A generalizable and targeted molecular biopsy approach for in situ cryogenic electron tomography of vitreous brain tissue

Cellular cryogenic electron tomography (cryo-ET) enables the capture of detailed structural information within a biologically relevant environment. However, information in more complex samples, such as multicellular specimens and tissues, is lacking. Importantly, these observations need to be set in the context of populations. Currently, imaging on the molecular scale is limited to a few observations in situ that struggle to be generalized. This is due to limitations in throughput and versatility employed by current instrumentation. Here, we utilize plasma focused ion beam milling to examine the molecular landscape of mouse hippocampus by cryo-ET. We reveal the complex organization of macromolecules in targeted regions across CA1 stratum pyramidale (sp) to radiatum (sr), representing a molecular atlas of hippocampal architecture in adult mice. The combination of instrumentation and application of technical advancements provides a framework to explore specific structural questions within other tissues in a targeted manner.