Super-resolution Imaging of Proteus mirabilis Biofilm by Expansion Microscopy

This article presents a comprehensive protocol for PmbExM, an Expansion Microscopy technique designed specifically for Proteus mirabilis biofilms. PmbExM utilizes a stepwise enzymatic treatment of biofilm samples to achieve an isotropic, 4.3-fold expansion, enabling super-resolution analysis of the spatial organization of cellular and subcellular structures within these sessile microbial communities.

The scope of our research is to provide the scientific community with an accessible super resolution method for studying proteus mirabilis biofilm architecture, assembly, and intercellular features beyond the refractory limit.

Quantitative microscopy of biofilms is challenging due to the limitations imposed by optical resolution. Proteus mirabilis biofilm expansion microscopy, or PmbExM will contribute towards an improved morphological and topological description and analysis of this complex microbial communities. Two main advantages of PmbExM is that, first, it allows the super resolution visualization of proteus mirabilis biofilms using conventional refraction-limited microscopes, and second, it does not rely on complex post-acquisition data processing routines.

Our method facilitate the nanoscale study of biofilm structure and organization to researchers lacking access to specialized super resolution equipment and with advanced experience in digital image processing or analysis.

PmbExM will allow the interrogation of proteus mirabilis biofilm architecture, assembly, cellular, and intracellular features at the nanoscale. Moreover, its malleability supports adaptation and codification for other biofilm species.

[Narrator] To begin, add 400 microliters of one-millimolar methacrylic acid N-hydroxysuccinimidyl ester, or MA-NHS in PBS to the stained biofilm samples, and incubate them at room temperature for one hour with mild agitation. After one hour, gently wash the stained biofilm samples three times with 300 microliters of PBS at room temperature for 10 minutes each. Remove the PBS, and add 300 microliters of monomer solution to the samples. Incubate overnight at four degrees Celsius. To construct the gelation pre-chambers, use a glass slide as a base, then cut, arrange, and attach two pieces of folded-over double-sided tape on the slide to act as 400-micrometer spacers. Arrange wet chambers to protect samples from desiccation during polymerization. Now, prepare a fresh stock volume of gelling solution by mixing monomer solution, 10% tetramethyl ethylenediamine, 0.5% 4-Hydroxy-TEMPO, and 10% ammonium persulfate in a 47 to 1 to 1 to 1 ratio. Immediately after preparing the gelling solution, remove the monomer solution from the samples, and replace it with 300 microliters of the gelling solution. Incubate the samples at four degrees Celsius for five minutes. Meanwhile, remove the remaining protective cover from the double-sided tape strips arranged on the glass slides. Pipette 40 microliters of gelling solution between the spacers. Once the incubation of the sample is complete, use tweezers to lift each biofilm-bearing cover slip, and place it on top of the 40-microliter drop of gelling solution on the slide. Orient the cover slip so that the biofilm, on its surface, contacts the gelling solution. Finish constructing the gelation chamber by gently pressing the biofilm-bearing cover slip with tweezers to ensure adhesion to the tape spacers. Place the assembled gelation chamber inside a wet chamber to allow polymerization, and incubate at 37 degrees Celsius without agitation for two hours. After two hours, disassemble the gelation chambers, and use a surgical blade to trim the excess gel around the biofilm sample's region of interest. Place the cover slips carrying the trimmed gels into a new 24-well plate with the gel side facing upwards. After enzymatic digestion of the gelled samples with proteinase K solution, remove the proteinase solution, and transfer each gel to a separate 60-millimeter Petri dish for the expansion of the samples. Fill each Petri dish with excess deionized water so the gel is fully submerged, and incubate under gentle agitation at room temperature for 20 minutes. After completing the fifth water exchange, remove the deionized water covering the gel. Use a small flat brush to gently push the sample onto a 24-by-50-millimeter glass cover slip. Remove the excess water from the gel using tissue paper. Carefully place the gel onto the glass surface of the imaging chamber, ensuring no air bubbles are trapped between the gel and the glass. Finally, add deionized water to the imaging chamber until the gel is fully submerged. Quantitative and spatial analyses were used to evaluate the expansion fidelity and morphological preservation of proteus mirabilis biofilms following proteus mirabilis biofilm expansion microscopy, or PmbExM treatment. The PmbExM technique expanded the biofilm structures by approximately 4.3 fold while maintaining both cellular morphology and topological organization. This expansion significantly increased the visual resolution of bacterial cells, making individual cell structures sharper and more defined. With the enhanced resolution, PmbExM also enabled the clear identification of multi-layered bacterial arrangements within the biofilm that were previously unresolved. PmbExM also facilitated the visualization of previously unresolvable subcellular structures, including distinct patterns of DNA organization. These organizational patterns were confirmed by line-intensity profiles, which revealed multiple distinct fluorescence peaks in expanded samples compared to the single peak observed in non-expanded cells.