Blue Autofluorescence Research Articles

Multicolour imaging in central serous chorioretinopathy

BackgroundMulticolour imaging (MI) is a novel, non‐invasive retinal imaging technology. Its sensitivity for detecting the clinical features in central serous chorioretinopathy (CSCR) has not been previously described. The aim of this study is to evaluate the accuracy of MI compared to fluorescein angiography and colour fundus photography in CSCR, and to describe the imaging features of MI.MethodsIn this retrospective study at a tertiary referral centre, 63 consecutive eyes with CSCR (both acute and chronic) were included after obtaining permission from the institutional review board. Multimodal imaging with colour fundus photography, optical coherence tomography, MI and fluorescein angiography/indocyanine green angiography and near‐infrared and blue wavelength autofluorescence was analysed to identify the clinical findings in CSCR. Sensitivity and specificity values were computed for the different clinical features for each imaging modality.ResultsOn comparison with fluorescein angiography, MI was found to be more effective in identifying the extent of subretinal fluid (78 per cent versus 13 per cent). MI was equally capable in identifying pigment epithelium detachment (100 per cent versus 100 per cent) and retinal pigment epithelial changes (100 per cent versus 100 per cent). Focal leaks were identified in 84 per cent and 97 per cent of eyes using MI and fluorescein angiography imaging, respectively. The sensitivity of MI in identifying focal retinal pigment epithelial leaks was higher compared to near‐infrared autofluorescence (84 per cent versus 34 per cent) and blue wavelength autofluorescence (84 per cent versus 18 per cent) imaging.ConclusionMI is a useful, non‐invasive imaging modality for detecting clinical features in CSCR. In the future, MI has the potential to substitute for fluorescein angiography and colour fundus photography as the imaging modality of choice.

Multimodal Imaging Correlation of the Concentric Macular Rings Sign in Foveal Hypoplasia: A Distinctive Henle Fiber Layer Geometry

To expand the multimodal imaging correlation of the concentric macular rings (CMR) sign seen on ultra-widefield fundus photography in patients with foveal hypoplasia. Retrospective case series. Thirty-two patients with foveal hypoplasia who demonstrated the CMR sign on ultra-widefield fundus photography. Inclusion criterion was the presence of a CMR sign detected on ultra-widefield fundus photography. Noninvasive multimodal retinal imaging, including ultra-widefield fundus photography, structural OCT, near-infrared reflectance, and blue fundus autofluorescence, were investigated. Horizontal dense B-scans and en face OCT images were acquired. Evaluation of all patients (n= 32 patients) demonstrated a CMR sign on ultra-widefield fundus photography. Structural OCT scans were consistent with foveal hypoplasia in all patients. En face OCT images acquired at the level of the Henle fiber layer highlighted similar concentric rings around the location of the incipient fovea. The series of concentric rings was not visible at any other level of the macula. A significant correlation was found between the horizontal diameter of the largest outer ring and foveal hypoplasia grades (P < 0.0001). The CMR sign seen on ultra-widefield fundus imaging may be a distinctive feature of foveal hypoplasia and can support this diagnosis, especially in patients in whom OCT cannot be performed (patients with poor fixation or nystagmus or young children). Multimodal imaging correlation suggests that these concentric rings occur exclusively at the level of the Henle fiber layer. This distinctive Henle fiber layer geometry may reflect an arrested development stage in the timeline of foveal maturation.

