Polymer-supported cryo-FIB milling enables near-native structural analysis of mature bacterial spores

Bacterial spores exhibit remarkable resistance to environmental stresses, posing challenges for food safety and sterilization. In Bacillus subtilis var. natto , spores are embedded within a viscous extracellular matrix rich in γ-polyglutamic acid (γ-PGA), yet their internal membrane architecture and spatial relationships with the surrounding matrix remain poorly understood. Here, we develop polymer-supported cryogenic focused ion beam (PolyS-FIB) milling and combine it with cryogenic transmission electron microscopy (cryo-TEM) and cryo-electron tomography (cryo-ET) to investigate mature spores under near-native conditions. The polymer support improves sample adhesion and lamella stability, enabling two- and three-dimensional visualization of spores embedded within the extracellular matrix. We identify morphologically diverse sub-core membranes (SCMs), including single- and double-layered configurations, complex membrane structures associated with invaginations of the inner membrane (IM), together with vesicle-like structures within these regions. Quantitative analysis reveals a significant difference in apparent membrane thickness between the IM and the inner layer of double-layered SCMs. Three-dimensional imaging further resolves the multilayered spore coat and membrane-like structures at the spore periphery, while complementary cryo-ultramicrotomy reveals spatial heterogeneity in the surrounding extracellular matrix. Together, these findings provide new insights into the membrane architecture of mature bacterial spores and establish PolyS-FIB as a sample preparation strategy for investigating biological structures embedded within complex extracellular environments.