Super-resolution Imaging of Proteus mirabilis Biofilm by Expansion Microscopy

Accessing detailed visual information and quantitative data from microbiological samples using conventional optical microscopy is limited by the diffraction barrier. One solution to enhance resolution is Expansion Microscopy (ExM), an innovative and cost-effective super-resolution technique that physically enlarges samples by approximately four times their original size. For successful expansion, it is essential to homogenize the mechanical properties of the biological material. Biofilms are bacterial communities, adhering to a surface and embedded in an extracellular matrix they produce; they require ExM protocols to be adapted to accommodate their unique structural components. This article presents Proteus mirabilis biofilm ExM (PmbExM), a specialized variant of ExM that enables super-resolution visualization of P. mirabilis biofilms grown for 48 h. The protocol focuses on the targeted degradation of key structural components of samples through serial enzymatic digestions, optimized near their theoretical conditions. PmbExM utilizes a combination of enzymes, including α-amylase, cellulase, and lyticase glycoside-hydrolases for polysaccharide hydrolysis; mutanolysin for peptidoglycan hydrolysis; and proteinase K for protein hydrolysis. These digestion procedures are independent of the gelation process, allowing modifications to meet specific homogenization requirements in different biofilm models. This adaptability offers great potential for application across various bacterial species and growth conditions. ExM has been applied to different biofilm species with overall suboptimal expansion factors. In contrast, PmbExM achieves the theoretical maximum expansion factor of the standard acrylamide-acrylate ExM hydrogel, without significant distortion of morphology or topology. The aim of this work is to provide an accessible super-resolution protocol for visualizing the architecture, assembly, and cellular and intracellular features of P. mirabilis biofilms.