High-Resolution Imaging of Dried and Living Single Bacterial Cell Surfaces: Artifact or Not?

Abstract

When studying highly resolved scanning electron microscope images of cell surfaces, the question arises, whether the observed patterns are real or just artifacts of the cell preparation process. The following steps are usually necessary for preparation: fixation, drying, and metal coating. Each step might introduce different artifacts. Clever techniques have been developed to dry cells as gently as possible, for example critical point drying with different organic solvents and CO2. Instrument manufacturers also have taken account of this issue, for example, through the realization of the environmental scanning electron microscope (ESEM), operating with a low-vacuum environment saturated with water so that samples might stay hydrated. Another approach is the extreme high-resolution scanning electron microscope (XHR SEM), where the electron beam is decelerated shortly before reaching the sample. This technique requires no metal coating of the sample. Cryo-SEM also may be used, where no sample preparation is required beyond freezing in a high-pressure freezer or other cryo-fixation device. Then the cell can be examined in the frozen, hydrated state using a cryostage. However, at least some kind of preparation is necessary for SEM imaging, and we wanted to find out what changes the preparation makes on the cell surface.