MP10-19 MATURE RANDALL’S PLAQUE CONTAINS CELL NUCLEI: OBSERVATIONS USING HIGH-RESOLUTION MICROSCOPY

You have accessJournal of UrologyStone Disease: Basic Research & Pathophysiology II (MP10)1 Apr 2020MP10-19 MATURE RANDALL’S PLAQUE CONTAINS CELL NUCLEI: OBSERVATIONS USING HIGH-RESOLUTION MICROSCOPY Victor Hugo Canela*, Sharon B. Bledsoe, Glenn S. Gerber, Elaine M. Worcester, James E. Lingeman, Tarek M. El-Achkar, and James C. Williams Victor Hugo Canela*Victor Hugo Canela* More articles by this author , Sharon B. BledsoeSharon B. Bledsoe More articles by this author , Glenn S. GerberGlenn S. Gerber More articles by this author , Elaine M. WorcesterElaine M. Worcester More articles by this author , James E. LingemanJames E. Lingeman More articles by this author , Tarek M. El-AchkarTarek M. El-Achkar More articles by this author , and James C. WilliamsJames C. Williams More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000000830.019AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: The formation of CaOx stones on Randall’s plaque is a common phenomenon (perhaps 25% of all stones), and stones that have grown on Randall’s plaque can be easily identified using simple microscopy, yet this mechanism of stone formation is still poorly understood. The objective of this study was to identify differences in the structure and composition of Randall’s plaque that differentiate it from the CaOx overgrowth region and to shed light on possible mechanisms for plaque formation. METHODS: Randall’s plaque stones were mounted in gel along with fiducial markers and scanned by micro CT to document orientation. Following demineralization, the stone was embedded in paraffin and sectioned for histology and fluorescence imaging. RESULTS: Demineralized and sectioned Randall’s plaque displayed the same autofluorescence in the far-blue region that we have previously documented in mineralized plaque. Microscopic analysis demonstrates that the plaque autofluoresces in the far-blue range as opposed to the CaOx overgrowth which appears mostly with green or yellow autofluorescence. Further, high-resolution imaging of the sectioned plaque showed that this blue autofluorescence was present throughout the demineralized plaque, but that its intensity was diminished in oval regions of the right size to be nuclei of cells. Subsequent staining of sections with the DNA marker, 7-aminoactinomycin D (7-AAD), confirmed the presence of nuclei within the demineralized Randall’s plaque. CONCLUSIONS: This is the first demonstration of the presence of cells within areas of mature Randall’s plaque. The nature of cells and their role in plaque formation are yet to be determined. Our data suggest that these cells contain ordinary nuclear morphology and are preserved within the calcium phosphate plaque. The presence of nuclei in the mature plaque means that apoptosis and/or necrosis is not part of the normal process of the formation of fully mineralized Randall’s plaque. Furthermore, the nuclei were exclusively detected in the plaque area and not in the CaOx overgrowth. Proteomic analysis in the different regions along with the interface region will provide invaluable information in understanding the growth of CaOx on Randall’s plaque. Source of Funding: NIDDK P01 56788 © 2020 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 203Issue Supplement 4April 2020Page: e133-e133 Advertisement Copyright & Permissions© 2020 by American Urological Association Education and Research, Inc.MetricsAuthor Information Victor Hugo Canela* More articles by this author Sharon B. Bledsoe More articles by this author Glenn S. Gerber More articles by this author Elaine M. Worcester More articles by this author James E. Lingeman More articles by this author Tarek M. El-Achkar More articles by this author James C. Williams More articles by this author Expand All Advertisement PDF downloadLoading ...

Validation of multicolor imaging signatures of central serous chorioretinopathy lesions vis-a-vis conventional color fundus photographs.

Purpose:The current study compares the ability of multicolor imaging (MCI) to detect the lesions of central serous chorioretinopathy against conventional color fundus photographs (CFP).Methods:It was a retrospective, observational case series of 93 eyes of 58 patients of central serous chorioretinopathy who underwent MCI and CFP. MCI and spectral-domain optical coherence tomography (SD-OCT) were performed using Spectralis SD-OCT system (HRA + OCT). CFP was obtained using FF 450 Plus fundus camera (Carl Zeiss Meditec, Jena, Germany). SD-OCT was considered gold standard for subretinal fluid (SRF) and retinal pigment epithelium detachment (PED). CFP was considered confirmatory investigation for fibrin and blue autofluorescence image (BAF) was considered gold standard to detect retinal pigment epithelium (RPE) atrophy.Results:CFP could detect SRF in 41 (44.1%) eyes. MCI detected SRF in 43 (46.2%) eyes. The sensitivity and specificity of MCI to detect SRF were 70.7% and 94.3%, respectively. PED was detected by CFP in 21 (22.6%) eyes and MCI in 27 (29%) eyes. The sensitivity and specificity of MCI to detect PED were 70% and 97.7% respectively. CFP could pick RPE atrophy in 52 (55.9%) eyes whereas MCI was picked it in 78 (83.9%) of eyes.Conclusion:Both MCI and CFP were inferior to a gold standard in identifying the SRF, PED, and RPE atrophy. However, MCI was better than CFP in comparison with gold standard for these clinical findings in CSC. Thus, MCI seems to be a more valuable imaging tool compared to CFP.

