Histology-directed imaging mass spectrometry (IMS) is a spatially targeted IMS acquisition method informed by expert annotation that provides rapid molecular characterization of select tissue structures. The expert annotations are usually determined on digital whole slide images of histological stains where the staining preparation is incompatible with optimal IMS preparation, necessitating serial sections: one for annotation, one for IMS. Registration is then used to align staining annotations onto the IMS tissue section. Herein, we report a next-generation histology-directed platform implementing IMS-compatible autofluorescence (AF) microscopy taken prior to any staining or IMS. The platform enables two histology-directed workflows, one that improves the registration process between two separate tissue sections using automated, computational monomodal AF-to-AF microscopy image registration, and a registration-free approach that utilizes AF directly to identify ROIs and acquire IMS on the same section. The registration approach is fully automated and delivers state of the art accuracy in histology-directed workflows for transfer of annotations (∼3-10 μm based on 4 organs from 2 species) while the direct AF approach is registration-free, allowing targeting of the finest structures visible by AF microscopy. We demonstrate the platform in biologically relevant case studies of liver stage malaria and human kidney disease with spatially targeted acquisition of sparsely distributed (composing less than one tenth of 1% of the tissue section area) malaria infected mouse hepatocytes and glomeruli in the human kidney case study.

An understanding of how cells respond to perturbation is essential for biological applications; however, most approaches for profiling cellular response are limited in scope to pre-established targets. Global analysis of molecular mechanism will advance our understanding of the complex networks constituting cellular perturbation and lead to advancements in areas, such as infectious disease pathogenesis, developmental biology, pathophysiology, pharmacology, and toxicology. We have developed a high-throughput multiomics platform for comprehensive, de novo characterization of cellular mechanisms of action. Platform validation using cisplatin as a test compound demonstrates quantification of over 10"000 unique, significant molecular changes in less than 30 days. These data provide excellent coverage of known cisplatin-induced molecular changes and previously unrecognized insights into cisplatin resistance. This proof-of-principle study demonstrates the value of this platform as a resource to understand complex cellular responses in a high-throughput manner.