Immobilisation of living bacteria for AFM imaging under physiological conditions

Atomic force microscopy (AFM) holds great potential for studying the nanoscale surface structures of living cells, and to measure their interactions with abiotic surfaces, other cells, or specific biomolecules. However, the application of AFM in microbiology is challenging due to the difficulty of immobilising bacterial cells to a flat surface without changing the cell surface properties or cell viability. We have performed an extensive and thorough study of how to functionalise surfaces in order to immobilise living bacteria for AFM studies in liquid environments. Our aim was to develop a scheme which allows bacterial cells to be immobilised to a flat surface with sufficient strength to avoid detachment during the AFM scanning, and without affecting cell surface chemistry, structure, and viability. We compare and evaluate published methods, and present a new, reproducible, and generally applicable scheme for immobilising bacteria cells for an AFM imaging.Bacterial cells were immobilised to modified glass surfaces by physical confinement of cells in microwells, physisorption to positively charged surfaces, covalent binding to amine- or carboxyl-terminated surfaces, and adsorption to surfaces coated with highly adhesive polyphenolic proteins originating from the mussel Mytilus edulis. Living cells could be immobilised with all of these approaches, but many cells detached when immobilised by electrostatic interactions and imaged in buffers like PBS or MOPS. Cells were more firmly attached when immobilised by covalent binding, although some cells still detached during AFM imaging. The most successful method revealed was immobilisation by polyphenolic proteins, which facilitated firm immobilisation of the cells. Furthermore, the cell viability was not affected by this immobilisation scheme, and adhesive proteins thus provide a fast, reproducible, and generally applicable scheme for immobilising living bacteria for an AFM imaging