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

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 <i>Escherichia coli</i> 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 <i>Pseudomonas aeruginosa</i> and <i>Acinetobacter baumannii</i> outer membranes, which lack the characteristic protein trimers observed in model <i>E. coli</i>.