Autofluorescence imaging of human RPE cell granules using structured illumination microscopy

Ach, Thomas; Best, Gerrit; Roßberger, Sabrina; Heintzmann, Rainer; Cremer, Christoph; Dithmar, Stefan

doi:10.1136/bjophthalmol-2012-301547

Cited by 23 publications

(21 citation statements)

References 17 publications

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“…Both lipofuscin and melanolipofuscin have autofluorescent properties and show a specific topographic distribution (Ach et al. , , ). Fundus autofluorescence (AF) of lipofuscin has long been thought to originate from bisretinoid fluorophores in the RPE cells, particularly A2E (Sparrow et al.…”

Section: Introductionmentioning

confidence: 99%

“…Lipofuscin redistribution, loss of photoreceptors and changes in RPE cells as well as changes in the cytoskeleton can be observed in the course of diseases such as AMD (Curcio et al 1996;Adler et al 1999;Rossberger et al 2013;Ach et al 2015). Both lipofuscin and melanolipofuscin have autofluorescent properties and show a specific topographic distribution (Ach et al 2012(Ach et al , 2014(Ach et al , 2015. Fundus autofluorescence (AF) of lipofuscin has long been thought to originate from bisretinoid fluorophores in the RPE cells, particularly A2E Yamamoto et al 2012), but more recently this most studied bisretinoid (and hence its derivatives) has been found in low quantities in the macula of humans (Ablonczy et al 2013).…”

Section: Introductionmentioning

confidence: 99%

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Repeatability and reliability of quantitative fundus autofluorescence imaging in patients with early and intermediate age‐related macular degeneration

Reiter

Told

Baratsits

et al. 2018

Acta Ophthalmologica

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Purpose Quantification of fundus autofluorescence has only recently become available. We report our findings on the evaluation of the repeatability and reliability of quantitative fundus autofluorescence (qAF) measurements in patients with early and intermediate age‐related macular degeneration (AMD), using the first approved and commercially available instrument. Methods A total of 43 eyes of 22 patients (aged between 52 and 84 years) diagnosed with early and intermediate AMD were included. All eyes were imaged at day 1, 3 months and 6 months using a modified scanning laser ophthalmoscope, equipped with an internal fluorescent reference. Mean qAF values were calculated for the fovea and for each concentric ring of the Delori pattern. Repeatability and reliability were calculated using Bland–Altman analysis and intraclass correlation (ICC). Results The mean patient age was 73.5 ± 7.9 years. Sixteen patients (73%) were female. qAF repeatability of the eight segments in the middle ring of the Delori pattern (qAFM8) for between sessions was ±8.2%. Agreement at 3‐ and 6‐month follow‐up in eyes without retinal changes was ±8.3% and ±9.8%, respectively. Reliability of qAFM8 was high for all images acquired [ICC = 0.98 (CI: 0.96–0.99), 0.97 (0.93–0.99) and 0.98 (0.92–0.99)]. Agreement at 3‐ and 6‐month follow‐up in eyes with retinal changes was ±18.1% and ±20.2%, respectively. Intraclass correlation (ICC) was slightly lower in eyes with retinal changes at 0.93 (0.84–0.97) and 0.96 (0.91–0.98), respectively. Conclusions Quantitative autofluorescence shows excellent repeatability and reliability as well as follow‐up agreement in patients with early and intermediate AMD without retinal changes. This is relevant when conducting longitudinal studies using qAF.

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Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Repeatability and reliability of quantitative fundus autofluorescence imaging in patients with early and intermediate age‐related macular degeneration

Reiter

Told

Baratsits

et al. 2018

Acta Ophthalmologica

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“…4). Thus, the four RPE sources all localized to areas surrounding the nuclei in a manner characteristic of lipofuscin and melanolipofuscin in organelles that are known to be autofluorescent [13,30]. Confirmation of subcellular fluorophore attribution awaits further investigation with higher resolution microscopy techniques.…”

Section: Step 3: Nmf Of Single Excitation Hyperspectral Data Setsmentioning

confidence: 99%

Simultaneous decomposition of multiple hyperspectral data sets: signal recovery of unknown fluorophores in the retinal pigment epithelium

Rjh

Post

Johri

et al. 2014

Biomed. Opt. Express

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Abstract:Upon excitation with different wavelengths of light, biological tissues emit distinct but related autofluorescence signals. We used nonnegative matrix factorization (NMF) to simultaneously decompose coregistered hyperspectral emission data from human retinal pigment epithelium/Bruch's membrane specimens illuminated with 436 and 480 nm light. NMF analysis was initialized with Gaussian mixture model fits and constrained to provide identical abundance images for the two excitation wavelengths. Spectra recovered this way were smoother than those obtained separately; fluorophore abundances more clearly localized within tissue compartments. These studies provide evidence that leveraging multiple coregistered hyperspectral emission data sets is preferential for identifying biologically relevant fluorophore information.

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“…Multi-coloured 3D-SIM can also be achieved by illuminating the specimen with three beams of interfering light and observing the interference pattern along x , y and z axes [17]. SIM has been used to gain new insights into the organization features of the pigment granules of retinal pigment epithelial cells [18] and the membrane network and membrane-mediated pathways critical for the establishment of infection of erythrocytes by the malaria parasite, Plasmodium falciparum [19]. …”

Section: Super-resolution Light Microscopy: Resolving Nanomoleculamentioning

confidence: 99%

Imaging and Quantitation Techniques for Tracking Cargo along Endosome-to-Golgi Transport Pathways

Chia

Gleeson

2013

Cells

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Recent improvements in the resolution of light microscopy, coupled with the development of a range of fluorescent-based probes, have provided new approaches to dissecting membrane domains and the regulation of membrane trafficking. Here, we review these advances, as well as highlight developments in quantitative image analysis and novel unbiased analytical approaches to quantitate protein localization. The application of these approaches to endosomal sorting and endosome-to-Golgi transport is discussed.

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Autofluorescence imaging of human RPE cell granules using structured illumination microscopy

Abstract: SIM is a useful tool for examining AF signals within single LF and MLF granules in RPE cells. This allows new insights into RPE autofluorescence patterns.

Cited by 23 publications

References 17 publications

Repeatability and reliability of quantitative fundus autofluorescence imaging in patients with early and intermediate age‐related macular degeneration

Repeatability and reliability of quantitative fundus autofluorescence imaging in patients with early and intermediate age‐related macular degeneration

Simultaneous decomposition of multiple hyperspectral data sets: signal recovery of unknown fluorophores in the retinal pigment epithelium

Imaging and Quantitation Techniques for Tracking Cargo along Endosome-to-Golgi Transport Pathways

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