Autofluorescent Research Articles

Are the Hypo-Reflective Clumps Associated With Age-Related Macular Degeneration in Adaptive Optics Ophthalmoscopy Autofluorescent?

Hypo-reflective clumps (HRCs) are structures associated with age-related macular degeneration (AMD) that were identified using flood-illumination adaptive optics ophthalmoscopy (FIAO) and hypothesized to be either macrophages that have accumulated melanin through the phagocytosis of retinal pigmented epithelial (RPE) cell organelles or transdifferentiated RPE cells. HRCs may be autofluorescent (AF) in the near infrared (NIR) but clinical NIR autofluorescence imaging lacks the resolution to answer this question definitively. Here, we used near infrared autofluorescence (NIRAF) imaging in fluorescence adaptive optics scanning laser ophthalmoscopy (AOSLO) to determine whether HRCs are AF. Patients with AMD and HRCs underwent imaging with FIAO, optical coherence tomography (OCT), and multi-modal AOSLO (confocal, NIRAF, and non-confocal multi-offset detection using a fiber bundle). HRCs were segmented on FIAO and images, co-registered across modalities, and HRC morphometry and AF were quantified. Eight patients participated (mean age = 79 years, standard deviation [SD] = 5.7, range = 69-89 years, and 5 female patients). Most HRCs (86%, n = 153/178) were autofluorescent on AOSLO. HRC AF signal varied but most uniformly dark HRCs on FIAO showed corresponding AF on AOSLO, whereas heterogeneous HRCs showed a smaller AF area or no AF. These findings are consistent with the hypothesis that HRCs contain AF RPE organelles. A small proportion of HRCs were not AF; these may represent macrophages that have not yet accumulated enough organelles to become AF. HRCs may have clinical significance but further study is needed to understand the interplay among HRCs, RPE cells, and macrophages, and their relationship to geographic atrophy (GA) progression in AMD.

Nonlinear Reduction in Hyperautofluorescent Ring Area in Retinitis Pigmentosa

To report baseline dimension of the autofluorescent (AF) ring in a large cohort of retinitis pigmentosa (RP) patients and to evaluate models of ring progression. Cohort study PARTICIPANTS: Four hundred and forty-five eyes of 224 patients with clinical diagnosis of RP. AF rings from near-infrared AF (NIRAF) and short wavelength AF (SWAF) imaging modalities in RP eyes were segmented with ring area and horizontal extent extracted for each image for cross-sectional and longitudinal analyses. In longitudinal analysis, for each eye, ring area, horizontal extent and natural logarithm of the ring area were assessed as the best dependant variable for linear regression by evaluating R2 values. Linear mixed-effects modelling was utilized to account for inter-eye correlation. AF ring size characteristics at baseline and ring progression rates. A total of 439 eyes had SWAF imaging at baseline with the AF ring observed in 206 (46.9%) eyes. Mean (95% CI) of ring area and horizontal extent were 7.85 (6.60 to 9.11) mm2 and 3.35 (3.10 to 3.60) mm, respectively. In NIRAF, the mean ring area and horizontal extent were 7.74 (6.60 to 8.89) mm2 and 3.26 (3.02 to 3.50) mm, respectively in 251 out of 432 eyes. Longitudinal analysis showed mean progression rates of -0.57 mm2/year and -0.12 mm/year in SWAF using area and horizontal extent as the dependent variable, respectively. When ln(Area) was analysed as the dependant variable, mean progression was -0.07 ln(mm2)/year which equates to 6.80% decrease in ring area per year. Similar rates were found in NIRAF (area: -0.59 mm2/year, horizontal extent: -0.12mm/year and ln(Area): -0.08 ln(mm2)/year equating to 7.75% decrease in area per year). Analysis of R2 showed that the dependent variable ln(Area) provided the best linear model for ring progression in both imaging modalities, especially in eyes with large overall area change. Our data suggests that using an exponential model to estimate progression of the AF ring area in RP is more appropriate than the models assuming linear decrease. Hence the progression estimates provided in this study should provide more accurate reference points in designing RP clinical trials.

Brain injury accelerates the onset of a reversible age-related microglial phenotype associated with inflammatory neurodegeneration.

