Aging and Alzheimer’s Disease (AD) differentially affect the hippocampal subfields. A robust, reliable imaging tool is needed to evaluate the hippocampal subfields and distinguish early AD from normal aging. Though recent neuroimaging studies have harmonized the labeling of the whole hippocampus with success, multiple groups have parcellated the hippocampus further into subfields and highly variable results emerged. Conflicting reports exist about atrophy changes along the aging-Alzheimer’s continuum. The hippocampus contains selective vulnerable cell populations. Identifying pyramidal cell profiles for all subicular cortices, CA1, CA2, and CA3 is essential to develop rigorous, cell-validated definitions for the hippocampal subregions and to corroborate these as reliable biomarkers is a critical step to determine AD vulnerability. Histology is required to differentiate subfields accurately. Our overall goal is to characterize the hippocampus subregions based on cells, not layers or microanatomical features. The objective of this proposal is to create twelve histologically-validated hippocampal subfields based on novel, cellular criteria (size, pattern, directionality measures) and apply this neuroimaging map to in vivo populations. This regionally integrated map will establish new cellular readouts for the hippocampal formation that represent the subpopulations most vulnerable in AD. The project will combine high-resolution ex vivo MRI, a preclinical brain tissue collection, and our neuroanatomical expertise. Aim 1 will establish a novel neuroimaging tool that segments twelve hippocampal subfields in FreeSurfer. Aim 2 will validate hippocampal subregions histologically with Nissl staining corresponding to the same cases and establish novel anatomical and cellular frameworks for each subregion: pyramidal cell diameter measures, pyramidal neuronal pattern profiles from cell directionality measures and our triple “c” protocol to compute architectonic boundaries. Aim 3 will apply the hippocampus subfield segmentation tool to 3 T in vivo MRI data including the Harvard Aging Brain Study, the Human Connectome Project, Alzheimer’s disease Neuroimaging Initiative and validate against established biomarkers. Our regionally integrated subfield map is needed to disambiguate aging from AD in terms of specificity, sensitivity and reliability in early prediction of AD. The work – at the microscale – will generate well-validated standards that will help to disentangle cellular and network vulnerabilities in healthy aging from AD in future in vivo functional, longitudinal, or therapeutic studies.