Distinctive Mechanisms and Patterns of Exudative Versus Tractional Intraretinal Cystoid Spaces as Seen With Multimodal Imaging

To determine clear-cut distinctions between tractional and exudative intraretinal cystoid spaces subtypes. Retrospective, multicenter, observational case series. A cohort of patients diagnosed with intraretinal cystoid spaces and imaged with optical coherence tomography (OCT), fluorescein angiography (FA), blue fundus autofluorescence (BFAF), en face OCT, and OCT angiography (OCT-A) was included in the study. All images were qualitatively and quantitatively evaluated. In this study were included 72 eyes of 69 patients. Exudative intraretinal cystoid spaces (36/72 eyes, 50%) displayed a "petaloid" morphology as seen with en face OCT, FA, and BFAF. Tractional intraretinal cystoid spaces (24/72 eyes, 33.3%), displayed a radial "spoke-wheel" en face OCT pattern. There was no leakage with FA and BFAF did not reveal specific patterns. Eyes with full-thickness macular hole (FTMH, 12/72 eyes, 16.7%) displayed a "sunflower" en face OCT appearance. FTMH showed OCT, OCT-A, and BFAF features of both exudative and tractional cystoid spaces, but without any FA leakage. Inner nuclear layer (INL) thickness was significantly lower in tractional cystoid spaces (P < .001). There were a greater number of INL cystoid spaces in both the exudative and FTMH subgroups (P= .001). The surface area of INL cystoid spaces was significantly lower in the tractional subgroup (P < .001). There was a significant reduction of the microvascular density in eyes with exudative vs tractional (P= .002) and FTMH (P < .001) subgroups. Exudative and tractional intraretinal cystoid spaces displayed characteristic multimodal imaging features and they may represent 2 different pathologic conditions with equally different clinical implications.

Dynamic in vivo quantification of rod photoreceptor degeneration using fluorescent reporter mouse models of retinitis pigmentosa

Imaging techniques have revolutionised the assessment of retinal disease in humans and animal models. Here we describe a novel technique for the in vivo visualisation of rod photoreceptors which permits semiquantitative assessment of outer retinal degeneration, and validate this approach in two mouse models of retinitis pigmentosa (RP).Transgenic mice carrying an Nrl-EGFP allele and homozygous for either knock-out of rhodopsin (Nrl-EGFP, Rho−/−) or heterozygous for knock-in of P23H mutant rhodopsin (Nrl-EGFP, RhoP23H/+) were used in this study. These novel strains have green fluorescent rods which undergo a progressive degeneration. Fundus imaging was performed at three-weekly intervals by near infrared reflectance (NIR) and blue light autofluorescence (BAF) confocal scanning laser ophthalmoscopy (cSLO). Mean grey values (mGV), which quantify fluorescence levels within such images, were compared for degenerate and age-matched non-degenerate (Nrl-EGFP, Rho+/+) controls.Mean grey value significantly decreased over time in the Rho−/− and RhoP23H/+ groups but was maintained in Rho+/+ mice (P < 0.001, two-way ANOVA). This corresponded to outer nuclear layer (ONL) thinning as observed by histology. The mGV of superior retina was significantly greater than that of inferior retina in RhoP23H/+ (P = 0.0024) but not in age-matched Rho+/+ (P = 0.45) or Rho−/- (P = 0.65) mice reflecting histological findings. Focal loss of rods could be visualised and mapped in vivo with this technique following a toxic insult, with thinning of the ONL being confirmed in hypofluorescent regions by spectral domain ocular coherence tomography (OCT).Fluorescence labelling of rods permits in vivo characterisation of models of RP and may provide new insights into patterns of degeneration, or rescue effect after treatment. mGV can be used in such cases as a semiquantitative metric of ONL degeneration, and can be used to identify regional variations in photoreceptor loss.

