Development of a high throughput system for molecular imaging of different cell types in mouse brain tissues Mass spectrometry imaging (MSI) is a powerful tool for developing detailed molecular maps of biological tissues with high specificity and sensitivity. This label-free technique enables simultaneous imaging of multiple classes of molecules including lipids, metabolites, and proteins thereby advancing the understanding of tissue organization and function. In this project, we will advance brain cell census research by developing an innovative platform for the acquisition of a comprehensive spatially-resolved cell-specific atlas of lipids, metabolites, and proteins in mouse brain tissue. The platform will combine MSI with immunofluorescence imaging to place the detailed molecular maps into the spatial cellular context within the brain tissue, which will facilitate image registration to the common coordinate system. Furthermore, we will develop deep learning and data mining approaches to enable automated assignment of molecular signatures to different cell types and efficient data sharing and comparison with other techniques. This research will address the existing bottlenecks in the experimental throughout and molecular coverage of the current MSI technologies along with the limitations of data analysis tools. Furthermore, our approach will compensate for signal suppression during ionization also called ‘matrix effects’, which is particularly severe in imaging of brain tissue. Such matrix effects common to all the MSI modalities including commercial MALDI MSI instruments interfere with the accurate measurement of the spatial localization of molecules. To overcome these challenges, we will advance the capabilities of nanospray desorption electrospray ionization (nano-DESI) - an ambient ionization technique, which efficiently compensates for matrix effects and thereby enables accurate and sensitive imaging of chemical gradients for hundreds of metabolites and lipids in biological tissue sections. Nano-DESI MSI does not require sample pretreatment, has a sub-femtomole sensitivity, and high spatial resolution. The new nano-DESI MSI system will provide a 5-fold increase in the experimental throughput and improve the spatial resolution of protein imaging in whole tissue sections from ~80 µm to ~10 µm. Coupling nano-DESI MSI with a high- resolution ion mobility mass spectrometer will substantially enhance molecular coverage. Meanwhile, co- registration of MSI with immunofluorescence data will be used to generate comprehensive 3D molecular maps of the mouse brain tissue. Collectively, these efforts will establish a robust imaging platform, which will transform our ability to generate detailed molecular maps of different cell types in brain tissues.

Project Narrative This research is focused on the development of a transformative technology for the high- throughput cell-specific imaging of molecules in brain tissue using mass spectrometry, immunofluorescence microscopy, and advanced data mining and deep learning tools. This novel correlative imaging approach will substantially enhance the range of biomolecules in the brain cell census data.