Ischemic stroke remains a significant cause of morbidity and mortality, with limited rational therapeutic options, despite years of experimental animal modelling and empiric trials in living patients. Translational progress requires better cellular characterization directly in human postmortem tissue, particularly of the ischemic penumbra as potentially “salvageable” in the acute and subacute clinical setting. Our comprehensive multimodal approach employs premortem and postmortem magnetic resonance imaging (MRI), followed by whole-brain histology. Though heretofore done only in fetal specimens, we adapted the technique for definition of the ischemic core and penumbra in 2 adult donors with 3- and 16-day hospital courses. The fixed brains were cryoprotected, sectioned at 20 µm, and serially stained for markers of neurons, glial cells, tissue hypoxia, blood-brain barrier disruption, axonal injury and regeneration, followed by 3-dimensional (3D) digital reconstruction of sections. Acute infarcts showed blood-brain-barrier disruption, negligible in distant zones and unaffected hemisphere. Extensive subcortical white matter axonal injury and regeneration were found not only in the infarcts but also in peri-infarct and distant brain regions on immunohistochemistry for amyloid precursor protein and growth-associated protein-43. Surprisingly, distant cortical regions had acute hypoxic changes detectable only on HIF1a immunohistochemistry. Digital 3D reconstruction enabled mapping of cellular changes across neuroanatomical regions to provide a more accurate, detailed rendering of infarct, peri-infarct (penumbral), and distant zones directly in human brain tissue. We propose as “proof of principle” that our multimodal-multiscale approach precisely bridges between neuroimaging and neuropathology, offering novel insights into the spatial and temporal progression of ischemic stroke including the penumbra and “normal-appearing” areas, greater than appreciated with standard analyses, and potentially amenable to therapeutic intervention.