Red fluorescent AuNDs with conjugation of cholera toxin subunit B (CTB) for extended-distance retro-nerve transporting and long-time neural tracing

A retrograde transportation nerve probe, Au nanodots-cholera toxin B subunit (AuNDs-CTB), are prepared and fully characterized, which emit bright red fluorescence and show high quantum yield (7.2%) and good stability. The fluorescence emitted by the AuNDs is constant across a wide pH range (4–10) and after prolonged UV irradiation (>4 h). Previously, CTB has shown targeting characteristic for nerve cells with high sensitivity and effectiveness. After linking CTB to AuNDs through amidation reactions, AuNDs-CTB are obtained with excellent fluorescence property, nerve target characteristic, and, particularly, neural retrograde transportation feature. The red emission of the AuNDs-CTB is well distinguished from the blue autofluorescence of normal tissues, which provides potential for detection by naked eyes. Further, the fluorescence emission intensity maintains for 10 days in vivo, suggesting great utility for long-time monitoring and sensing of the nerve tissue. Furthermore, the AuNDs-CTB with bright red fluorescence can travel through the peripheral nerve to the spinal cord rapidly by retrograde transportation. The transportation occurs for a long distance (>5 cm) within only 2 days after injection of the AuNDs-CTB into the sciatic nerve. The present study exhibits a novel method for nerve visualization and drug delivery. Statement of significanceAu nanodots (AuNDs) conjugated with cholera toxin subunit B (CTB) have been developed for nerve labeling and neural retro-transporting. The red fluorescence from AuNDs-CTB is stable in vitro (pH 4–10 and 4 h UV irradiation) and in vivo (for a long time, more than 10 days). When injecting AuNDs-CTB into the sciatic nerve located at the midpiece of the thigh, the targeted nerve emits bright red fluorescence under UV light. Furthermore, the nerve can retrograde transport the AuNDs-CTB to the spinal cord for a distance of more than 5 cm just in 2 days. This work exhibits a novel method for nerve visualization by naked eyes and demonstrates the potential for intraoperative navigation.

Cannabis glandular trichomes alter morphology and metabolite content during flower maturation.

The cannabis leaf is iconic, but it is the flowers of cannabis that are consumed for the psychoactive and medicinal effects of their specialized metabolites. Cannabinoid metabolites, together with terpenes, are produced in glandular trichomes. Superficially, stalked and sessile trichomes in cannabis only differ in size and whether they have a stalk. The objectives of this study were: to define each trichome type using patterns of autofluorescence and secretory cell numbers, to test the hypothesis that stalked trichomes develop from sessile-like precursors, and to test whether metabolic specialization occurs in cannabis glandular trichomes. A two-photon microscopy technique using glandular trichome intrinsic autofluorescence was developed which demonstrated that stalked glandular trichomes possessed blue autofluorescence correlated with high cannabinoid levels. These stalked trichomes had 12-16 secretory disc cells and strongly monoterpene-dominant terpene profiles. In contrast, sessile trichomes on mature flowers and vegetative leaves possessed red-shifted autofluorescence, eight secretory disc cells and less monoterpene-dominant terpene profiles. Moreover, intrinsic autofluorescence patterns and disc cell numbers supported a developmental model where stalked trichomes develop from apparently sessile trichomes. Transcriptomes of isolated floral trichomes revealed strong expression of cannabinoid and terpene biosynthetic genes, as well as uncharacterized genes highly co-expressed with CBDA synthase. Identification and characterization of two previously unknown and highly expressed monoterpene synthases highlighted the metabolic specialization of stalked trichomes for monoterpene production. These unique properties and highly expressed genes of cannabis trichomes determine the medicinal, psychoactive and sensory properties of cannabis products.

Molecular and biochemical basis for the loss of bioluminescence in the dinoflagellate Noctiluca scintillans along the west coast of the USA.

