Imaging mass spectrometry (IMS) yields high-dimensional and large data sets commonly exceeding 100,000 pixels, each reporting a mass spectrum of 200,000 intensity values or more. Reducing the dimensionality and size of IMS data is often necessary to enable downstream analysis, and matrix-factorization-based approaches are often used for this purpose. However, the model underlying most of these techniques, decomposing measurements into the sum of a low-rank term (presumed signal) and a small entry-wise residual term (presumed noise), is often not optimal for IMS. For example, while spatially or spectrally sparse signals are common in IMS data, they can heavily distort the low-rank approximation. Therefore, we propose capturing the IMS data structure using low-rank models that, in addition to a dense residual, allow for sparse variation to be captured separately. We implement two such methods, principal component pursuit (PCP) and stable principal component pursuit (SPCP), apply them to IMS data, and compare them to a classical factorization method, principal component analysis (PCA). We investigate their dimensionality and noise reduction performance on MALDI Q-TOF IMS measurements of human cornea and retina tissue since the human eye is a complex organ with lots of small, tightly packed tissue substructures that are spatially sparse. Our results suggest that if parameters are set adequately, PCP and SPCP enable stronger dimensionality reduction and higher compression of IMS data compared to PCA, while concurrently reducing signal overestimation.

Purpose: Mutations in the gene that encodes the enzyme acid sphingomyelinase (ASMase) are associated with Niemann-Pick disease, a lysosomal storage disorder. Mice that lack ASMase (ASMase-/-) exhibit age-related retinal degeneration and large increases in accumulation of lipofuscin in the retinal pigment epithelium (RPE). We examined which lipid species accumulate in the retina and the RPE of ASMase-/- mice and whether the retinal degeneration is associated with impaired photoreceptor metabolism and retinyl chromophore processing. Methods: NADPH availability and all-trans retinol formation after rhodopsin bleaching were measured in isolated single rod photoreceptors with fluorescence imaging; sphingolipid levels in retinas and RPEs were measured with LC/MS; relative abundances of different lipid species in different retinal layers were measured with MALDI imaging mass spectrometry. Results: There was no detectable difference in the kinetics of all-trans retinol formation or the NADPH-generating capacity between ASMase-/- and wild-type mice. Sphingomyelin levels were much higher in the retinas and RPEs of ASMase-/- animals compared to wild type, but there were no significant differences for ceramides. There was a large increase in the abundance of bis(monoacylglycero)phosphates (BMPs) in ASMase-/- mice, indicative of lysosomal dysfunction, but no substantial changes were detected for the bis-retinoid A2E. Conclusions: Lysosomal dysfunction and retinal degeneration in ASMase-/- mice are not associated with defects in rod photoreceptor metabolism that affect all-trans retinol formation and availability of NADPH. Lysosomal dysfunction in ASMase-/- mice is not associated with bis-retinoid A2E accumulation.

Introduction: Age related macular degeneration (AMD) causes legal blindness worldwide, with few therapeutic targets in early disease and no treatments for 80% of cases. Extracellular deposits, including drusen and subretinal drusenoid deposits (SDD; also called reticular pseudodrusen), disrupt cone and rod photoreceptor functions and strongly confer risk for advanced disease. Due to the differential cholesterol composition of drusen and SDD, lipid transfer and cycling between photoreceptors and support cells are candidate dysregulated pathways leading to deposit formation. The current study explores this hypothesis through a comprehensive lipid compositional analysis of SDD. Methods: Histology and transmission electron microscopy were used to characterize the morphology of SDD. Highly sensitive tools of imaging mass spectrometry (IMS) and nano liquid chromatography tandem mass spectrometry (nLC-MS/MS) in positive and negative ion modes were used to spatially map and identify SDD lipids, respectively. An interpretable supervised machine learning approach was utilized to compare the lipid composition of SDD to regions of uninvolved retina across 1873 IMS features and to automatically discern candidate markers for SDD. Immunohistochemistry (IHC) was used to localize secretory phospholipase A2 group 5 (PLA2G5). Results: Among the 1873 detected features in IMS data, three lipid classes, including lysophosphatidylcholine (LysoPC), lysophosphatidylethanolamine (LysoPE) and lysophosphatidic acid (LysoPA) were observed nearly exclusively in SDD while presumed precursors, including phosphatidylcholine (PC), phosphatidylethanolamine (PE) and phosphatidic acid (PA) lipids were detected in SDD and adjacent photoreceptor outer segments. Molecular signals specific to SDD were found in central retina and elsewhere. IHC results indicated abundant PLA2G5 in photoreceptors and retinal pigment epithelium (RPE). Discussion: The abundance of lysolipids in SDD implicates lipid remodeling or degradation in deposit formation, consistent with ultrastructural evidence of electron dense lipid-containing structures distinct from photoreceptor outer segment disks and immunolocalization of secretory PLA2G5 in photoreceptors and RPE. Further studies are required to understand the role of lipid signals observed in and around SDD.

Neurofibromatosis type 1 (NF1) is a common neurogenetic disorder, in which affected individuals develop tumors of the nervous system. Children with NF1 are particularly prone to brain tumors (gliomas) involving the optic pathway that can result in impaired vision. Since tumor formation and expansion requires a cooperative tumor microenvironment, it is important to identify the cellular and acellular components associated with glioma development and growth. In this study, we used 3-D matrix assisted laser desorption ionization imaging mass spectrometry (MALDI IMS) to measure the distributions of multiple molecular species throughout optic nerve tissue in mice with and without glioma, and to explore their spatial relationships within the 3-D volume of the optic nerve and chiasm. 3-D IMS studies often involve extensive workflows due to the high volume of sections required to generate high quality 3-D images. Herein, we present a workflow for 3-D data acquisition and volume reconstruction using mouse optic nerve tissue. The resulting 3-D IMS data yield both molecular similarities and differences between glioma-bearing and wild-type (WT) tissues, including protein distributions localizing to different anatomical subregions. Biological significance The current work addresses a number of challenges in 3-D MALDI IMS, driven by the small size of the mouse optic nerve and the need to maintain consistency across multiple 2-D IMS experiments. The 3-D IMS data yield both molecular similarities and differences between glioma-bearing and wild-type (WT) tissues, including protein distributions localizing to different anatomical subregions, which could then be targeted for identification and related back to the biology observed in gliomas of the optic nerve.