Atomic Force Microscopy: A New Look at Pathogens

Microbial cells are highly complex and heterogeneous systems. In general, cell populations contain subgroups of cells which exhibit differences in growth rate as well as resistance to stress and drug treatment [1]. In addition, individual cells are spatially organized and heterogeneous, and this cellular heterogeneity is used to perform key functions [2]. This complexity emphasizes the need for single-cell analysis techniques in microbial research. Fluorescence imaging is a powerful tool to localize molecules in single cells [3,4], but the resolution remains limited to the wavelength of the light source. On the other hand, high-resolution images of microbial structures can be obtained by electron microscopy techniques. In particular, cryo-electron tomography—or three-dimensional (3-D) electron microscopy—provides images of whole bacterial cells, at resolutions that are one to two orders of magnitude higher than those obtained with light microscopy [5]. In the past 20 years a new form of microscopy, atomic force microscopy (AFM), has revolutionized the way researchers probe the microbial cell surface. Instead of using an incident beam, AFM measures the minute forces acting between a sharp tip and the sample [6–8]. To generate a topographic image, the tip is attached to a cantilever that bends under force and is moved in threedimensions using a piezoelectric scanner. While scanning the sample surface, cantilever’s bending is measured by a laser beam focused on the free end of the cantilever and reflected into a photodiode. Unlike other microscopy techniques, 3-D images of cells and membranes are obtained at high resolution without staining, labelling or fixation, thus in physiological conditions. AFM is much more than a surface-imaging tool in that it also measures the localization and mechanical properties of the individual cell surface molecules. In this modality, known as single-molecule force spectroscopy, the cantilever deflection is recorded as a function of the vertical displacement of the scanner (as the sample is pushed towards the tip and it retracts) [6,7]. This results in a cantilever deflection vs. scanner displacement curve, which is transformed into a force-distance curve using appropriate corrections. The characteristic adhesion force between tip and sample measured during retraction is used to probe the distribution and mechanics of single molecules, such as cell surface receptors. These novel AFM techniques complement traditional methods used to analyse microbial cell walls and provide new opportunities for understanding cell surface interactions. In this review, we provide a flavour of the various applications offered by AFM in microbiology, and we highlight some of the key breakthroughs the technique has enabled in pathogen research.