Scanning electron microscopy (SEM) is widely used to visualize the fine surface structures of bacteria and other microorganisms. However, biological samples contain water and therefore must be dried before being introduced into the SEM vacuum chamber. Conventional sample preparation procedures commonly involve chemical fixation, graded ethanol dehydration, and either critical-point drying or freeze-drying after replacement with an organic solvent such as t-butyl alcohol. These steps carry the risk of extracting lipids and soluble components and may cause shrinkage, cracking, or collapse, making it difficult to distinguish native structures from preparation-induced artifacts.
A collaborative research team comprising Yoichi Yamada and Wakano Ogawa of Shujitsu University; Toshinobu Suzaki of Kobe University; Kazuyoshi Murata of National Institute for Physiological Sciences; Hideki Ishida of Shimane University, Japan; Liudmyla Gaponova of Institute for Evolutionary Ecology of the National Academy of Sciences of Ukraine; Andrii Kolosiuk of Institute of Physics of the National Academy of Sciences of Ukraine, Ukraine; and Chihong Song of Pusan National University, South Korea, has successfully applied Water Freeze-Drying (WFD) to bacterial specimens. The study has been published in Frontiers in Microbiology.
In the WFD procedure (Fig. 1), immobilized microorganisms are collected on a membrane filter and rinsed with ultrapure water to remove residual salts. A copper block pre-cooled to -80°C is then brought gently into contact with the specimen to initiate freezing. The frozen sample is transferred to a freeze-dryer while kept cold, where the ice is removed directly via sublimation. Because the method avoids ethanol dehydration and organic-solvent replacement, it can reduce the physical and chemical stresses associated with specimen preparation.
The researchers compared E. coli cells prepared by conventional t-butyl alcohol freeze-drying method with those prepared by WFD method. Specimens prepared by the conventional method showed conspicuous cracking and large-scale shrinkage across the bacterial layer. Specimens prepared by the WFD remained largely continuous and intact appearance. At higher magnification observation, cells prepared by the conventional method displayed mostly rounded, dome-shaped poles, whereas those prepared by WFD presented a heterogeneous population including both rounded poles and sharply truncated, flat ends (Fig. 2). These results suggest that conventional dehydration methods, which make cell poles appear more uniformly rounded, may mask transient or physiologically meaningful structural states. The authors emphasize that further investigation is requires to understand the biological significance of the flat poles and acknowledge that WFD-specific artifacts cannot yet be completely excluded.
A key practical advantage of WFD is its accessibility. The research team confirmed that WFD can be performed not only with specialized freeze-drying equipment designed for electron microscopy but also with a general-purpose laboratory freeze-dryer. This method therefore has a potential to lower both technical and economic barriers to high-fidelity SEM specimen preparation. Potential applications include studies of microbial cell architecture, antimicrobial-induced surface damage, and the three-dimensional organization of biofilms.