Lipofuscin is an autofluorescent (AF) pigment formed by lipids and misfolded proteins, which accumulates in postmitotic cells with advanced age. Here, we immunophenotyped microglia in the brain of old C57BL/6 mice (>18 months old) and demonstrate that in comparison to young mice, one-third of old microglia are AF, characterized by profound changes in lipid and iron content, phagocytic activity, and oxidative stress. Pharmacological depletion of microglia in old mice eliminated the AF microglia following repopulation and reversed microglial dysfunction. Age-related neurological deficits and neurodegeneration after traumatic brain injury (TBI) were attenuated in old mice lacking AF microglia. Furthermore, increased phagocytic activity, lysosomal burden, and lipid accumulation in microglia persisted for up to 1 year after TBI, were modified by APOE4 genotype, and chronically driven by phagocyte-mediated oxidative stress. Thus, AF may reflect a pathological state in aging microglia associated with increased phagocytosis of neurons and myelin and inflammatory neurodegeneration that can be further accelerated by TBI.

Isolation-free measurement of single urinary extracellular vesicles by imaging flow cytometry

Urinary extracellular vesicles (uEVs) are promising biomarkers for various diseases. However, many tools measuring uEVs rely on time-consuming uEV isolation methods, which could induce sample bias. This study demonstrates the detection of single uEVs without isolation using imaging flow cytometry (IFCM). Unstained urine samples contained auto-fluorescent (A-F) particles when characterized with IFCM. Centrifugation successfully removed A-F particles from the unprocessed urine. Based on the disappearance of A-F particles, a gate was defined to distinguish uEVs from A-F particles. The final readouts of IFCM were verified as single EVs based on detergent treatment and serial dilutions. When developing this protocol to measure urine samples with abnormally high protein levels, 25 mg/mL dithiothreitol (DTT) showed improved uEV recovery over 200 mg/mL DTT. This study provides an isolation-free protocol using IFCM to quantify and phenotype single uEVs, eliminating the hindrance and influence of A-F particles, protein aggregates, and coincidence events.

Cerium oxide nanoparticles reduce the accumulation of autofluorescent deposits in light-induced retinal degeneration: Insights for age-related macular degeneration

Accumulation of lipofuscin deposits in the retinal pigment epithelium (RPE) is one of the main events involved in age-related macular degeneration and its increase together with RPE dysfunction, blood retinal barrier disruption and photoreceptors death progressively leads to blindness. Lipofuscin is the main autofluorescent (AF) component of the retina and therapies to counteract its deposition are a main goal to be achieved, since effective treatments have not yet been identified. Here, we first investigated the spatio-temporal pattern of AF deposits accumulation in the light-damage model of age-related macular degeneration. Afterward, we tested the ability of cerium oxide nanoparticles, a well known anti-oxidant agent, to counteract AF granules accumulation. The treatment was performed both before and after the induction of the degeneration. AF granules were quantified by confocal microscopy on whole mounted retinas. We demonstrated that the acute light-damage increases the accumulation of AF deposits in the hot spot retina in terms of number of granules and percentage of occupied area, with a peak 7 days after the exposure. Remarkably, cerium oxide nanoparticles showed a strong efficacy in preventing the formation of AF deposits when they were injected 3 days before light exposure. Moreover, when the treatment was performed 7 days after light exposure, nanoceria activity was found to be effective also in reducing the amount of the AF granules still deposited up to 60 days. These important results represent the very first evidence about the ability of cerium oxide nanoparticles to counteract AF deposits accumulation in retinal degeneration, laying the foundations for the development of a new therapy possibly targeting lipofuscin in AMD.

In vivo degradation kinetics of 3D scaffolds traced with a fluorescence marker

In vivo scaffold degradation is one of the main points in tissue engineering. There are many studies on the in vitro estimation of scaffold degradation kinetics (Zhu et al. 2013; Costa-Pinto et al. 2014; Oliveira et al. 2014). However, in vivo assessment of this parameter remains a problem; and a new in vivo non-invasive imaging method is needed to reduce the number of surgical procedures. There are several approaches to solving this problem: biopolymer crosslinking with fluorescent dyes or nanoparticles, fluorophore encapsulation into material, development of auto fluorescent polymers. Auto fluorescent biodegradable materials are the most attractive because this approach requires no use of additional fluorescent agents, which cannot be homogeneously distributed within scaffolds. This leads to incorrect degradation kinetics values. Moreover, fluorescent agents are toxic and increase the burden on normally functioning organs and tissues (liver, kidney, spleen, etc.). We showed that two-photon polymerization (2PP) enables the fabrication of auto fluorescent biodegradable scaffolds, which can be observed in real-time via in vivo non-invasive methods. We used successfully this approach in our study and revealed that polylactide 2PP scaffolds possessed auto fluorescence and were implanted after cell seeding into C57/B16 mice. The in vivo degradation rates were assessed due to changes in fluorescence intensity in real time.

