Blue-Light Fundus Autofluorescence Imaging following Ruthenium-106 Brachytherapy for Choroidal Melanoma.

PURPOSE: To describe changes in blue-light fundus autofluorescence (FAF) and corresponding alterations in optical coherence tomography (OCT) within the irradiation field after ruthenium-106 brachytherapy (RBT) for choroidal melanoma. METHODS: Consecutive patients with choroidal melanoma were included in a retrospective case series. Patients were treated with RBT at a single institution. As part of their routine examination patients underwent multimodal imaging including ultrasonography, fundus photography, OCT, and FAF imaging (excitation = 488 nm). FAF images were analysed for changes within the irradiation field. RESULTS: 31 patients (mean age 65.7 years) were treated with RBT for unilateral choroidal melanoma. Mean tumour height before therapy was 2.7 mm (SD 1.0). Mean follow-up time was 23.3 months (SD 13.3). Main FAF characteristics attributable to RBT emerged as increased FAF with speckled decreased FAF (FAF mottling) within the irradiation field and a rim of increased FAF at its border. OCT scans demonstrated loss of the ellipsoid zone and the external limiting membrane, thinning of the neurosensory retina, and alterations of the retinal pigment epithelium like clumping, migration, and atrophy. CONCLUSIONS: FAF changes in the irradiation field after RBT of choroidal melanomas follow a characteristic pattern that correlates with distinct OCT alterations. FAF and OCT imaging give additional information to monitor effects of RBT and, therefore, complement multimodal imaging techniques after plaque therapy.

[Fundus Autofluorescence Imaging: Clinical Application and Diagnostic Relevance]. / Fundusautofluoreszenz: Klinische Anwendung und diagnostische Bedeutung.

Non-invasive fundus autofluorescence (FAF) imaging visualizes fluorophores at the level of the photoreceptors, the subneurosensory space and the retinal pigment epithelium. It gives important information over and above other imaging techniques. FAF imaging has improved the pathophysiological understanding of various retinal diseases. In combination with fundus photography, fluorescence angiography, optical coherence tomography, and electrophysiological examinations, FAF imaging is useful with regard to diagnostics and monitoring both during the natural course and after therapies. This review highlights basic principles of FAF and its clinical application in various retinal diseases.

Blue-light fundus autofluorescence imaging of pigment epithelial detachments.

BACKGROUND: Pigment epithelial detachments (PEDs) occur in association with various chorioretinal diseases. With respect to the broad clinical spectrum of PEDs we describe fundus autofluorescence (FAF) characteristics of PEDs. METHODS: Ninety-three eyes of 66 patients (mean age 71.9 ± 11.1) with uni- or bilateral PED ( ≥ 350 µm) were included in a retrospective cross-sectional study. PEDs were secondary to age-related macular degeneration (n = 79), central serous chorioretinopathy (n = 7), polypoidal choroidal vasculopathy (n = 2), pattern dystrophy (n = 3) or idiopathic PED (n = 2). FAF images were recorded using confocal scanning laser ophthalmoscopy (488 nm excitation wavelength, detection of emission >500 nm). Diagnosis of PED was confirmed using spectral-domain optical coherence tomography. A qualitative FAF grading system was established, and grading was performed by two independent readers. RESULTS: PEDs showed highly variable characteristics on FAF imaging. FAF within the area of PED was found to be irregular/granular (n = 59, 63.4%), increased (n = 28, 30.1%), decreased (n = 3, 3.2 %), or normal (n = 3, 3.2%). Accompanying FAF changes included condensation of macular pigment (n = 67, 72.0%), focally increased FAF at the PED apex (n = 14, 15.1%) or elsewhere (n = 52, 55.9%), focally decreased FAF (n = 23, 24.7%), a cartwheel-like pattern (n = 10, 10.8%), a doughnut sign (n = 6, 6.5%), and a halo of decreased FAF encircling the PED (completely n = 20, 21.5% or incompletely n = 20, 21.5%). CONCLUSIONS: PEDs show a variety of abnormal patterns on FAF imaging. These changes in FAF signals may be secondary to morphological and metabolic alterations within corresponding retinal layers and do not necessarily correspond with the underlying PED subtype or a specific pathology.

Fundus Autofluorescence Imaging in Patients with Choroidal Melanoma.

