? TR&D PROJECT 1 Higher Throughput Multi-element Distributions & Quantitation at the Tissue Level Metals are crucial for physiological processes with highly regulated trafficking, localization, and homeostasis, where interruptions or slight variations lead to cell and tissue dysfunction and ultimately to disease. This technology research & development (TR&D) project will advance laser ablation inductively coupled plasma time-of-flight mass spectrometry (LA-ICP-TOFMS) techniques to develop increased throughput imaging capacity for metals in diverse biomedical applications. This approach will complement those being developed in TR&Ds 2 and 3, enabling biomedical researchers to balance sensitivity, selectivity, and spatial resolution needs to develop a wholistic understanding of the role metals play in fundamental biological processes. Using our Driving Biomedical Projects (DBP) as test beds, this TR&D will integrate LA-ICP-TOFMS components to develop workflows for acquiring rapid quantitative 2D multi-elemental maps derived from unique tissue types and different sample preparation methods. Use of a cryogenic LA cell will enable analysis of cryogenically preserved samples, ensuring tissue chemistry and structure are preserved. We will also develop standardized approaches for producing tissue-specific LA-ICP-MS calibration standards. Our standards will be validated using SXFM at beamline 8-BM. Once protocols for standard production and validation are developed, a set quantity of tissue specific SXFM-validated standards will be made available to the community as universal external LA-ICP-MS calibration standards. Finally, because metal hotspots in larger volumes can be misrepresented or missed in 2D images, we will develop 3D imaging LA-ICP-TOFMS techniques for whole organs and organisms. To produce 3D images we will use serial sectioning techniques facilitated by the rapid image acquisition capabilities of the TOFMS and incorporate multi-modality image analysis software (PyElements) being developed in TR&D 2. 3D imaging capabilities will support a wider applicability of bioimaging techniques for the biomedical community. All of these developments will take place in the context of our DBPs, with projects from all four themes (Metal homeostasis or dysregulation in brain function; metal modulation in host-pathogen interactions; metal fluxes controlling reproduction and development; and metal imbalances in metabolic pathology), supporting development of tissue-specific calibration standards and workflows for analyzing 2D sections of different tissue types. Development of 3D LA-ICP-TOFMS imaging technology will be most relevant to DBP Theme C (metal fluxes controlling reproduction and development). Once mature, the cryo-LA-ICP-TOFMS will be incorporated as a service line in Northwestern?s Quantitative Bioelement Imaging Center.