Evaluation of autofluorescent Eimeria maxima oocysts as a potential indicator of non-viability when enumerating oocysts.

Aging, poor oxygenation, or improper storage temperature can lead to variable Eimeria oocyst viability, which is not readily assessed microscopically. Under fluorescent microscopy, some aged Eimeria maxima (EM) oocysts were strongly autofluorescent (AF) within the oocystoplasm and sporocysts, whereas others were distinctly non-fluorescent, leading to the hypothesis that non-viable oocysts may be detectible using this simple approach. Using accelerated aging conditions at 45°C, two experiments were conducted to evaluate variable percentages of autofluorescent EM oocysts on body weight gain (BWG), lesion scores (LS), and total oocyst shedding (OS) per bird. Through oral gavage, EM oocysts were administered on d10, whereas LS and BWG were determined d5 post-inoculation. Experiment 1 groups consisted of non-challenged controls (n = 15), or 25,000 EM exhibiting 12.8% (n = 14), 61.1% (n = 10), or 93.3% (n = 10) autofluorescence. BWG in 12.8% AF group was lower (P = 0.054) than non-challenged control. LS were higher (P < 0.05) in 61.1% and 12.8% AF groups as compared to non-challenged control and 93.3% AF groups. Experiment 2 groups consisted of non-challenged controls (LS n = 20/BWG n = 40), or 22,500 EM exhibiting 7% AF (LS n = 20/BWG n = 40), 80.6% AF (LS n = 19/BWG n = 39), or 99% AF (LS n = 19/BWG n = 39). BWG in 7% AF group was lower (P < 0.05) than non-challenged control and 99% AF groups. LS were higher (P < 0.05) in 80.6% and 7% AF groups as compared to non-challenged control and 99% AF groups. OS from d5-8 post-inoculation was determined for each of five replicates per group (n = 20/group; n = 4/replicate), with higher (P < 0.05) OS in 80.6% and 7% AF groups than in non-challenged control or 99% AF groups. Taken together, data indicate lower LS, higher BWG, and reduced OS with higher %AF oocysts, consistent with the hypothesis of lowered viable challenge with this EM isolate.

Origin of fundus hyperautofluorescent spots and their role in retinal degeneration in a mouse model of Goldmann-Favre syndrome

SUMMARYGoldmann-Favre syndrome, also known as enhanced S-cone syndrome, is an inherited retinal degeneration disease in which a gain of photoreceptor cell types results in retinal dysplasia and degeneration. Although microglia have been implicated in the pathogenesis of many neurodegenerative diseases, the fundamental role of these cells in this disease is unknown. In the current study, sequential analyses suggest that microglia are recruited and appear after outer nuclear layer folding. By crossing rd7 mice (a model for hereditary retinal degeneration owing to Nr2e3 mutation) with mice carrying the macrophage Fas-induced apoptosis (Mafia) transgene, we generated double-mutant mice and studied the role of the resident retinal microglia. Microglial cells in these double-mutant mice express enhanced green fluorescent protein (EGFP) and a suicide gene that can trigger Fas-mediated apoptosis via systemic treatment with AP20187 (FK506 dimerizer). We demonstrated that more than 80% of the EGFP+ cells in retinas from rd7/rd7;Tg/Tg mice express Iba-1 (a microglial marker), and resident microglia are still present in the retina because AP20187 does not cross the blood-brain barrier. Hence, only circulating bone marrow (BM)-derived microglia are depleted. Depletion of circulating BM-derived microglia accelerates retinal degeneration in rd7 mice. An increased number of autofluorescent (AF) spots is a consequence of resident microglia proliferation, which in turn establishes an inflammatory cytokine milieu via the upregulation of IL-1β, IL-6 and TNFα expression. This inflammation is likely to accelerate retinal degeneration. This study not only identifies inflammation as a crucial step in the pathogenesis of retinal degeneration, but also highlights the involvement of specific cytokine genes that could serve as future treatment targets in retinal degenerations.

