Expansion microscopy (ExM) is a new super-resolution technique based on embedding the biological sample within a hydrogel and its physical expansion after swelling. This allows increasing its size by several times while preserving its structural details. Applied to prokaryotic cells, ExM requires digestion steps for efficient expansion as bacteria are surrounded by a rigid cell wall. Furthermore, bacteria can live in social groups forming biofilms, where cells are protected from environmental stresses by a self-produced matrix. The extracellular matrix represents an additional impenetrable barrier for ExM. Here we optimize the current protocols of ExM and apply them to mono- and dual-species biofilms formed by clinical isolates of Limosilactobacillus reuteri, Enterococcus faecalis, Serratia marcescens and Staphylococcus aureus. Using scanning electron microscopy for comparison, our results demonstrate that embedded bacteria expanded 3-fold. Moreover, ExM allowed visualizing the three-dimensional architecture of the biofilm and identifying the distribution of different microbial species and their interactions. We also detected the presence of the extracellular matrix after expansion with a specific stain of the polysaccharide component. The potential applications of ExM in biofilms will improve our understanding of these complex communities and have far-reaching implications for industrial and clinical research.

膨胀显微镜（ExM）是一种新的超分辨率技术，基于将生物样品嵌入水凝胶中及其膨胀后的物理膨胀。这允许将其尺寸增加数倍，同时保留其结构细节。应用于原核细胞时，ExM 需要消化步骤才能有效扩增，因为细菌被坚硬的细胞壁包围。此外，细菌可以生活在形成生物膜的社会群体中，其中细胞通过自身产生的基质免受环境压力的影响。细胞外基质是 ExM 的另一个难以穿透的屏障。在这里，我们优化了 ExM 的当前方案，并将其应用于由罗伊氏柠檬酸杆菌、粪肠球菌、粘质沙雷氏菌和金黄色葡萄球菌的临床分离株形成的单物种和双物种生物膜。使用扫描电子显微镜进行比较，我们的结果表明嵌入的细菌扩增了 3 倍。此外，ExM 可以可视化生物膜的三维结构，并识别不同微生物物种的分布及其相互作用。我们还检测了用多糖成分的特定染色剂扩增后细胞外基质的存在。ExM 在生物膜中的潜在应用将提高我们对这些复杂群落的理解，并对工业和临床研究产生深远的影响。