All cells require nutrient metal to carry out essential biochemical processes. This requirement is something that the vertebrate immune system has exploited as a strategy to defend against infection by restricting microbial access to nutrient metal. This process of nutrient restriction during infection is called “nutritional immunity”. Bacterial pathogens have evolved elaborate mechanisms to circumvent nutritional immunity and acquire metal during infection. This struggle for nutrient metal impacts both microbial virulence as well as the immune response of the host, profoundly impacting the outcome of the host-pathogen interactions. To study these interactions in more detail, we have developed a powerful imaging workflow that can be applied to murine models of infectious disease. All diseases, including infections, are characterized by distinct changes in tissue molecular distribution. Molecular analysis of intact tissues traditionally requires knowledge and reagents relevant to the targets of interest as well as destructive processing for downstream identification platforms. Tissue-based analyses therefore sacrifice discovery to gain spatial distribution of known targets, or sacrifice tissue architecture for discovery of unknown targets. To overcome these obstacles, we developed a multi-modality, three-dimensional imaging platform for discovery-based molecular histology. We have applied this platform to the study of multiple murine models of infection, leading to the discovery of infection-associated alterations in the distribution and abundance of macromolecules and elements in tissue. These data provide a three-dimensional analysis of how disease impacts the molecular architecture of complex tissues in infected animals, enable diagnosis of infection through imaging-based detection of bacterial and host analytes, and reveal molecular heterogeneity at the host-pathogen interface.