Visualizing melanosomes, lipofuscin, and melanolipofuscin in human retinal pigment epithelium using serial block face scanning electron microscopy

Pollreisz, Andreas; Messinger, Jeffrey D.; Sloan, Kenneth R.; Mittermueller, Tamara; Weinhandl, Alexandra Stephanie; Benson, Emily; Kidd, Grahame J.; Schmidt‐Erfurth, Ursula; Curcio, Christine A.

doi:10.1016/j.exer.2017.10.018

Cited by 62 publications

(86 citation statements)

References 54 publications

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“…However, a similar appearance was seen in peripheral zones in our oldest healthy subject (#4, Fig. 3 zoom at 10° temporal), so this may perhaps reflect an age-related aspect of fluorophore distribution [9] rather than an indication of retinal pathology. Future work is needed to better characterize and distinguish the changes that occur from normal aging from those that occur on the pathway to age related diseases such as AMD.…”

Section: Imaging the Rpe Mosaic With Niraf In Diseased Eyessupporting

confidence: 59%

“…Concentrations of intrinsic RPE fluorophores follow an inverse relation across the macula with melanin peaking in the fovea and lipofuscin in the periphery. In addition, melanolipofuscin forms with age [9] in quantities closer to that of lipofuscin than that of melanin. This means that both SWAF and NIRAF may potentially be generated from the same structures, containing both fluorophores.…”

Section: Introductionmentioning

confidence: 98%

“…However, NIRAF has garnered increased interest recently as it is comfortable for patients and provides complementary information [5]. In addition, considering recent findings regarding the high proportion of melanolipofuscin in aging eyes [9], the NIRAF signal may serve as a surrogate for SWAF and biomarker of RPE status in diseased eyes.…”

Section: Introductionmentioning

confidence: 99%

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In vivo near-infrared autofluorescence imaging of retinal pigment epithelial cells with 757 nm excitation

Grieve¹,

Gofas-Salas²,

Ferguson³

et al. 2018

Biomed. Opt. Express

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Pâques, et al.. In vivo near-infrared autofluorescence imaging of retinal pigment epithelial cells with 757 nm excitation.Abstract: We demonstrate near-infrared autofluorescence (NIRAF) imaging of retinal pigment epithelial (RPE) cells in vivo in healthy volunteers and patients using a 757 nm excitation source in adaptive optics scanning laser ophthalmoscopy (AOSLO). NIRAF excited at 757 nm and collected in an emission band from 778 to 810 nm produced a robust NIRAF signal, presumably arising from melanin, and revealed the typical hexagonal mosaic of RPE cells at most eccentricities imaged within the macula of normal eyes. Several patterns of altered NIRAF structure were seen in patients, including disruption of the NIRAF over a drusen, diffuse hyper NIRAF signal with loss of individual cell delineation in a case of non-neovascular age-related macular degeneration (AMD), and increased visibility of the RPE mosaic under an area showing loss of photoreceptors. In some participants, a superposed cone mosaic was clearly visible in the fluorescence channel at eccentricities between 2 and 6° from the fovea. This was reproducible in these participants and existed despite the use of emission filters with an optical attenuation density of 12 at the excitation wavelength, minimizing the possibility that this was due to bleed through of the excitation light. This cone signal may be a consequence of cone waveguiding on either the ingoing excitation light and/or the outgoing NIRAF emitted by fluorophores within the RPE and/or choroid and warrants further investigation. NIRAF imaging at 757 nm offers efficient signal excitation and detection, revealing structural alterations in retinal disease with good contrast and shows promise as a tool for monitoring future therapies at the level of single RPE cells.

