As a result, the current study suggests that EBL and PGPR together, can reduce CP-induced toxicity in B. juncea seedlings and recovering the seedling biomass.

Autofluorescence imaging for diagnosis and follow‐up of serpiginous choroiditis

AbstractPurpose: To report by multimodality imaging the follow‐up of a patient with posterior uveitis presenting serpiginous choroiditis (SC). To highlight the importance of autofluorescence in the follow‐up of patients with atrophic scarring due to CS.Methods: We present an observational case of SC documented photographically. Clinical data were gathered retrospectively from several visits. The data included corrected visual acuity (BCVA), fundus examination, optical coherence tomography (OCT) and autofluorescence (AF).Results: Periodic revision of a 49‐year‐old female being followed up for SC secondary to tuberculosis. Mantoux test was positive in 2010 and she was treated correctly. Twelve years later, BCVA was hand movements in the right eye and in the left eye 20/40. Fundus examination revealed no presence of vitritis and atrophic scars post choroiditis serpiginosa. OCT displayed disruption of outer retinal layers. AF revealed atrophy plaque with a new diffuse hypoautofluorescence halo surrounding hyperautofluorescent rim. Aqueous humour and vitreous sample results were negatives. Finally, the lesion was diagnosed as a serpiginous choroiditis. She was treated with corticosteroid and biological therapy to improve her visual acuity. It should suggest a reactivation of SC the appearance of a new atrophy plaque with ill‐defined creamy rim, early block and diffuse oozing at late times, choroidal hypoperfusion, diffuse hypoautofluorescence halo surrounding hyperautofluorescent rim and disruption of outer retinal layers with ellipsoid loss.Conclusions: A differential diagnosis should be made between SC and posterior uveitis secondary to tuberculosis. Multimodal imaging by autofluorescence is important for the diagnosis and follow‐up of patients with atrophic scarring due to SC. Differential diagnosis of cause posterior uveitis is essential for the monitoring and treatment. In inflammatory diseases always rule out infection prior to corticosteroid treatment or biological therapy.

SPATIAL PATTERN OF RETINAL PIGMENT EPITHELIUM TEAR DEVELOPMENT AND PROGRESSION AFTER ANTIVASCULAR ENDOTHELIAL GROWTH FACTOR THERAPY FOR NEOVASCULAR AGE-RELATED MACULAR DEGENERATION.

The aim of this study was to demonstrate the spatial pattern of retinal pigment epithelium (RPE) tear development and progression after antivascular endothelial growth factor therapy for neovascular age-related macular degeneration. We retrospectively reviewed six eyes with neovascular age-related macular degeneration that showed RPE tears after administration of intravitreal antivascular endothelial growth factor agents and were followed up for 12 months. The patterns of RPE tear development and progression were evaluated by analyzing positional relationships among the locations of the choroidal neovascularization membrane and pigment epithelial detachment (PED) area at baseline and the tear area using spectral-domain optical coherence tomography, color photography, fluorescein angiography, and fundus autofluorescence images. Pretear OCT images revealed fibrovascular PED in all eyes, one of which showed complications of hemorrhagic PED after treatment. In five eyes, RPE tears developed at the PED edge located on the opposite side of the choroidal neovascularization membrane. In the eye showing hemorrhagic PED, the RPE tear developed along the wide area of the PED edge. The torn RPE monolayer contracted toward the side of the choroidal neovascularization membrane in all eyes, and RPE loss involved the fovea in five eyes that showed significantly worse visual acuity (VA) after 12 months in comparison with the baseline value before the tear (logMAR VA; 0.3 vs. 1.29; P < 0.02). The location of choroidal neovascularization membrane in PED determines the spatial pattern of RPE tear development and progression and helps to predict the visual outcome after RPE tears.

Spectroscopic Study of Time-Varying Optical Redox Ratio in NADH/FAD Solution.

Autofluorescence imaging has been widely applied as advanced noninvasive diagnostics for in vivo and ex vivo tissues. The optical redox ratio (ORR), which is defined as the fluorescence intensity ratio between reduced nicotine adenine dinucleotide (NADH) and oxidized flavin adenine dinucleotide (FAD), has been used as a diagnostic parameter strongly, because NADH and FAD play an important role in energetic and respiratory metabolism as coenzymes. The ORR method has provided successful assessment in cancer diagnosis including breast, cervical, and oral cancer; few studies have been reported about optical and chemical interference between two molecules resulting in a change in ORR values. In this study, we investigated the variations in ORR values of NADH/FAD mixtures dissolved in tris(hydroxymethyl)aminomethane, phosphate buffer, and deionized water environments. In vitro solutions were prepared in various concentration ratios and the experimental and theoretical ORR values were obtained from fluorescence and absorption spectra in time series. Based on the spectroscopic analysis, we concluded that the inner filter effect causes an instant decrease in FAD fluorescence just after dissolution and that the oxidation-reduction coupled with oxygenation reaction results in time-varying decreases in NADH fluorescence and FAD emission.

