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Thomas Ach, Sungmin Hong, Rainer Heintzmann, Jost Hillenkamp, Kenneth R Sloan, Neel S Dey, Guido Gerig, Theodore Smith, Christine Curcio, Katharina Bermond; High-resolution and multispectral imaging of autofluorescent retinal pigment epithelium (RPE) granules. Invest. Ophthalmol. Vis. Sci. 2017;58(8):3382.
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© ARVO (1962-2015); The Authors (2016-present)
To image and analyze individual RPE melanosomes (M), lipofuscin (LF), and melanolipofuscin (MLF) granules using high-resolution structured illumination microscopy (hrSIM) and confocal multispectral laser scanning microscopy (cmLSM).
Human donor RPE-flatmounts (n=35; normal macular status: 9<51yrs, 9>80yrs; age-related macular degeneration (AMD): 17) were scanned apical to basal through RPE cells at the fovea, perifovea, and near periphery using hrSIM (Zeiss Elyra.S1; ex488 nm; em>510 nm; 100 nm step size) and cmLSM (Zeiss LSM780; ex488 nm; em 490-695 nm; 390 nm step size; 8.9 nm spectral channel width). The hrSIM and lower-resolution cmLSM images were co-registered by linear 3D registration and choice of mutual information as the image match criterion (PMID16545965). This results in a 1:1 mapping between the single channel hrSIM and multichannel cmLSM data. Individual granules were segmented from the hrSIM data by expert-guided 3D level-set segmentation. Via the hrSIM-cmLSM mapping, the spectra of individual granules can be extracted for quantitative analysis. M, LF, MLF granules/cell were also counted using a custom FIJI plugin.
HrSIM imaging and segmentation enables clear delineation and identification of M, LF, and MLF granules (Fig. A,B). Individual granules can be tracked in the z-direction, and size, shape, dimensions, and intracellular position can be monitored. Each cell contains several hundred granules. A cushion of M localizes apically, while LF/MLF prefer basolateral accumulation. Software-assisted mapping of corresponding z-sections (hrSIM/cmLSM) for spectral characterization (Fig. C,D) demonstrates spectral variability among granules..
With the combination of hrSIM and cmLSM imaging, individual autofluorescent RPE granules can be identified, localized in three-dimensions, and spectrally analyzed. The examination of spectral characteristics and changes related to intracellular and tissue localization, age, and disease status, on an individual granule basis, might reveal LF/MLF metabolism, and help elucidate LF`s role in human RPE physiology. Ongoing studies are examining RPE granule properties in aging and AMD.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.
A. Cropped (from an overview, yellow box) and mapped hrSIM and cmLSM images. B. Further zoom in with tagged LF (green) and MLF (red) granules. C. The spectral information from the cmLSM data set delivers spectra (D) for both granules.
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