Autofluorescence imaging of human RPE cell granules using structured illumination microscopy

Background/aimsTo characterise single autofluorescent (AF) granules in human retinal pigment epithelium (RPE) cells using structured illumination microscopy (SIM).MethodsMorphological characteristics and autofluorescence behaviour of lipofuscin (LF) and melanolipofuscin (MLF) granules of...

In vivo imaging of retinal inflammation in experimental autoimmune uveoretinitis

Abstract Purpose Experimental animal models are essential for us to understand the pathogenesis of human diseases. Posterior uveoretinitis can be modelled in mice with IRBP immunization (i.e. experimental autoimmune uveitis, EAU), whereas a number of mouse models are also available for age‐related macular degeneration (AMD). With the advancement in new technologies, it is now possible to image inflammatory retinal changes in experimental mice in vivo none invasively. The aim of the study is to clinical revisit the traditional retinal inflammation animal models with modern imaging techniques. Methods EAU was induced in C57B/6 mice with IRBP peptide 1‐20. Aged CCL2 knockout mice were used as an AMD model. Retinal inflammatory changes were imaged in vivo non‐invasively using topical endoscopic fundus imaging system and the scanning laser ophthalmoscopy (SLO) system. Results Inflammatory retinal changes in the early stages of EAU were characterised as retinal oedema, vascular sheathing, multiple small retinal infiltrates or large linear retinal infiltrates. “Snow‐ball”‐like vitreous infiltrates were observed in the inferior part of the fundus at the peak stage of EAU. Using SLO autofluorescent (AF)‐macrophages were detected at the peak stages of EAU and were located predominately around inflamed retinal venules. At the late stages of EAU, retinal scars and intraretinal neovascular membranes were observed. In the retina aged CCL2 KO mice, regional retinal atrophy and dursen‐like multiple lesions were observed. Dursen‐like changes were autofluorescent in SLO examination. Ex vivo confocal microscopy indicated that they were not dursen but subretinal lipofuscin‐loaded microglial cells. Conclusion EAU mimics many aspects of human posterior uveoretinitis including retinal vasculitis, multifocal choroiditis. Late stage EAU could be a good model for inflammation induced retinal neovascularisation. CCL2 KO mouse is a model of dry‐AMD.

Progressive myoclonic epilepsy: A clinical, electrophysiological and pathological study from South India

Progressive myoclonic epilepsy (PME) is a syndrome complex encompassing different diagnostic entities and often cause problems in diagnosis. We describe the clinical, electrophysiological and pathological features of 97 patients with the diagnosis of PME evaluated over 25 years. Case records of confirmed patients of Neuronal ceroid lipofuscinosis (NCL = 40), Lafora body disease (LBD = 38), Myoclonic epilepsy with ragged red fibers (MERRF = 10), and probable Unverricht–Lundberg disease (ULD = 9) were reviewed. The mean age at onset in patients with NCL ( n = 40) was 5.9 ± 9.1 years (M:F:: 28:12). Subtypes of NCL were: late infantile ( n = 19), infantile ( n = 8), juvenile ( n = 11) and adult ( n = 2) NCL. EEG ( n = 37) showed varying degree of diffuse slowing of background activity in 94.6% and epileptiform discharges in 81.1% of patients. Slow frequency photic stimulation evoked photo-convulsive response in 5 patients only. Giant SSEP was demonstrated in 7 and VEP study revealed a prolonged P100 (2) and absent waveform (7). Electrophysiological features of neuropathy were present in 3 patients. Presence of PAS and Luxol Fast Blue (LFB) positive, auto fluorescent (AF) ceroid material in brain tissue ( n = 12) and electron microscopy of brain ( n = 5), skin ( n = 28) and muscle ( n = 1) samples showing curvilinear and lamellar bodies established the diagnosis. Patients of LBD (mean age of onset at 14.4 ± 3.9 years, M:F:: 24:14) with triad of PME symptoms were evaluated. EEG ( n = 37) showed variable slowing of background activity in 94.6% and epileptiform discharges in 97.4%. Photosensitivity with fast frequency was observed only in 5 patients. CT ( n = 32) and MRI ( n = 4) revealed diffuse cortical atrophy. Giant SSEP was demonstrated in 24 patients of LBD while VEP study revealed a prolonged P100 (4) and absent waveform (8). Electrophysiological features of neuropathy were present in one patient. Diagnosis was established by the presence of PAS positive diastase resistant, Lugol's Iodine labeled inclusions in sweat glands of axillary skin ( n = 35), brain ( n = 2) and liver ( n = 1). Ten patients with MERRF (mean age at onset: 14.6 ± 5.8 years; M: F:: 3:2) had triad of PME symptoms. Muscle biopsy revealed oxidative reaction product and classical ragged red fibers. In nine patients of PME without cognitive decline, probable diagnosis of ULD (mean age at onset: 13.8 ± 9.5 years) was considered after biopsy of skin and/or muscle excluded other forms of PMEs. Neuronal ceroid lipofuscinosis and Lafora body diseases were the common causes of PME in the series from south India. This is one of the largest series from the Indian subcontinent to the best of our knowledge. Photosensitivity is notably less common in LBD/NCL in this series distinctly different from those reported in the literature. Further exploration is required to determine whether different genotype is responsible. Morphological changes were helpful in diagnosis and could be confirmed by biopsy of peripheral tissues like skin and muscle in majority (60%). Electron microscopy was helpful in the diagnosis NCL and MERRF.