Angiogenesis Assays for the Analysis of CCN Proteins.

Angiogenesis, the process of generating new blood vessels from an existing vasculature, is essential in normal developmental processes such as endochondral ossification and in numerous kinds of pathogenesis including tumor growth. A part from the actin of angiogenic factor or antiangiogenic factor, it is still unknown at which stage of the angiogenic cascade these agents affect angiogenesis. Here, we describe methods for the use of cellular communication network factor/connective tissue growth factor (CTGF/CCN2) and CCN2-neutralizing antibody in the currently used principal angiogenesis assays, including those in vitro ones for the proliferation, migration, adhesion, and tube formation of endothelial cells and in vivo assays such as those utilizing type I collagen implantation and the chick chorioallantoic membrane (CAM). In addition, we introduce an autofluorescence imaging of blood vessels in the subcutaneous tumor xenograft mouse model. These assays can be applied to studies on roles of CCN proteins in tumor metastasis and development of treatment strategies targeting CCN proteins.

Translational rapid ultraviolet-excited sectioning tomography for whole-organ multicolor imaging with real-time molecular staining.

Rapid multicolor three-dimensional (3D) imaging for centimeter-scale specimens with subcellular resolution remains a challenging but captivating scientific pursuit. Here, we present a fast, cost-effective, and robust multicolor whole-organ 3D imaging method assisted with ultraviolet (UV) surface excitation and vibratomy-assisted sectioning, termed translational rapid ultraviolet-excited sectioning tomography (TRUST). With an inexpensive UV light-emitting diode (UV-LED) and a color camera, TRUST achieves widefield exogenous molecular-specific fluorescence and endogenous content-rich autofluorescence imaging simultaneously while preserving low system complexity and system cost. Formalin-fixed specimens are stained layer by layer along with serial mechanical sectioning to achieve automated 3D imaging with high staining uniformity and time efficiency. 3D models of all vital organs in wild-type C57BL/6 mice with the 3D structure of their internal components (e.g., vessel network, glomeruli, and nerve tracts) can be reconstructed after imaging with TRUST to demonstrate its fast, robust, and high-content multicolor 3D imaging capability. Moreover, its potential for developmental biology has also been validated by imaging entire mouse embryos (~2 days for the embryo at the embryonic day of 15). TRUST offers a fast and cost-effective approach for high-resolution whole-organ multicolor 3D imaging while relieving researchers from the heavy sample preparation workload.

Ophthalmic Manifestations of ROSAH (Retinal Dystrophy, Optic Nerve Edema, Splenomegaly, Anhidrosis, and Headache) Syndrome, an Inherited NF κB–Mediated Autoinflammatory Disease with Retinal Dystrophy

We aimed to characterize the ocular phenotype of patients with ROSAH (retinal dystrophy, optic nerve edema, splenomegaly, anhidrosis, and headache) syndrome and their response to therapy. Single-center observational case study. Eleven patients with a diagnosis of ROSAH syndrome and mutation in ALPK1 were included. Patients with molecularly confirmed ROSAH syndrome underwent ophthalmic evaluation, including visual acuity testing, slit-lamp and dilated examinations, color fundus and autofluorescence imaging, fluorescein angiography, OCT, and electrophysiologic testing. Visual acuity, electrophysiology, fluorescein angiography, and OCT findings. Eleven individuals (6 female and 5 male patients) from 7 families ranging in age from 7.3 to 60.2 years at the time of the initial evaluation were included in this study. Seven patients were followed up for a mean of 2.6 years (range, 0.33-5.0 years). Best-corrected visual acuity at baseline ranged from 20/16 to no light perception. Variable signs or sequelae of intraocular inflammation were observed in 9 patients, including keratic precipitates, band keratopathy, trace to 2+ anterior chamber cells, cystoid macular edema, and retinal vasculitis on fluorescein angiography. Ten patients were observed to show optic disc elevation and demonstrated peripapillary thickening on OCT. Seven patients showed retinal degeneration consistent with a cone-rod dystrophy, with atrophy tending to involve the posterior pole and extending peripherally. One patient with normal electroretinography findings and visual evoked potential was found to have decreased Arden ratio on electro-oculography. Leveraging insights from the largest single-center ROSAH cohort described to date, this study identified 3 main factors as contributing to changes in visual function of patients with ROSAH syndrome: optic nerve involvement; intraocular inflammation, including cystoid macular edema; and retinal degeneration. More work is needed to determine how to arrest the progressive vision loss associated with ROSAH syndrome. Proprietary or commercial disclosure may be found after the references.