Water freeze-drying for high-resolution SEM: an accessible strategy for capturing native microorganism morphology...

Conventional scanning electron microscopy (SEM) preparation, relying on chemical dehydration, frequently induces structural artifacts such as shrinkage and distortion. While high-pressure freezing (HPF) offers superior preservation, its accessibility is limited by the requirement for specialized equipment. In this study, we evaluated a water freeze-drying (WFD) method using a general-purpose laboratory freeze-dryer for observing bacteria and protists. Our results demonstrate that WFD effectively preserves morphological integrity by minimizing dehydration-induced stress. Notably, WFD enabled the observation of the highly contractile ciliate Spirostomum ambiguum in its natural, extended state without chemical fixation, effectively bypassing the osmotic and physical stresses that typically trigger contraction. Furthermore, WFD revealed significant heterogeneity in Escherichia coli polar morphologies; while some cells maintained rounded ends, a substantial subset exhibited remarkably sharp, truncated geometries. In contrast, the Gram-positive bacterium Bacillus subtilis consistently retained dome-shaped poles regardless of the drying method, reflecting the superior mechanical rigidity of its cell wall. WFD also revealed substantial heterogeneity in E. coli pole morphology, suggesting that conventional dehydration masks physiologically relevant structural diversity. By enabling high-fidelity imaging with standard equipment, the WFD method provides an accessible and robust solution for high-resolution ultrastructural analysis across diverse microbial taxa.