Imaging live bacteria at the nanoscale: comparison of immobilisation strategies. Issue 23 (17th October 2019) - Benn, Georgina
Abstract : Different sample preparations are compared, to facilitate atomic force microscopy (AFM) of live Gram-negative bacteria. The obtained resolution is sufficient to resolve the proteinaceous network in the outer membrane. Abstract : Atomic force microscopy (AFM) provides an effective, label-free technique enabling the imaging of live bacteria under physiological conditions with nanometre precision. However, AFM is a surface scanning technique, and the accuracy of its performance requires the effective and reliable immobilisation of bacterial cells onto substrates. Here, we compare the effectiveness of various chemical approaches to facilitate the immobilisation of Escherichia coli onto glass cover slips in terms of bacterial adsorption, viability and compatibility with correlative imaging by fluorescence microscopy. We assess surface functionalisation using gelatin, poly-l -lysine, Cell-Tak™, and Vectabond®. We describe how bacterial immobilisation, viability and suitability for AFM experiments depend on bacterial strain, buffer conditions and surface functionalisation. We demonstrate the use of such immobilisation by AFM images that resolve the porin lattice on the bacterial surface; local degradation of the bacterial cell envelope by an antimicrobial peptide (Cecropin B); and the formation of membrane attack complexes on the bacterial membrane.