Exploring physiological structure and composition in bacteria with highresolution quantitative EUV ptychography

Table-top extreme ultraviolet (EUV) microscopy offers unique opportunities for label-free high resolution imaging of biological sample with micrometer-scale penetration depth and strong elemental contrast. In this work, we further advanced a state-of-the-art EUV ptychographic microscope and applied it to systematic studies of the structure and composition of two prokaryotic model bacteria, Escherichia coli and Bacillus subtilis. For the first time, a unique combination of quantitative amplitude and phase imaging with high precision and a record-sub-50 nm resolution has been achieved, which unveils the ultrastructure and composition of the bacteria at a nanometer scale. This capability allows for the clear distinction of two different bacterial types, despite their similar size and shape. Furthermore, B. subtilis and its endospores are examined, enabling visualization of their distinct morphology and composition as well as detailed observations of various stages of sporulation. We further visualized the impact of the antibiotic monazomycin on B. subtilis for the first time, revealing its disruptive effects on cellular structures and compositional alterations. This work demonstrates that EUV ptychography, with a straightforward, label-free sample preparation, can serve as a powerful, quantitative imaging modality with a unique sensitivity to chemical information at the nanoscale, advancing biomedical studies e.g. toward developing novel antimicrobial strategies.