In vivo multimodal retinal imaging of disease-related pigmentary changes in retinal pigment epithelium
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Abstract Melanosomes, lipofuscin, and melanolipofuscin are the three principal types of pigmented granules found in retinal pigment epithelium (RPE) cells. Changes in the density of melanosomes and lipofuscin in RPE cells are considered hallmarks of various retinal diseases, including Stargardt disease and age-related macular degeneration (AMD). Herein, we report the potential of an in vivo multimodal imaging technique based on directional back-scattering and short-wavelength fundus autofluorescence (SW-FAF) to study disease-related changes in the density of melanosomes and lipofuscin granules in RPE cells. Changes in the concentration of these granules in Abca4 −/− mice (a model of Stargardt disease) relative to age-matched wild-type (WT) controls were investigated. Directional optical coherence tomography (dOCT) was used to assess melanosome density in vivo, whereas the autofluorescence (AF) images and emission spectra acquired with a spectrometer-integrated scanning laser ophthalmoscope (SLO) were used to characterize lipofuscin and melanolipofuscin granules in the same RPE region. Subcellular-resolution ex vivo imaging using confocal fluorescence microscopy and electron microscopy was performed on the same tissue region to visualize and quantify melanosomes, lipofuscin, and melanolipofuscin granules. Comparisons between in vivo and ex vivo results confirmed an increased concentration of lipofuscin granules and decreased concentration of melanosomes in the RPE of Abca4 −/− mice, and provided an explanation for the differences in fluorescence and directionality of RPE scattering observed in vivo between the two mouse strains.Keywords:
Lipofuscin
Stargardt disease
Autofluorescence
Melanosome
Ex vivo
It is thought that lipofuscin plays a central role in the pathogenesis of age-related macular degeneration (AMD). The lack of histopathological material has been a severe limitation in our knowledge on lipofuscin in this disease. A new technique has been developed that allows in vivo imaging of fundus autofluorescence derived from lipofuscin in the retinal pigment epithelium (RPE) using a confocal Laser Scanning Ophthalmoscope (LSO). We studied the dynamics of lipofuscin accumulation and degradation in patients with AMD.Serial examinations of the spatial distribution of fundus autofluorescence were performed in 148 eyes of 74 patients with AMD using a LSO over a period of 1-3.5 years.Fundus autofluorescence changed over time in almost all eyes studied. Areas of increased autofluorescence occurred progressively during follow up in eyes with drusen and hyperpigmentation. The size of pathologic autofluorescence increased over time in almost all eyes with geographic atrophy, subretinal neovascularisations and disciform scars. Irregular autofluorescence was seen over most subretinal neovascularisations. Autofluorescence intensity decreased in old subretinal neovascularisations and disciform scars over time.Changes of the distribution of autofluorescence occur in eyes with AMD over time. Fundus autofluorescence imaging allows in vivo analysis of the dynamics of accumulation and degradation of lipofuscin in the RPE in eyes with AMD and documentation of metabolic activity of the RPE.
Lipofuscin
Autofluorescence
Stargardt disease
Fundus (uterus)
Drusen
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The retinal pigment epithelium contains three major types of pigment granules; melanosomes, lipofuscin and melanolipofuscin. Melanosomes in the retinal pigment epithelium (RPE) are formed during embryogenesis and mature during early postnatal life while lipofuscin and melanolipofuscin granules accumulate as a function of age. The difficulty in studying the formation and consequences of melanosomes and lipofuscin granules in RPE cell culture is compounded by the fact that these pigment granules do not normally occur in established RPE cell lines and pigment granules are rapidly lost in adult human primary culture. This review will consider options available for overcoming these limitations and permitting the study of melanosomes and lipofuscin in cell culture and will briefly evaluate the advantages and disadvantages of the different protocols.
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Melanosome
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Abstract Melanosomes, lipofuscin, and melanolipofuscin are the three principal types of pigmented granules found in retinal pigment epithelium (RPE) cells. Changes in the density of melanosomes and lipofuscin in RPE cells are considered hallmarks of various retinal diseases, including Stargardt disease and age-related macular degeneration (AMD). Herein, we report the potential of an in vivo multimodal imaging technique based on directional back-scattering and short-wavelength fundus autofluorescence (SW-FAF) to study disease-related changes in the density of melanosomes and lipofuscin granules in RPE cells. Changes in the concentration of these granules in Abca4 −/− mice (a model of Stargardt disease) relative to age-matched wild-type (WT) controls were investigated. Directional optical coherence tomography (dOCT) was used to assess melanosome density in vivo, whereas the autofluorescence (AF) images and emission spectra acquired with a spectrometer-integrated scanning laser ophthalmoscope (SLO) were used to characterize lipofuscin and melanolipofuscin granules in the same RPE region. Subcellular-resolution ex vivo imaging using confocal fluorescence microscopy and electron microscopy was performed on the same tissue region to visualize and quantify melanosomes, lipofuscin, and melanolipofuscin granules. Comparisons between in vivo and ex vivo results confirmed an increased concentration of lipofuscin granules and decreased concentration of melanosomes in the RPE of Abca4 −/− mice, and provided an explanation for the differences in fluorescence and directionality of RPE scattering observed in vivo between the two mouse strains.
