Enhanced laterally resolved ToF-SIMS and AFM imaging of the electrically conductive structures in cable bacteria

ABSTRACT Cable bacteria are electroactive bacteria that form a long, linear chain of ridged cylindrical cells. These filamentous bacteria perform centimeter-scale long-range electron transport through parallel, interconnected conductive pathways of which the detailed chemical and electrical properties are still unclear. Here, we combine ToF-SIMS (time of flight secondary ion mass spectrometry) and AFM (atomic force microscopy) to investigate the structure and composition of this naturally-occurring electrical network. The enhanced lateral resolution achieved allows differentiation between the cell body and the cell-cell junctions that contain a conspicuous cartwheel structure. Three ToF-SIMS modes were compared in the study of so-called fiber sheaths (i.e., the cell material that remains after removal of cytoplasm and membranes and which embeds the electrical network). Among these, fast imaging delayed extraction (FI-DE) was found to balance lateral and mass resolution, thus yielding multiple benefits in the study of structure-composition relations in cable bacteria: (i) it enables the separate study of the cell body and cell-cell junctions, (ii) by combining FI-DE with in-situ AFM, the depth of Ni-containing protein – key in the electrical transport – is determined with greater precision, and (iii) this combination prevents contamination, which is possible when using an ex-situ AFM. Our results imply that the interconnects in extracted fiber sheaths are either damaged during extraction, or that their composition is different from fibers, or both. From a more general analytical perspective, the proposed methodology of ToF-SIMS in FI-DE-mode combined with in-situ AFM holds great promise for studying the chemical structure of other biological systems.