Artifact-free preparation of biological samples for SEM by optimized water freeze-drying.

Water freeze-drying has traditionally been considered an inappropriate drying method for biological samples for scanning electron microscopy (SEM) because ice crystals can potentially form and damage cell structures. Chemical fixation is also considered essential for the SEM observation of soft cells, such as protozoa and animal cells. We succeeded in preparing specimens of freshwater protists for SEM using simple water freeze-drying without chemical fixation. Specifically, a suspension of live cells in water was frozen by contact with a − 80 °C copper block using an outer cylinder that ensured precise planar contact. This significantly reduced artifact formation and increased the success rate from ~ 10% to 20–30%. Transmission electron microscopy (TEM) observations confirmed that intracellular ice crystal formation was significantly suppressed in approximately 5% of Paramecium cells, representing a notable improvement over conventional methods. This method also shortens preparation time to 2–3 h and enables high-resolution SEM imaging of various organisms. Overall, based on these findings, we standardized a simple and low-artifact method using water freeze-drying as a novel and improved technique for SEM sample preparation.