Microscopy-Directed Imaging Mass Spectrometry for Rapid High Spatial Resolution Molecular Imaging of Glomeruli

The glomerulus is a multicellular functional tissue unit(FTU)of the nephron that is responsible for blood filtration. Each glomeruluscontains multiple substructures and cell types that are crucial fortheir function. To understand normal aging and disease in kidneys,methods for high spatial resolution molecular imaging within theseFTUs across whole slide images is required. Here we demonstrate aworkflow using microscopy-driven selected sampling to enable 5 & mu;mpixel size matrix-assisted laser desorption/ionization imaging massspectrometry (MALDI IMS) of all glomeruli within whole slide humankidney tissues. Such high spatial resolution imaging entails largenumbers of pixels, increasing the data acquisition times. AutomatingFTU-specific tissue sampling enables high-resolution analysis of criticaltissue structures, while concurrently maintaining throughput. Glomeruliwere automatically segmented using coregistered autofluorescence microscopydata, and these segmentations were translated into MALDI IMS measurementregions. This allowed high-throughput acquisition of 268 glomerulifrom a single whole slide human kidney tissue section. Unsupervisedmachine learning methods were used to discover molecular profilesof glomerular subregions and differentiate between healthy and diseasedglomeruli. Average spectra for each glomerulus were analyzed usingUniform Manifold Approximation and Projection (UMAP) and k-means clustering, yielding 7 distinct groups of differentiated healthyand diseased glomeruli. Pixel-wise k-means clusteringwas applied to all glomeruli, showing unique molecular profiles localizedto subregions within each glomerulus. Automated microscopy-driven,FTU-targeted acquisition for high spatial resolution molecular imagingmaintains high-throughput and enables rapid assessment of whole slideimages at cellular resolution and identification of tissue featuresassociated with normal aging and disease.