The globally distributed heterotrophic dinoflagellate Noctiluca scintillans (Macartney) Kofoid & Swezy is well known for its dense blooms and prominent displays of bioluminescence. Intriguingly, along the west coast of the USA its blooms are not bioluminescent. We investigated the basis for the regional loss of bioluminescence using molecular, cellular and biochemical analyses of isolates from different geographic regions. Prominent differences of the non-bioluminescent strains were: (1) the fused luciferase and luciferin binding protein gene (lcf/lbp) was present but its transcripts were undetectable; (2) lcf/lbp contained multiple potentially deleterious mutations; (3) the substrate luciferin was absent, based on the lack of luciferin blue autofluorescence and the absence of luciferin derived metabolites; (4) although the cells possessed scintillons, the vesicles that contain the luminescent chemistry, electron microscopy revealed additional scintillon-like vesicles with an atypical internal structure; (5) cells isolated from the California coast were 43% smaller in size than bioluminescent cells from the Gulf of Mexico. Phylogenetic analyses based on the large subunit of rDNA did not show divergence of the non-bioluminescent population in relation to other bioluminescent N. scintillans from the Pacific Ocean and Arabian Sea. Our study demonstrates that gene silencing and the lack of the luciferin substrate have resulted in the loss of a significant dinoflagellate functional trait over large spatial scales in the ocean. As the bioluminescence system of dinoflagellates is well characterized, non-bioluminescent N. scintillans is an ideal model to explore the evolutionary and ecological mechanisms that lead to intraspecific functional divergence in natural dinoflagellate populations.

Multimodal Imaging of Multiple Evanescent White Dot Syndrome: A New Interpretation

ABSTRACT Purpose To investigate the pathogenesis of Multiple Evanescent White Dot Syndrome (MEWDS) using multimodal imaging (MMI). Methods Retrospective case series of 7 patients with acute MEWDS. Each patient underwent: near-infrared reflectance (IR), blue and near-infrared autofluorescence (FAF and NIRAF), fluorescein angiography (FA), indocyanine green angiography (ICGA), enhanced depth imaging optical coherence tomography (EDI-OCT) on Spectralis, and optical coherence tomography angiography on OCTA Spectralis, XR Avanti or Plex Elite 9000. Results OCTA and FA findings of early hyperfluorescence depict an unaffected choriocapillaris. On ICGA early to late hypofluorescent lesions corresponded to the hyporeflectivity on IR, consistent with altered reflectivity of the RPE. The SDI-OCT showed ellipsoid zone disruption as confirmed by FAF hyperautofluorescence. Some lesions showed a hypertransmission sign underneath the RPE, possibly due to changes in RPE intracellular melanin as suggested by NIRAF hypoautofluorescence. Conclusions The MMI findings of MEWDS are secondary to RPE reflectivity changes, suggesting its pivotal role.

Red Fluorescent AuNDs-CTB for Extended-Distance Retro-Nerve Transporting and Long-Time Neural Tracing

Retrograde transportation nerve probe, Au nanodots-cholera toxin B subunit (AuNDs-CTB) are prepared and fully characterized, which possess bright red fluorescence, high quantum yield (7.2%) and good stability. The fluorescence provided by the AuNDs keep constant across a wide pH range (4~10) and after prolonged UV irradiation (> 4 h). Previously, CTB shows targeting characteristics for nerve cells with high sensitivity and effectiveness. After linking CTB to AuNDs through amidation reactions, AuNDs-CTB are obtained with excellent fluorescence property, nerve target characteristic and especially neural retrograde transportation feature. The red emission of the AuNDs-CTB is well distinguished from the blue auto fluorescence of normal tissue, allowing for unambiguous discrimination. Also, the fluorescence emission intensity maintains in vivo for 10 days, suggesting great utility for long-time monitoring and sensing of nerve tissue. Meanwhile, the AuNDs-CTB with bright red fluorescence can retrograde transported rapidly from the peripheral nerve to the spinal cord. Retrograde transportation occurs over a long distance (> 5 cm) after injection of AuNDs-CTB into the sciatic nerve with only 5 days. Attractively, the present study exhibits a novel method for nerve visualization and medicine transportation.

Blue autofluorescence in protein aggregates “lighted on” by UV induced oxidation

Oxidation of amino acid side chains in protein structure can be induced by UV irradiation leading to critical changes in molecular structure possibly modifying protein stability and bioactivity.Here we show, by using a combination of multiple spectroscopic techniques and Fluorescence Lifetime Imaging, that UV-light exposure induces irreversible oxidation processes in Ubiquitin structure. In particular, the growth of a new autofluorescence peak in the blue region is detected, that we attribute to tyrosine oxidation products. Blue autofluorescence intensity is found to progressively increase also during aggregation processes leading to the formation of aggregates of non-amyloid nature.Significantly, analogous spectral modifications are found in amyloid fibrils from human insulin and Amyloid-β peptide grown under UV exposure. Experimental results reveal a substantial overlap between the fluorescence signal here attributed to tyrosine oxidation and the one referred in literature as “Amyloid autofluorescence”. These findings clearly represent a caveat about the specificity of the blue fluorescence peak measured for amyloids, especially when grown in conditions in which tyrosine residues may be oxidized.Moreover, our results once again highlight the close link between the formation of amyloid aggregates and protein damage resulting from oxidative stress, as these neurotoxic aggregate species are found to contain damaged residues.