Lipofuscin
Stargardt disease
Autofluorescence
Melanosome
Ex vivo
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Lipofuscin
Melanosome
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We measured autofluorescence of the macula with fluorophotometry to evaluate age-related changes in human retinal pigment epithelium. Examined in this study were 35 aphakic eyes of 25 patients, ranging in age from 52 to 87 years, after uneventful intracapsular cataract extraction and 21 normal phakic eyes of 20 patients, ranging in age from 9 to 29 years. Autofluorescence at the macula of aphakic eyes increased in an age-dependent manner (r = 0.514; p less than 0.01) as follows: 15.0 ngEq/ml for the sixth decade (n = 1), 17.2 +/- 4.2 for the seventh decade (n = 11), 21.3 +/- 3.6 for the eighth decade (n = 16) and 24.6 +/- 2.7 for the ninth decade (n = 7). We believe that the autofluorescence originates mainly from lipofuscin in the retinal pigment epithelium, and that the autofluorescence enhanced with age reflects the accumulation of lipofuscin.
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We report on our investigation of the spatial distribution and autofluorescence lifetime characterization of lipofuscin and oxidized melanin in the retinal pigment epithelium cells of the pig eye using a two-photon excitation fluorescence lifetime imaging microscopy (TPE-FLIM) system, which that is based on a time-correlated single photon counting technique. In particular, we analyzed the difference of autofluorescence lifetimes of these pigment granules in light-induced oxidizing environment. The experimental results showed that the fluorescence lifetime imaging can provide an effective differentiation of multi-component fluorophores, and fluorescence decay can be used to distinguish normal from abnormal fluorescence. TPE-FLIM has the potential to provide a high sensitive imaging instrument for the clinical diagnosis and pathological studies in ophthalmology, and is also of significance to the study of aging mechanism of cells in the fundus.
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The fluorescent pigment lipofuscin accumulates within the cytoplasm of cells of most of the organs. Because of its broad excitation and emission spectra, the presence of lipofuscin autofluorescence complicates the use of fluorescence microscopy. In this study, we examined chemical treatments of tissue sections with two concentration of CuSO4 for their ability to reduce or eliminate lipofuscin-like autofluorescence without adversely affecting other fluorescent labels. We found that 4mM CuSO4 in 50 mM ammonium acetate buffer mildly eliminated lipofuscin autofluorescence in sections of human tonsil or mouse synovial tissue, whereas, at higher concentration (10mM) the autofluorescence was almost completely abolished. We found that this treatment had no effect on the fluorescence intensity of target tissue. We conclude that treatment of tissue with CuSO4 provides a reasonable compromise between reduction of lipofuscin-like fluorescence and maintenance of specific fluorescent labels.
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Lipofuscin, a by-product of the vision cycle of photoreceptors, is the major source of the fundus auto-fluorescence (FAF) in the Retinal Pigment Epithelium (RPE). In other words, a complex mixture of partially digested lipids and protein components in the retinal pigment epithelium (RPE) cells, is a major source of fundus autofluorescence (FAF). FAF hence is a natural biomarker that carries the information of lipofuscin content, and quantification of FAF signal could be used to assess the amount of lipofuscin in the RPE for diagnosis and monitoring disease progression. FAF imaging has been used in ophthalmology clinics for many years. For example, hyper-autofluorescence is positively correlated with the progression of AMD and Stargate’s macular dystrophy. In the case of geographic atrophy (GA), the late stage of dry AMD, advanced RPE alterations exhibit clinically recognizable patterns of hyper-autofluorescence, which is positively correlated with the rate of GA progression and can be analysed semi-automatically with newly developed software by non-expert graders.
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Fundus (uterus)
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Lipofuscin is the autofluorescent material, which accumulates with aging in the cells of various tissues. However, its autofluorescence characteristics are different among tissues. In the present study, the autofluorescence features of lipofuscin in the brain and adrenal were compared. In 18–21-month-old rats, the brain lipofuscin was granular and its autofluorescence was bright whitish-yellow to bright orange. On the contrary, the adrenal lipofuscin was not demarkated as granules, and its autofluorescence was subdued orange. The emission maximum of the bright whitish-yellow brain lipofuscin was 540 nm to 570 nm and that of the adrenal lipofuscin was 640 nm to 660 nm, when excited at 330 nm to 380 nm. When the spectra were drawn after correcting the wavelength-dependent bias of microspectrofluorometer, the autofluorescence spectra were consistent with microscopically observable tint. To conclude, the present results showed that the autofluorescence features of the bright whitish-yellow brain lipofuscin and the adrenal lipofuscin were quite different.
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Abstract Melanosomes, lipofuscin, and melanolipofuscin are the three principal types of granular pigmented organelles found in the retinal pigment epithelium (RPE) cells. Changes in the density of melanosomes and lipofuscin granules in RPE cells are considered hallmarks of various retinal diseases, including Stargardt disease and age-related macular degeneration (AMD). Herein, we report the potential of an in vivo multimodal imaging technique based on directional back-scattering and short-wavelength fundus autofluorescence (SW-FAF) to study the disease-related changes in the density of melanosomes and lipofuscin granules in RPE cells. Changes in the concentration of these granules in Abca4 -/- mice (a model of Stargardt disease) relative to age-matched wild-type (WT) controls were investigated. Directional optical coherence tomography (dOCT) was used to assess the melanosome density in vivo , whereas the AF images and emission spectra acquired with a spectrometer-integrated scanning laser ophthalmoscope (SLO) were used to characterize the lipofuscin and melanolipofuscin granules in the same RPE region. Subcellular-resolution ex vivo imaging using confocal fluorescence microscopy and electron microscopy was performed on the same tissue region to visualize and quantify melanosomes, lipofuscin, and melanolipofuscin granules. Comparisons between in vivo and ex vivo results confirmed an increased concentration of lipofuscin granules and decreased number of melanosomes in the RPE of Abca4 -/- mice and provided an explanation for the differences in fluorescence and directionality of RPE scattering observed in vivo in the two mouse strains.
Lipofuscin
Stargardt disease
Melanosome
Ex vivo
Autofluorescence
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