Auto fluorescence of intervertebral disc tissue: a new diagnostic tool

The paper reports on auto fluorescence phenomena of inter-vertebral human discs. It systematically investigates the auto fluorescence effects of ex vivo disc specimen and reports on surgical cases to demonstrate the potential value of the new method. The paper offers biologic explanations of the phenomenon and discusses the potential value of the UV auto fluorescence technique as a diagnostic tool. Intra- and postoperative observations are made by a surgical microscope with an integrated UV light source. Quantitative measurements were carried out using a photon counter and a spectrometer ex vivo. The auto fluorescence phenomenon allows the differentiation of traumatized and degenerated disc tissue intraoperatively in some cases, it allows the differentiation of bony and collagen endplate in cervical disc surgery. The source of the auto fluorescent light emission are amino acids of the collagen molecules. The proteoglycan components and the liquid components of the disc do not show relevant auto fluorescence. Emission wavelength of disc material is equivalent to color perception. It differs due to different collagen composition of the intervertebral disc components from yellow-green to blue-green and can be visualized in situ by naked eye.UV-auto fluorescence of inter-vertebral discs is a new clinical tool that has the potential to differentiate disc material from the anatomical surrounding, to distinguish between different fractions of the disc and to give information on the quality and status of the disc material. Since the technology has just emerged, it needs further investigations to quantify the clinical observations reported in this paper.

Neuronal ceroid lipofuscinosis: a clinicopathological study

We report the clinical, electrophysiological, radiological and morphological features in a series of 12 patients of histopathologically confirmed cases (infantile, juvenile and adult onset) of neuronal ceroid lipofuscinosis (NCL) observed from 1979 to 1998 at National Institute of Mental Health & Neurosciences (NIMHANS), Bangalore (South India). The commonest type of NCL was juvenile ( n=8, 67%) while infantile and adult forms were two each ( n=2, 16.8%). The age at presentation ranged from 2 to 45 years (mean—12.6, 14.3 years; median—7 years; M:F ratio of 2:1). Four patients (33%) had positive family history and five patients had history of consanguineous parentage (41.6%). The commonest presenting symptoms were regression of milestones (83.3%) and/or seizures, myoclonus (83.8%) followed by involuntary choreiform movements (50%), visual loss (41.6%), ataxia (33.3%) and abnormal behaviour (16.6%). Neuro-ophthalmological abnormalities like optic atrophy (50%), macular degeneration (33.3%) and retinitis pigmentosa (8.3%) were seen in two thirds. Nerve conduction studies ( n=4) revealed abnormalities in two, suggestive of sensorimotor neuropathy. Scalp EEG ( n=9) showed slowing of background activity (BGA) of varying degrees with paroxysmal bursts of seizure discharges in majority. Cranial CT scan ( n=4) revealed varying degrees of diffuse atrophy. Diagnostic brain biopsy was carried out in 11 and brain was examined at autopsy in 1 case. Histological examination revealed characteristic PAS and Luxol Fast Blue (LFB) positive, autofluorescent (AF) intracellular ceroid material, both in neurons and astrocytes in the grey matter. Electron microscopy ( n=5) revealed curvilinear ( n=4), lamellar ( n=2) and electron dense ( n=2) inclusions in neurons, astrocytes and vascular endothelial cells. To conclude, this neurodegenerative disease had varied but characteristic clinical presentations and required histopathological confirmation of diagnosis.

