Microscopy Insights as an Invaluable Tool for Studying Antimicrobial Interactions with Bacterial Membranes

Abstract The bacterial cell envelope is a complex structure that acts as a barrier to many small molecules, surfactants, and antibiotics. Microscopic understanding of how these molecules interact with the bacterial cell envelope is crucial for the development of new antimicrobials and antibiotics. Advanced microscopy techniques, such as confocal and super‐resolution microscopy, atomic force microscopy (AFM), and electron microscopy (EM), enable high‐resolution examination of membrane packing, pore formation, membrane dynamics, and related mechanisms, both before and after antimicrobial treatment. These methods provide valuable insights into the morphological and dynamical alternations of bacterial cell envelopes during the interaction with antimicrobials/antibiotics. In this review, we highlight findings from various studies utilizing fluorescence correlation spectroscopy (FCS), EM, and AFM to investigate bacterial membrane interactions with antimicrobial molecules. We investigate potential pathways through which antimicrobial molecules disrupt the bacterial cell envelope, using lipid diffusivity as a marker. Using AFM, the morphological and dynamical changes in the bacterial membrane were investigated, uncovering structural alterations induced by antimicrobial exposure, including variations in topography, roughness, and other key features. Taken together, these findings illustrate how microscopy‐based techniques have evolved into powerful tools for unraveling molecular membrane interactions, offering significant potential to guide the development of novel antimicrobials and more effective strategies for treating bacterial infections.