The glomerulus is a multicellular functional tissue unit (FTU) of the nephron that is responsible for blood filtration. Each glomerulus contains multiple substructures and cell types that are crucial for their function. To understand normal aging and disease in kidneys, methods for high spatial resolution molecular imaging within these FTUs across whole slide images is required. Here we demonstrate a workflow using microscopy-driven selected sampling to enable 5 μm pixel size matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI IMS) of all glomeruli within whole slide human kidney tissues. Such high spatial resolution imaging entails large numbers of pixels, increasing the data acquisition times. Automating FTU-specific tissue sampling enables high-resolution analysis of critical tissue structures, while concurrently maintaining throughput. Glomeruli were automatically segmented using coregistered autofluorescence microscopy data, and these segmentations were translated into MALDI IMS measurement regions. This allowed high-throughput acquisition of 268 glomeruli from a single whole slide human kidney tissue section. Unsupervised machine learning methods were used to discover molecular profiles of glomerular subregions and differentiate between healthy and diseased glomeruli. Average spectra for each glomerulus were analyzed using Uniform Manifold Approximation and Projection (UMAP) and k-means clustering, yielding 7 distinct groups of differentiated healthy and diseased glomeruli. Pixel-wise k-means clustering was applied to all glomeruli, showing unique molecular profiles localized to subregions within each glomerulus. Automated microscopy-driven, FTU-targeted acquisition for high spatial resolution molecular imaging maintains high-throughput and enables rapid assessment of whole slide images at cellular resolution and identification of tissue features associated with normal aging and disease.@en

Gangliosides are acidic glycosphingolipids, containing ceramide moieties and oligosaccharide chains with one or more sialic acid residue(s) and are highly diverse isomeric structures with distinct biological roles. Matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI IMS) enables the untargeted spatial analysis of gangliosides, among other biomolecules, directly from tissue sections. Integrating trapped ion mobility spectrometry with MALDI IMS allows for the analysis of isomeric lipid structures in situ. Here, we demonstrate the gas-phase separation and identification of disialoganglioside isomers GD1a and GD1b that differ in the position of a sialic acid residue, in multiple samples, including a standard mixture of both isomers, a biological extract, and directly from thin tissue sections. The unique spatial distributions of GD1a/b (d36:1) and GD1a/b (d38:1) isomers were determined in rat hippocampus and spinal cord tissue sections, demonstrating the ability to structurally characterize and spatially map gangliosides based on both the carbohydrate chain and ceramide moieties.@en

We demonstrate the utility of combining silicon nanopost arrays (NAPA) and trapped ion mobility imaging mass spectrometry (TIMS IMS) for high spatial resolution and specificity mapping of neutral lipid classes in tissue. Ionization of neutral lipid species such as triglycerides (TGs), cholestryl esters (CEs), and hexosylceramides (HexCers) from biological tissues has remained a challenge for imaging applications. NAPA, a matrix-free laser desorption ionization substrate, provides enhanced ionization efficiency for the above-mentioned neutral lipid species, providing complementary lipid coverage to matrix-assisted laser desorption ionization (MALDI). The combination of NAPA and TIMS IMS enables imaging of neutral lipid species at 20 μm spatial resolution while also increasing molecular coverage greater than 2-fold using gas-phase ion mobility separations. This is a significant improvement with respect to sensitivity, specificity, and spatial resolution compared to previously reported imaging studies using NAPA alone. Improved specificity for neutral lipid analysis using TIMS IMS was shown using rat kidney tissue to separate TGs, CEs, HexCers, and phospholipids into distinct ion mobility trendlines. Further, this technology allowed for the separation of isomeric species, including mobility resolved isomers of Cer(d42:2) (m/z 686.585) with distinct spatial localizations measured in rat kidney tissue section. @en

We demonstrate the utility of combining silicon nanopost arrays (NAPA) and trapped ion mobility imaging mass spectrometry (TIMS IMS) for high spatial resolution and specificity mapping of neutral lipid classes in tissue. Ionization of neutral lipid species such as triglycerides (TGs), cholestryl esters (CEs), and hexosylceramides (HexCers) from biological tissues has remained a challenge for imaging applications. NAPA, a matrix-free laser desorption ionization substrate, provides enhanced ionization efficiency for the above-mentioned neutral lipid species, providing complementary lipid coverage to matrix-assisted laser desorption ionization (MALDI). The combination of NAPA and TIMS IMS enables imaging of neutral lipid species at 20 μm spatial resolution while also increasing molecular coverage greater than 2-fold using gas-phase ion mobility separations. This is a significant improvement with respect to sensitivity, specificity, and spatial resolution compared to previously reported imaging studies using NAPA alone. Improved specificity for neutral lipid analysis using TIMS IMS was shown using rat kidney tissue to separate TGs, CEs, HexCers, and phospholipids into distinct ion mobility trendlines. Further, this technology allowed for the separation of isomeric species, including mobility resolved isomers of Cer(d42:2) (m/z 686.585) with distinct spatial localizations measured in rat kidney tissue section. @en