UV laser induced fluorescence of unirradiated and irradiated low density polyethylene

The auto fluorescence of unirradiated and γ-rays, thermal neutrons, argon ions (Ar +) irradiated low density polyethylene (LDPE) films was investigated using a spectroscopic system incorporating a pulsed nitrogen (N 2) laser and optical multichannel analyzer (OMA). LDPE film was found to be auto fluorescent in the visible range (639 nm) after being pumped by pulsed N 2 laser in the ultraviolet (UV) range (337 nm). The fluorescence spectra was found to be affected remarkably by the type of irradiation and also on the absorbed dose showing different behavior either in the peak position or height. This behavior showed that LDPE film could be used as a fluorescence solar energy collector in the application of solar energy or laser dyes where it emits in a very important region in the visible region, which is having many applications especially for the photodynamic therapy.

Accumulation of a high molecular weight glycoprotein during in vitro ageing and contact inhibition of growth

A 240 000 molecular weight protein was found to accumulate in sorted autofluorescent (AF) cell, and during growth inhibition and in vitro ageing of cultures of human skin fibroblasts. Vitamin E, a lipophilic free radical scavenger which suppressed completely the formation of cellular autofluorescence, did not affect the accumulation of this protein. So, this accumulation is not related to cellular autofluorescence and lipid peroxidation, the major cause of this autofluorescence. This protein was also found in cells from a patient with the Spielmeyer-Vogt syndrome with a high percentage of maximal lifespan (MLS), while it was completely absent from all cells of a patient with Werner's syndrome. On two-dimensional gel electrophoresis the protein showed a heterogenous acidic isoelectric point (IEP) of around 5.3. Neuraminidase treatment caused the IEP of this protein to shift towards a less acidic pH value (5.85). Upon differential centrifugation of a cell homogenate the protein was found to be located in the microsomal pellet and the cytosol. Chromatography on gelatin-sepharose revealed that the protein was not fibronectin. It is concluded that in human skin fibroblasts a high molecular weight glycoprotein accumulates as a result of impaired proliferation and that this accumulation is not related to cellular lipid peroxidation.

Isolation of autofluorescent ‘aged’ human fibroblasts by flow sorting: Morphology, enzyme activity and proliferative capacity

In cultures of human fibroblasts the percentage of bright autofluorescent (AF) cells increases with increasing passage number. These autofluorescent cells were isolated using a FACS II cell sorter and compared with sorted non-fluorescent (NF) cells. The AF cells showed an increase in population doubling time (2.3-fold), cell protein (1.9-fold), and in specific activities of the lysosomal enzymes: β-hexosaminidase (4.2-fold), β-galactosidase (3.8-fold) and acid phosphatase (2.5-fold). The specific activities of two non-lysosomal enzymes glucose-6-phosphate dehydrogenase and lactate dehydrogenase had increased only slightly (1.1-fold) respectively (1.5-fold). The autofluorescence in the AF cells was restricted to small round organelles. The distribution and size of these autofluorescence granules were similar to the acid phosphatase-containing granules in the cytochemically stained cells. Electronmicroscopical examination showed that these AF cells contained a large amount of small electron-dense granules containing amorphosmophilic material. These granules which were positive for the acid phosphatase reaction, were classified as secondary lysosomes. The low percentage of the sorted AF cells which incorporate [ 3H]thymidine during a 24 h test period (19%) as compared with the labelling percentage of sorted NF cells (73%) from the same culture, indicate that the autofluorescent cells in a ‘young’ culture have a very limited remaining proliferative capacity. The results imply, that by flow sorting it is possible to isolate ‘aged’ cells with characteristics of ‘phase III’ cells out of non-aged fibroblast cultures.