Nanometer-resolution imaging of living bacteria across species and cellular orientations

Abstract The bacterial cell envelope is a complex superstructure that is an important target and a major barrier for antibiotics. To date, highest-resolution images of native cell envelopes have been obtained by atomic force microscopy (AFM), but its use is limited by challenges in bacterial immobilization. We establish glutaraldehyde-functionalized glass surfaces for reversible covalent attachment, enabling robust adhesion across species and cellular orientations while preserving cell viability and growth. This approach allowed imaging of Escherichia coli both expressing O-antigen and fully lacking lipopolysaccharide (LPS) oligosaccharides, revealing that AFM contrast depends on LPS complexity and confirming previously observed outer membrane domains as LPS-enriched islands. It also enabled imaging of Pseudomonas aeruginosa and Acinetobacter baumannii outer membranes, which lack the characteristic protein trimers observed in model E. coli . Finally, we discovered growth-phase dependent alterations in E. coli pole morphology, where trimer networks typical of exponential-phase cells were unresolved and morphological defects appeared.