Objectives: Our long term aim is to develop methodologies for imaging bacteria using AFM, to enable molecular mapping of the cell surface. The specific aims of this work were to 1) identify a suitable means of immobilisation of bacteria for AFM with minimum alteration to cell surface properties and 2) to develop a method for optimisation of AFM tips to use as molecular probes for molecular mapping of bacterial surfaces.
Methods: We employed the AFM in amplitude modulation mode (tapping mode) under phosphate buffered saline to image bacteria in near natural conditions with minimal distortion of the subject. A range of immobilisation techniques (including chemical adhesives and mechanical entrapment) were tested using Streptococcus salivarius, Streptococcus sanguinis and Escherichia coli as living substrates. Tip functionalisation was carried out using silicon models treated first with ethanolamine or aminopropyltriethoxysilane (APTES), followed by attachment of bifunctional polyethyleneglycol (PEG) which terminated in succinimide and maleimide groups. The maleimide group was then reacted with mercaptoethanol to check the orientation of the PEG. X-Ray Photoelectron Scattering (XPS) was used to validate functionalisation.
Results: Mechanical entrapment using polycarbonate filters proved to be the optimum method for imaging coccoid micro-organisms. Preliminary XPS data confirmed flat silicon models for AFM tips can be successfully functionalised with correctly oriented PEG cross-linkers following APTES coating.
Conclusions: Obtaining high quality, stable images of bacteria under fluid underpins the development of more advanced force microscopy techniques. We will now use tip functionalisation with PEG bound to specific ligands to map the surfaces of bacteria with a degree of precision that cannot be achieved using current microscopy methods.
Funding: Supported by an MRC studentship.