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Section: Imaging the Rpe Mosaic With Niraf In Diseased Eyessupporting

confidence: 59%

Section: Introductionmentioning

confidence: 98%

See 1 more Smart Citation

In vivo near-infrared autofluorescence imaging of retinal pigment epithelial cells with 757 nm excitation

Grieve¹,

Gofas-Salas²,

Ferguson³

et al. 2018

Biomed. Opt. Express

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show abstract

“…A2E was reported in the range of 200 to 800 ng per eye [27]. In another study, A2E was quantified as 7.8 × 10 −20 mol per lipofuscin granule in the human RPE [28], leading to an estimation of 55 ng A2E per eye, given that a human eye has 3.56 million RPE cells [29] and that each cell contains 300 or so lipofuscin granules on average (in samples from individuals > 70-years-old) [30]. The significant difference in A2E levels between our study and those reported by others could be due to the different methodologies used.…”

Section: Discussionmentioning

confidence: 99%

A2E Distribution in RPE Granules in Human Eyes

Guan

Jiao

et al. 2020

Molecules

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A2E (N-retinylidene-N-retinylethanolamine) is a major fluorophore in the RPE (retinal pigment epithelium). To identify and characterize A2E-rich RPE lipofuscin, we fractionated RPE granules from human donor eyes into five fractions (F1–F5 in ascending order of density) by discontinuous sucrose density gradient centrifugation. The dry weight of each fraction was measured and A2E was quantified by liquid chromatography/mass spectrometry (LC/MS) using a synthetic A2E homolog as a standard. Autofluorescence emission was characterized by a customer-built spectro-fluorometer system. A significant A2E level was detected in every fraction, and the highest level was found in F1, a low-density fraction that makes up half of the total weight of all RPE granules, contains 67% of all A2E, and emits 75% of projected autofluorescence by all RPE granules. This group of RPE granules, not described previously, is therefore the most abundant RPE lipofuscin granule population. A progressive decrease in autofluorescence was observed from F2 to F4, whereas no autofluorescence emission was detected from the heavily pigmented F5. The identification of a novel and major RPE lipofuscin population could have significant implications in our understanding of A2E and lipofuscin in human RPE.

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“…At the larger end of the scale the technique was used to image and model the entire silk spinneret system, including glands, nerves and other tissues, of a tanaid crustacean (Kaji et al, 2016). Structures which are dozens of microns in length such as axons (Kornfeld et al, 2017) and entire collagen fibrils (Svensson et al, 2017) can be imaged as well as large populations of cells in one automated run (Pollreisz et al, 2018;Salo et al, 2018). At the level of whole cells, SFBFEM has been used to image whole glomerular podocytes (Lausecker et al, 2018) and parasitic T. brucei cells (Hughes et al, 2017).…”

Section: Serial Block Face Scanning Electron Microscopymentioning

confidence: 99%

Serial block face scanning electron microscopy in cell biology: Applications and technology

Smith

Starborg

2019

Tissue and Cell

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Three-dimensional electron microscopy (3DEM) is an imaging field containing several powerful modalities such as serial section transmission electron microscopy and electron tomography. However, large-scale 3D studies of biological ultrastructure on a cellular scale have historically been hampered by the difficulty of available techniques. Serial block face scanning electron microscopy (SBFSEM) is a 3DEM technique, developed in 2004, which has greatly increased the reliability, availability and throughput of 3DEM. SBFSEM allows for 3D imaging at resolutions high enough to resolve membranes and small vesicles whilst having the capability to collect data with a large field of view. Since its introduction it has become a major tool for ultrastructural investigation and has been applied in the study of many biological fields, such as connectomics, cellular and matrix biology. In this review, we will discuss biological SBFSEM from a technical standpoint, with a focus on cellular applications and also subsequent image analysis techniques.

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Visualizing melanosomes, lipofuscin, and melanolipofuscin in human retinal pigment epithelium using serial block face scanning electron microscopy

Cited by 62 publications

References 54 publications

In vivo near-infrared autofluorescence imaging of retinal pigment epithelial cells with 757 nm excitation

In vivo near-infrared autofluorescence imaging of retinal pigment epithelial cells with 757 nm excitation

A2E Distribution in RPE Granules in Human Eyes

Serial block face scanning electron microscopy in cell biology: Applications and technology

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