Choroidal melanocytic lesions require reliable and precise clinical examination and diagnosis to differentiate benign choroidal nevi from choroidal melanoma, as the latter may become life-threatening through metastatic disease. To come to an accurate diagnosis, as well as for monitoring, and to assess the efficacy of therapy, various imaging modalities may be used, one of which is non-invasive fundus autofluorescence (FAF) imaging using novel high-resolution digital imaging technology. FAF imaging is based on the visualization of intrinsic fluorophores in the ocular fundus. Lipofuscin and melanolipofuscin within the postmitotic retinal pigment epithelium (RPE) cells represent the major fluorophores that contribute to the FAF signal. In addition, the presence or loss of absorbing molecular constituents may have an impact on the FAF signal. A choroidal melanoma can cause secondary retinal and RPE alterations that affect the FAF signal (e.g., occurrence of orange pigment). Therefore, FAF imaging supports multimodal imaging and gives additional information over and above conventional imaging modalities regarding retinal metabolism and RPE health status. This article summarises the features of FAF imaging and the role of FAF imaging in the context of choroidal melanoma, both before and following therapeutic intervention.

Fundus autofluorescence imaging.

Fundus autofluorescence (FAF) imaging is an in vivo imaging method that allows for topographic mapping of naturally or pathologically occurring intrinsic fluorophores of the ocular fundus. The dominant sources are fluorophores accumulating as lipofuscin in lysosomal storage bodies in postmitotic retinal pigment epithelium cells as well as other fluorophores that may occur with disease in the outer retina and subretinal space. Photopigments of the photoreceptor outer segments as well as macular pigment and melanin at the fovea and parafovea may act as filters of the excitation light. FAF imaging has been shown to be useful with regard to understanding of pathophysiological mechanisms, diagnostics, phenotype-genotype correlation, identification of prognostic markers for disease progression, and novel outcome parameters to assess efficacy of interventional strategies in chorio-retinal diseases. More recently, the spectrum of FAF imaging has been expanded with increasing use of green in addition to blue FAF, introduction of spectrally-resolved FAF, near-infrared FAF, quantitative FAF imaging and fluorescence life time imaging (FLIO). This article gives an overview of basic principles, FAF findings in various retinal diseases and an update on recent developments.

Progression of geographic atrophy and impact of fundus autofluorescence patterns in age-related macular degeneration.

PURPOSE: To test if fundus autofluorescence (FAF) patterns around geographic atrophy (GA) have an impact on GA progression rates over time in atrophic age-related macular degeneration (AMD). DESIGN: Prospective longitudinal multicenter natural history study. METHODS: Standardized digital FAF images were obtained from 195 eyes of 129 patients with GA using confocal scanning laser ophthalmoscopy (excitation 488 nm, emission >500 nm). Areas of GA were quantified and patterns of abnormal FAF in the junctional zone were classified. Repeated FAF images were obtained over a median follow-up period of 1.80 years (interquartile range [IQR], 1.28 to 3.34). RESULTS: Areas of GA (median, 7.04 mm(2) at baseline; IQR, 3.12 to 10.0) showed a median enlargement of 1.52 mm(2)/year (IQR, 0.81 to 2.33). Progression rates in eyes with the banded (median 1.81 mm(2)/year) and the diffuse FAF pattern (1.77 mm(2)/year) were significantly higher compared to eyes without FAF abnormalities (0.38 mm(2)/year) and focal FAF patterns (0.81 mm(2)/year, P < .0001). Within the group of the diffuse pattern, eyes with a diffuse trickling pattern could be identified that exhibited an even higher spread rate (median 3.02 mm(2)/year) compared to the other diffuse types (1.67 mm(2)/year, P = .001). CONCLUSIONS: The results indicate that distinct phenotypic FAF patterns have an impact on disease progression in eyes with atrophic AMD and may therefore serve as prognostic determinants. The findings underscore the relevance of FAF imaging and the pathogenetic role of excessive retinal pigment epithelium (RPE) lipofuscin (LF) accumulation in GA. Natural history data and identification of high-risk characteristics will be helpful to design interventional studies aiming at slowing the spread of atrophy.

Age-related structural abnormalities in the human retina-choroid complex revealed by two-photon excited autofluorescence imaging.

