Microscopy Methods for Biofilm Imaging: Focus on SEM and VP-SEM Pros and Cons

Simple Summary Bacterial biofilms cause infections that are often resistant to antibiotic treatments. Research about the formation and elimination of biofilms cannot be undertaken without detailed imaging techniques. In this review, traditional and cutting-edge microscopy methods to study biofilm structure, ultrastructure, and 3-D architecture, with particular emphasis on conventional scanning electron microscopy and variable pressure scanning electron microscopy, are addressed, with the respective advantages and disadvantages. When ultrastructural characterization of biofilm matrix and its embedded bacterial cells is needed, as in studies on the effects of drug treatments on biofilm, scanning electron microscopy with customized protocols such as the osmium tetroxide (OsO4), ruthenium red (RR), tannic acid (TA), and ionic liquid (IL) must be preferred over other methods for the following: unparalleled image quality, magnification and resolution, minimal sample loss, and actual sample structure preservation. The first step to make a morphological assessment of the effect of the various pharmacological treatments on clinical biofilms is the production of images that faithfully reflect the structure of the sample. The extraction of quantitative parameters from images, possible using specific software, will allow for the scanning electron microscopy morphological evaluation to no longer be considered as an accessory technique, but a quantitative method to all effects. Several imaging methodologies have been used in biofilm studies, contributing to deepening the knowledge on their structure. This review illustrates the most widely used microscopy techniques in biofilm investigations, focusing on traditional and innovative scanning electron microscopy techniques such as scanning electron microscopy (SEM), variable pressure SEM (VP-SEM), environmental SEM (ESEM), and the more recent ambiental SEM (ASEM), ending with the cutting edge Cryo-SEM and focused ion beam SEM (FIB SEM), highlighting the pros and cons of several methods with particular emphasis on conventional SEM and VP-SEM. As each technique has its own advantages and disadvantages, the choice of the most appropriate method must be done carefully, based on the specific aim of the study. The evaluation of the drug effects on biofilm requires imaging methods that show the most detailed ultrastructural features of the biofilm. In this kind of research, the use of scanning electron microscopy with customized protocols such as osmium tetroxide (OsO4), ruthenium red (RR), tannic acid (TA) staining, and ionic liquid (IL) treatment is unrivalled for its image quality, magnification, resolution, minimal sample loss, and actual sample structure preservation. The combined use of innovative SEM protocols and 3-D image analysis software will allow for quantitative data from SEM images to be extracted; in this way, data from images of samples that have undergone different antibiofilm treatments can be compared.