PROJECT SUMMARY The immune system plays a significant role in aging and in autoimmune, neurodevelopmental, and neurodegenerative diseases. Functionally diverse immune cells change with disease progression and shape disease pathology through complex mechanisms. Current molecular imaging techniques do not allow for simultaneous interrogation of multiple biomarkers from a single tissue sample. In the proposed preclinical study, I will develop a nanoparticle-based Raman imaging approach capable of highly multiplexed molecular spatial profiling of brain histology sections of Alzheimer’s disease (AD) patients. There is a critical need to improve our understanding of brain immunity to offer intervention tools for designing new therapeutic strategies for treating and preventing brain pathologies. My new multiplexed imaging platform will identify and capture the underlying cellular and phenotypic diversity in intricate neural tissues within the spatial context of the brain. Sharp and unique spectral barcodes of the carefully selected ensemble of surface-enhanced Raman scattering (SERS) nanoparticles (NPs) will ensure reliable and accurate spectral unmixing. During the K99 mentored phase, I will design NP constructs exhibiting strong and unique Raman scattering signals (SA1). Unlike traditional imaging techniques, the developed SERS NPs will be capable of visualizing at least 30 biomarkers in a single imaging acquisition. I will validate the high spectral specificity and quantitative accuracy of the spectral unmixing. In SA2, the NPs will be decorated with biotargeting species to ensure highly specific recognition of immune-brain biomarkers in cell culture and tissue sections. An innovative strategy will be used to stain and image archived patient samples starting with single plex Raman imaging and correlated with the gold standard IHC histology. As an independent investigator, I will use targeted SERS NPs to produce highly multiplexed proteomic images of brain tissue. To confirm the sensitivity and specificity of the Raman imaging approach, the adjacent tissue sections will be assessed by IHC and conventional immunofluorescence imaging. These highly multiplexed molecular maps of the brain-immune spatial biology will pave the way for future studies focused on improving our ability to understand, prevent, and treat AD. I have a unique set of expertise in SERS imaging and believe I am highly qualified to lead and conduct the proposed project at the University of Southern California. I also have identified the following key areas which require additional training to support my transition to independence: (1) quantitative spectral unmixing and image processing; (2) active cell targeting; (3) histopathological evaluation of postmortem brain tissue with a solid knowledge base in AD biology. I have deliberately chosen and carefully assembled a world-class committee of mentors and advisors including my primary mentor Dr. Cristina Zavaleta and co-mentor Dr. Peter Y. Wang. My mentors are in full support of my research and career goals. I am committed to the research proposed here and strongly believe that it will help me gain the necessary training required to enable my long-term career goal of being an established investigator in the field of molecular imaging.

PROJECT NARRATIVE Immune cells have recently been recognized as vital players in Alzheimer’s disease, as they change with disease progression and can shape the disease pathology through complex mechanisms. We require a new imaging tool to closely study various immune cell populations simultaneously within the spatial context of human brain tissue. The complete molecular mapping of the brain-immune relationship, enabled by our Raman imaging approach, will pave the way for improving our ability to understand, prevent, and treat Alzheimer’s disease.