The intensive metabolism of photoreceptors is delicately maintained by the retinal pigment epithelium (RPE) and the choroid. Dysfunction of either the RPE or choroid may lead to severe damage to the retina. Two-photon excited autofluorescence (TPEF) from endogenous fluorophores in the human retina provides a novel opportunity to reveal age-related structural abnormalities in the retina-choroid complex prior to apparent pathological manifestations of age-related retinal diseases. In the photoreceptor layer, the regularity of the macular photoreceptor mosaic is preserved during aging. In the RPE, enlarged lipofuscin granules demonstrate significantly blue-shifted autofluorescence, which coincides with the depletion of melanin pigments. Prominent fibrillar structures in elderly Bruch's membrane and choriocapillaries represent choroidal structure and permeability alterations. Requiring neither slicing nor labeling, TPEF imaging is an elegant and highly efficient tool to delineate the thick, fragile, and opaque retina-choroid complex, and may provide clues to the trigger events of age-related macular degeneration.

Two-photon-excited fluorescence imaging of human RPE cells with a femtosecond Ti:Sapphire laser.

PURPOSE: To record the distribution and spectrum of human retinal pigment epithelial cell lipofuscin (LF) by two-photon-excited fluorescence (TPEF) and confocal laser scanning microscopy. METHODS: Ex vivo TPEF imaging of the human retinal pigment epithelium (RPE) of human donor eyes was conducted with a multiphoton laser scanning microscope that employs a femtosecond Ti:sapphire laser as an excitation laser source. The spectrum of autofluorescence of LF granules was analyzed with a confocal laser scanning microscope coupled to a UV argon laser. RESULTS: TPEF examination allowed for imaging of RPE cell morphology and intracellular distribution of LF granules with high-contrast and submicrometer resolution. Variations in cell size and shape as well as in autofluorescence spectra of individual LF granules were recorded. The typical diameter of LF granules was found to be below 1 mum, with some RPE cells possessing larger granules. Remarkably, enhanced blue-green autofluorescence was observed from these larger LF granules. CONCLUSIONS: TPEF imaging represents a novel tool for the investigation of morphologic and spectral characteristics of human RPE cells. Spectral variations of individual LF granules may indicate differences in the complex molecular composition. Compared to conventional single-photon excited autofluorescence, TPEF with a tunable laser source allows for reduced photo damage and deeper sensing depth. It may help to elucidate further the pathophysiological role of LF accumulation as a common downstream pathogenetic pathway in retinal diseases. With the proof of principle from this ex vivo study, further work is now planned to evaluate the safety of TPEF RPE imaging in RPE cultures and animal models.

Correlation between the area of increased autofluorescence surrounding geographic atrophy and disease progression in patients with AMD.

PURPOSE: To test the hypothesis that the extension of areas with increased fundus autofluorescence (FAF) outside atrophic patches correlates with the rate of spread of geographic atrophy (GA) over time in eyes with age-related macular degeneration (AMD). METHODS: The database of the multicenter longitudinal natural history Fundus Autofluorescence in AMD (FAM) Study was reviewed for patients with GA recruited through the end of August 2003, with follow-up examinations within at least 1 year. Only eyes with sufficient image quality and with diffuse patterns of increased FAF surrounding atrophy were chosen. In standardized digital FAF images (excitation, 488 nm; emission, >500 nm), total size and spread of GA was measured. The convex hull (CH) of increased FAF as the minimum polygon encompassing the entire area of increased FAF surrounding the central atrophic patches was quantified at baseline. Statistical analysis was performed with the Spearman's rank correlation coefficient (rho). RESULTS: Thirty-nine eyes of 32 patients were included (median age, 75.0 years; interquartile range [IQR], 67.8-78.9); median follow-up, 1.87 years; IQR, 1.43-3.37). At baseline, the median total size of atrophy was 7.04 mm2 (IQR, 4.20-9.88). The median size of the CH was 21.47 mm2 (IQR, 15.19-28.26). The median rate of GA progression was 1.72 mm2 per year (IQR, 1.10-2.83). The area of increased FAF around the atrophy (difference between the CH and the total GA size at baseline) showed a positive correlation with GA enlargement over time (rho=0.60; P=0.0002). CONCLUSIONS: FAF characteristics that are not identified by fundus photography or fluorescein angiography may serve as a prognostic determinant in advanced atrophic AMD. As the FAF signal originates from lipofuscin (LF) in postmitotic RPE cells and since increased FAF indicates excessive LF accumulation, these findings would underscore the pathophysiological role of RPE-LF in AMD pathogenesis.

