Mass spectrometry imaging is a cutting-edge molecular technology that enables simultaneous analysis of multiple molecular components directly from single cells, tissues, and organs. In combination with histological methods, this technique provides information about the spatial distribution of molecules in various biological tissues. Particularly, MALDI-MS imaging increases the coverage of metabolites by using different matrices and ion modes. We have recently developed and optimized spatial metabolomics and lipidomics approaches to image small molecules and lipids in human kidneys and biopsy material. By capitalizing on our joint expertise in spatial metabolomics at UTHSA, PNNL and EMBL, we have established methods for identifying metabolites in human kidneys, employed ultra-high mass resolution MS imaging for tissue analysis, and developed a bioinformatics resource (METASPACE) to annotate metabolites for anatomical localization and 3-D reconstruction.MALDI-FTICR-MS offers the highest attainable mass spectrometer performance, with mass resolution >120,000 and mass measurement accuracy 100,000 FWHM at m\/z 200 with mass accuracy < 1 ppm. In combination with a novel elevated pressure MALDI\/ESI interface (Spectroglyph, LLC), the UTHSA Q Exactive HF-X MS imaging platform has increased sensitivity characterized to a limit of detection of \u223c400 zmol, when using a laser repetition rate of 2 kHz, a laser wavelength of 355 nm, and a laser energy of 2 \u03bcJ in a 5 ns pulse. The use of the combined MALDI\/ESI interface with Q Exactive Orbitrap mass spectrometer offers high sensitivity, high mass and spatial resolution, and high mass accuracy and represents a robust and reproducible approach for MSI of complex biological tissues. UTHSA MALDI-Q Exactive HF-X MSI is complementary with PNNL 15T MALDI-FTICR-MSI to increase the coverage of metabolites, validate data obtained from different MS imaging platforms, and increase the spatial resolution.The METASPACE cloud platform is used by us to find molecules in the generated big MALDI-MSI data, annotate small molecules and lipids with confidence, exchange the information between the involved partners, share and provide results to involved experimental and computational scientists, store metadata, and overlay MALDI-MSI data with microscopy images. METASPACE also used for dissemination of the MALDI-MSI results obtrained in the KPMP project within the project as well as within the broader scientific community. Significance of the data\/analysis generated:1. Generated high quality MSI data at high spatial resolution (e.g., 25 \u03bcm and 20 \u03bcm).2. Identified new lipid marker (e.g., sphingomyelin d34:1) for localization of glomeruli.3. Increased the coverage of metabolites.4. Developed protocols for detection and mapping of compounds (e.g., ATP, ADP, and AMP) indicative of tissue state.5. Integration of spatial metabolomics and snDrop-seq data identified new glomerulus-specific gene markers.