Imaging of biofilms using cryo-scanning electron microscopy

Biofilms are challenging samples for microscopy, because they are usually large samples with small features of interest, a fragile nature due to their high water content, and they may be grown on a variety of substrates. Cryo-scanning electron microscopy (cryo-SEM) has long been used to image microbiological components in plant-pathogen interactions, food products, and soil. The main advantage, compared with the more widely available room temperature SEM, is that the rapid sample preparation results in a native-like structure. It has been used to study environmental biofilms, but rarely for medically relevant biofilms. The cryo-SEM workflow starts with freezing the sample, either by high-pressure freezing, plunge-freezing, or slush nitrogen freezing. This is followed by fracture, sublimation, and coating, before the cryo-SEM imaging takes place. The present review aims to give new potential users an overview of the workflow and give examples of the equipment available, while discussing advantages and limitations of specific steps and their suitability for the research of various biofilms.