Improved Diagnosis of Retinal Laser Injuries Using Near-Infrared Autofluorescence

To assess whether near infrared autofluorescence (NIR-AF) imaging is a useful imaging modality in the diagnosis of handheld laser retinal injuries. Retrospective observational case series. Twelve patients identified to have handheld laser retinal injuries were included at 2 academic centers. Patients underwent ophthalmic assessment and retinal imaging including fundus photography, optical coherence tomography (OCT), conventional blue autofluorescence (B-AF), and NIR-AF imaging. In all cases, lesions consistent with retinal laser injury were detected by NIR-AF imaging. The lesions showed a characteristic appearance with central hyperfluorescence and surrounding hypofluorescence, although the number and extent of lesions varied between patients. Findings using other imaging modalities were variable: on color fundus photography these included localized pigmentary changes and on OCT imaging an ellipsoid zone interruption or outer nuclear layer changes. Only subtle changes were evident on B-AF imaging. Other macular conditions, such as poppers retinopathy or solar maculopathy, which may have similar findings on OCT imaging as laser damage, can be differentiated using NIR-AF imaging. An increased incidence of retinal injuries secondary to handheld lasers has been reported in recent years. We show that the diagnosis and full extent of retinal laser injuries is best demonstrated by NIR-AF, as other modalities give variable results. We propose that NIR-AF should be included when investigating patients suspected of macular injury secondary to exposure to handheld lasers.

Imaging and Spectral Characteristics of Amyloid Plaque Autofluorescence in Brain Slices from the APP/PS1 Mouse Model of Alzheimer's Disease.

Amyloid deposits are one of the hallmark pathological lesions of Alzheimer's disease (AD). They can be visualized by thioflavin-S, silver impregnation, Congo red staining, and immunohistochemical reactions. However, that amyloid deposits generate blue autofluorescence (auto-F) has been ignored. Here, we report that visible light-induced auto-F of senile plaques (SPs) was detected and validated with conventional methods. Brain slices from APP/PS1 (amyloid precursor protein/presenilin 1) transgenic mice were mounted on slides, rinsed, coverslipped and observed for details of the imaging and spectral characteristics of the auto-F of SPs. Then the slices were treated with the above classic methods for comparative validation. We found that the SP auto-F was greatest under blue-violet excitation with a specific emission spectrum, and was much easier, more sensitive, and reliable than the classic methods. Because it does not damage slices, observation of auto-F can be combined with all post-staining techniques in slices and for brain-wide imaging in AD.

Multimodal imaging including optical coherence tomography angiography in patients with type B Niemann-Pick disease.

To evaluate accumulation patterns of deposits in retinal layers of type B Niemann-Pick patients by multimodal imaging. Seven patients with type B Niemann-Pick disease were included in this study. All participants underwent a complete ophthalmologic evaluation, high-resolution digital colour imaging, spectral-domain optical coherence tomography, blue light fundus autofluorescence and optical coherence tomography angiography (OCTA). We demonstrated different accumulation patterns in the retinal ganglion cell layer, the retinal nerve fibre layer and the subfoveolar region by multimodal imaging. Local retinal capillary nonflow areas in the superficial plexus, increased vascular tortuosity and deformed foveal avascular areas were shown in OCTA scans. Multimodal imaging including OCTA is a useful technique for the identification of different types of accumulation patterns, diagnosis and follow-up of type B Niemann-Pick patients.

IMAGING OF VITELLIFORM MACULAR LESIONS USING POLARIZATION-SENSITIVE OPTICAL COHERENCE TOMOGRAPHY.

