Variable pressure SEM and conventional high vacuum SEM protocols for biofilm imaging

Several microscopic methodologies have been used in biofilm studies, each one giving its contribution to deepening the knowledge of biofilm structure. This chapter illustrates two electron microscopy techniques, scanning electron microscopy (SEM) and variable-pressure SEM (VP-SEM). SEM needs sample dehydration and metal coating, with its operating conditions of high vacuum and high voltage. Variable-pressure SEM works at low vacuum and low voltage, so samples can be imagined in their natural hydrated state. Each one of these two techniques has its advantages and disadvantages; for this reason, a new protocol for sample observation in high-voltage and high-vacuum operating conditions, named OsO4-RR-TA-IL, was developed. The best, perfect protocol to observe biofilms does not exist but is possible to choose the most suitable one, depending on what kind of study has to be carried on. The choice of the most appropriate protocol has to be done carefully, based on the specific aim of the study. If the characterization of new biofilm types has to be performed, OsO4-RR-TA-IL has to be preferred. It allows observation of the most detailed ultrastructural features of the biofilm, due to the properties of every single reagent used. This customized protocol for biofilm uses osmium tetroxide (OsO4), ruthenium red (RR), tannic acid (TA), and ionic liquid (IL), and it is unrivaled for its image quality, magnification, resolution, minimal sample loss, and actual sample structure preservation. If instead, the research has to examine the drug effects on a biofilm, or if a low amount of sample is available and magnification up to 8000 × is sufficient, VP-SEM has to be preferred. It is fast and has a few-step protocol that ensures very minimal sample loss giving images at sufficiently good resolution.