A high contrast method of unstained biological samples under a thin carbon film by scanning electron microscopy

The contrast of biological samples in scanning electron microscopy (SEM) is very weak. To examine a biological specimen by SEM, many steps and/or special equipment are required to prepare the sample. Here, we describe a method using an unstained biological sample under a 40 nm carbon film to give a high contrast image, where the image is detected by the secondary electron (SE) signal at a low accelerating voltage of 1.5 kV. Under these conditions, it is hard to detect a direct signal from a biological specimen. The high contrast image is created by the SEs from the lower surface of the thin carbon film. Therefore, the damage to the sample from the electron beam is very low. Our method can be utilized to observe various biological samples of bacteria, viruses, and protein complexes.