To examine the involvement of the retinal pigment epithelium (RPE) in the presence of vitelliform macular lesions (VML) in Best vitelliform macular dystrophy (BVMD), autosomal recessive bestrophinopathy, and adult-onset vitelliform macular degeneration using polarization-sensitive optical coherence tomography (PS-OCT). A total of 35 eyes of 18 patients were imaged using a PS-OCT system and blue light fundus autofluorescence imaging. Pathogenic mutations in the BEST1 gene, 3 of which were new, were detected in all patients with BVMD and autosomal recessive bestrophinopathy. Polarization-sensitive optical coherence tomography showed a characteristic pattern in all three diseases with nondepolarizing material in the subretinal space consistent with the yellowish VML seen on funduscopy with a visible RPE line below it. A focal RPE thickening was seen in 26 eyes under or at the edge of the VML. Retinal pigment epithelium thickness outside the VML was normal or mildly thinned in patients with BVMD and adult-onset vitelliform macular degeneration but was diffusely thinned or atrophic in patients with autosomal recessive bestrophinopathy. Patients with autosomal recessive bestrophinopathy showed sub-RPE fibrosis alongside the subretinal VML. Polarization-sensitive optical coherence tomography was more reliable in assessing the localization and the integrity of the RPE than spectral domain OCT alone. On spectral domain OCT, identification of the RPE was not possible in 19.4% of eyes. Polarization-sensitive optical coherence tomography allowed for definite identification of the location of VML in respect to the RPE in all eyes, since it provides a tissue-specific contrast. Polarization-sensitive optical coherence tomography confirms in vivo the subretinal location of VML and is useful in the assessment of RPE integrity.

Blue Autofluorescence Fundus Imaging for Monitoring Retinal Degeneration in Royal College of Surgeons Rats.

PurposeDevelopment of a method for noninvasive longitudinal follow-up of retinal degeneration in the whole retina for Royal College of Surgeons (RCS) rats, a commonly used model of retinitis pigmentosa associated with mutations in the MER-proto-oncogene tyrosine kinase (MERTK) gene.MethodsPigmented RCS rats at postnatal (p) days p28 to p84 were subjected to a biweekly spectral-domain optical coherence tomography (SD-OCT), blue laser fundus autofluorescence (BL-FAF) imaging, and multicolor fundus imaging. Wild-type (WT; Long Evans) rats were tested as control.ResultsHyperautofluorescence developed throughout the fundus at p42, concomitant with a significant increase in SD-OCT thickness and reflectivity of the debris zone (DZ) layer as well as thinning of the photoreceptor outer nuclear layer (ONL). From p56 to p84, discrete hypofluorescent lesions surrounded by hyperfluorescent flecks were demonstrated around the optic disc that gradually spread throughout the retina. The hypofluorescent lesions were associated with loss of ONL and gradual thinning of the DZ layer. No hypofluorescent BL-FAF lesions were observed in WT rats.ConclusionsThis study suggests that BL-FAF imaging may present a new method for noninvasive longitudinal follow-up of retinal degeneration in nearly the whole retina in RCS rats.Translational RelevanceA clinical test was developed that may be implemented in translational studies in the RCS rat model of MERTK-associated retinitis pigmentosa.

Retinal sensitivity and photoreceptor arrangement changes secondary to congenital simple hamartoma of retinal pigment epithelium

BackgroundThe congenital simple hamartoma of the retinal pigment epithelium is a benign lesion and previous observations with noninvasive imaging have detected potential photoreceptor abnormalities and retinal function interplay.Case presentationA 35-year-old woman was found to have an asymptomatic, solitary, circumscribed, pigmented lesion in her left eye. The patient underwent ophthalmic examination including multimodal evaluation with fluorescein angiography, near-infrared reflectance scanning laser ophthalmoscopy, blue autofluorescence, enhanced-depth imaging spectralis B-scan optical coherence tomography (EDI-SBOCT), en face OCT angiography (OCT-A) and microperimetry plus adaptive optics imaging. Ophthalmoscopic examination revealed a juxtafoveolar pigmented lesion with feeding retinal arteriole, consistent with congenital simple hamartoma of RPE. There was no macular edema, exudation, hemorrhage, traction or subretinal fluid. Multimodal imaging of the mass using fluorescein angiography revealed intra-lesion late staining, near-infrared reflectance imaging demonstrated intrinsic hyperreflectivity, short-wavelength autofluorescence and red-free filter photography revealed blocked signal, and SBOCT showed abrupt shadowing. On OCT-A, an exclusive ring-shaped vascular circuit with increased foveal avascular zone was noted. Adaptive optics revealed cell density arrangement and retinal sensitivity correlations on microperimetry.ConclusionThese findings suggest that this hamartomatous lesion might cause specific cellular changes that impact retinal sensitivity response and potentially result from vasculature malnourishment to the outer retinal layers.