Two-photon excited autofluorescence imaging of human retinal pigment epithelial cells.

Degeneration of retinal pigment epithelial (RPE) cells severely impairs the visual function of retina photoreceptors. However, little is known about the events that trigger the death of RPE cells at the subcellular level. Two-photon excited autofluorescence (TPEF) imaging of RPE cells proves to be well suited to investigate both the morphological and the spectral characteristics of the human RPE cells. The dominant fluorophores of autofluorescence derive from lipofuscin (LF) granules that accumulate in the cytoplasm of the RPE cells with increasing age. Spectral TPEF imaging reveals the existence of abnormal LF granules with blue shifted autofluorescence in RPE cells of aging patients and brings new insights into the complicated composition of the LF granules. Based on a proposed two-photon laser scanning ophthalmoscope, TPEF imaging of the living retina may be valuable for diagnostic and pathological studies of age related eye diseases.

A subgroup of age-related macular degeneration is associated with mono-allelic sequence variants in the ABCA4 gene.

Purpose. Age-related macular degeneration (AMD) is a heterogeneous condition of high prevalence and complex etiology involving genetic as well as environmental factors. By fundus autofluorescence (FAF) imaging, AMD can be classified into several distinct phenotypes, with one subgroup characterized by fine granular pattern with peripheral punctate spots (GPS[+]). Some features of GPS[+] overlap with Stargardt disease (STGD1), a recessive macular dystrophy caused by biallelic sequence variants in the ATP-binding cassette transporter 4 (ABCA4) gene. The aim of this study was to investigate the role of ABCA4 in GPS[+]. Methods. The ABCA4 gene was sequenced in 25 patients with the GPS[+] phenotype and 29 with geographic atrophy (GA)-AMD but no signs of GPS (GPS[-]). In addition, frequencies of risk-increasing alleles at three known AMD susceptibility loci, including complement factor H (CFH), age-related maculopathy susceptibility 2 (ARMS2), and complement component 3 (C3), were evaluated. Results. We demonstrate that GPS[+] is associated significantly with monoallelic ABCA4 sequence variants. Moreover, frequencies of AMD risk-increasing alleles at CFH, ARMS2, and C3 are similar in GPS[+] and STGD1 patients, with risk allele frequencies in both subcategories comparable to population-based control individuals estimated from 3,510 individuals from the NHLBI Exome Sequencing Project. Conclusions. Our data suggest that the GPS[+] phenotype is accounted for by monoallelic variants in ABCA4 and unlikely by the well-established AMD risk-increasing alleles at CFH, ARMS2, and C3. These findings provide support for a complex role of ABCA4 in the etiology of a minor proportion of patients with AMD.

Concordance of disease progression in bilateral geographic atrophy due to AMD.

PURPOSE: To determine the degree of concordance for progression rate, size of atrophy, and visual acuity in patients with bilateral geographic atrophy (GA) due to age-related macular degeneration (AMD). METHODS: Analysis was performed in 156 eyes of 78 patients with bilateral GA. Best corrected visual acuity was determined with ETDRS charts. GA was quantified in digital fundus autofluorescence images (excitation, 488 nm; emission, >500 nm) by semiautomated imaging analysis. A linear, two-level, random-effects model was used to assess the natural course of disease. The concordance correlation coefficient (CCC) was calculated to assess the degree of agreement between disease characteristics of the left and right eyes. Bland-Altman plots were applied to compare measurements in the eyes. RESULTS: CCC between the eyes was 0.310 (95% CI, 0.097-0.495) for visual acuity, 0.706 (95% CI, 0.575-0.801) for GA size, and 0.756 (95% CI, 0.644-0.837) for GA progression rate. Although Bland-Altman plots revealed high concordance for the progression rate, there was considerable discrepancy between both eyes for GA size. [corrected] CONCLUSIONS: GA progression in bilateral atrophic AMD is a symmetrical process; however, GA size may differ substantially between the eyes. High concordance in intraindividual disease progression in the presence of a high degree of interindividual variability indicates an influence by genetic and/or environmental factors rather than nonspecific ageing changes. The relatively small concordance of GA size in this cohort may indicate asymmetric evolution of the disease in affected individuals. The results may be useful in the design of future clinical trials designed to slow the rate of GA progression (Clinical Trials.gov number, NCT00393692).