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David Anderson, Jerry Messinger, Nathan H Patterson, Jeffrey M Spraggins, Christine A. Curcio, Kevin L Schey; High Resolution Imaging Mass Spectrometry of Human Donor Eyes with and without Age-Related Macular Degeneration (AMD). Invest. Ophthalmol. Vis. Sci. 2018;59(9):3211. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: The purpose of this study is to develop high spatial resolution and high mass resolution imaging mass spectrometry (IMS) methods for imaging molecular changes in chorioretinal tissue from donors with AMD and with normal aged maculas. We sought to compare signal intensities for small molecules, including lipids, and image quality from specimens that are fixed or fresh-frozen.
Methods: From each of 2 pairs of donor eyes (<6 hr after death), the left eye was frozen without prior fixation, and the right eye was preserved in 4% paraformaldehyde. Macula and periphery of both eyes in each pair were cryo-sectioned together at 12 µm thickness and placed on a MALDI target slide. Adjacent sections were stained with Periodic acid-Schiff and Hematoxylin for morphological analysis. IMS was performed at 15 µm spatial resolution in both positive and negative ion modes on a Bruker solarix 9.4T FTICR instrument with a modified MALDI source designed for high spatial resolution imaging experiments. Tissue autofluorescence and reflectance images were also acquired from sections subject to IMS, before and after IMS experiments, to enable high precision registration of IMS and optical signals, allowing high-confidence localization of IMS signals to even single cell layer histological features. Low abundance signals were enhanced in FTICR imaging experiments using continuous accumulation of selected ions (CASI) of defined mass regions. IMS data were analyzed using SCiLS lab and FIJI ImageJ software.
Results: Specific lipid signals observed in IMS images correlate with tissue morphological features, i.e. different retina cell layers, retinal pigment epithelium (RPE), and choroid, as well as features of AMD pathology. Specifically, high spatial resolution IMS combined with registration to autofluorescent signals enabled detection of distinct lipid signals in RPE and thick basal laminar deposits. High mass accuracy measurements allowed tentative identifications to be made including for PE-ceramides and glucosylceramides. RPE molecular markers previously identified as bis(monoacylglycero)phosphate lipids (BMPs), were also detected.
Conclusion: High spatial resolution IMS technology provides spatially-resolved molecular analysis of AMD-related pathological features in human donor eyes. Fixation preserves morphology, but not all lipid signals detectable by IMS.
This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.
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