Blue light versus green light fundus autofluorescence in normal subjects and in patients with retinochoroidopathy secondary to retinal and uveitic diseases

PurposeThe aim of this study is to evaluate the differences in the fundus autofluorescence (FAF) signal between the blue light autofluorescence (BAF) from Spectralis® (Heidelberg, CA) and green light autofluorescence (GAF) 200TxTM (OPTOS, UK, in normal subjects and in patients with retinochoroidopathies (RC).MethodsIn this prospective study, FAF was performed using BL (λ = 488 nm) and GL (λ = 532 nm) on normal subjects and patients with RC. The corresponding pairs of BAF and GAF images from both groups were analyzed using Photoshop. The strength of the FAF signal was measured on a gray scale, where optic disc was a standard to indicate absence of AF. In addition, gray values obtained from three identical points (foveal center, and points of hypo and hyper autofluorescence) in the corresponding BAF and GAF images of normal and RC subjects were divided by the optic disc value to calculate autofluorescence signal ratio (R). The R values at fovea (R1), hypoautofluorescent point (R2), and hyperautofluorescent point (R3) were compared between BAF and GAF modalities, in normal and in RC subjects separately.ResultsOne hundred six pairs (106 eyes) of FAF images analyzed (37 pairs: normal and 69 pairs: RC subjects). In normal subjects, the mean R1, R2, and R3 values for BAF were (1.5 ± 0.88, 1.23 ± 0.58, and 4.73 ± 2.85, respectively) and for GAF were (0.78 ± 0.20, 0.78 ± 0.20, and 1.62 ± 0.39, respectively). Similarly, in subjects with RC, the mean R1, R2, and R3 values for BAF were (1.68 ± 1.02, 1.66 ± 1.15, and 7.75 ± 6.82, respectively) and for GAF were (0.95 ± 0.59, 0.79 ± 0.45, and 2.50 ± 1.65, respectively). The mean difference in the R1, R2, and R3 ratios between BAF and GAF in normal and in RC subjects was statistically significant (p < 0.001). The strength of the correlation (r) between ratios for BAF and GAF was weak or not statistically significant in both normal and RC subjects (p > 0.05).ConclusionThe distribution and intensity of the AF signal differ in BAF and GAF and cannot be used interchangeably. In BAF, optic disc signal is always weaker than in other areas, which was not true for GAF where optic disc signal was stronger than fovea and hypoautofluorescent point in both groups.

Carbon Nitride Dots: A Selective Bioimaging Nanomaterial.

In contrast to the recent immense attention in carbon nitride quantum dots (CNQDs) as a heteroatom-doped carbon quantum dot (CQD), their biomedical applications have not been thoroughly investigated. Targeted cancer therapy is a prominently researched area in the biomedical field. Here, the ability of CNQDs as a selective bioimaging nanomaterial was investigated to assist targeted cancer therapy. CNQDs were first synthesized using four different precursor sets involving urea derivatives, and the characteristics were compared to select the best candidate material for bioapplications. Characterization techniques such as UV-vis, luminescence, X-ray photoelectron spectroscopy, nuclear magnetic resonance spectroscopy, and transmission electron microscopy were used. These CNQDs were analyzed in in vitro studies of bioimaging and labeling using pediatric glioma cells (SJGBM2) for possible selective biolabeling and nanodistribution inside the cell membrane. The in vitro cellular studies were conducted under long-wavelength emission without the interference of blue autofluorescence. Thus, excitation-dependent emission of CNQDs was proved to be advantageous. Importantly, CNQDs selectively entered SJGBM2 tumor cells, while it did not disperse into normal human embryonic kidney cells (HEK293). The distribution studies in the cell cytoplasm indicated that CNQDs dispersed into lysosomes within approximately 6 h after the incubation. The CNQDs exhibited great potential as a possible nanomaterial in selective bioimaging and drug delivery for targeted cancer therapy.