Comparison of Atomic Force Microscopy Interaction Forces between Bacteria and Silicon Nitride Substrata for Three Commonly Used Immobilization Methods

Abstract

Atomic force microscopy (AFM) has emerged as a powerful technique for mapping the surface morphology of biological specimens, including bacterial cells. Besides creating topographic images, AFM enables us to probe both physicochemical and mechanical properties of bacterial cell surfaces on a nanometer scale. For AFM, bacterial cells need to be firmly anchored to a substratum surface in order to withstand the friction forces from the silicon nitride tip. Different strategies for the immobilization of bacteria have been described in the literature. This paper compares AFM interaction forces obtained between Klebsiella terrigena and silicon nitride for three commonly used immobilization methods, i.e., mechanical trapping of bacteria in membrane filters, physical adsorption of negatively charged bacteria to a positively charged surface, and glutaraldehyde fixation of bacteria to the tip of the microscope. We have shown that different sample preparation techniques give rise to dissimilar interaction forces. Indeed, the physical adsorption of bacterial cells on modified substrata may promote structural rearrangements in bacterial cell surface structures, while glutaraldehyde treatment was shown to induce physicochemical and mechanical changes on bacterial cell surface properties. In general, mechanical trapping of single bacterial cells in filters appears to be the most reliable method for immobilization.During recent years, atomic force microscopy (AFM) has been increasingly used in the biosciences (5, 16). Theoretically, it combines the two most important aspects of studying structure-function relationships of biological specimens: it performs high-resolution imaging with a high signal-to-noise ratio on a molecular or submolecular scale and has the ability to operate in aqueous environments, allowing the observation of dynamic molecular events in real time and under physiological conditions. The AFM is surprisingly simple in its concept. A sharp tip located at the free end of a flexible cantilever scans over a surface. Interaction forces between the tip and the sample surface subsequently cause the cantilever to deflect. The deflection signal is acquired and digitized to provide a threedimensional image of the surface.Several biological specimens have been imaged, with lateral and vertical resolution on a nanometer and a subnanometer scale, respectively (9, 14, 23). However, when living microbial cell surfaces are imaged, the softness of the cell surface together with the high pressure over the contact area between the tip and the cell can prevent high-resolution imaging. Image contrast is indeed influenced by the probe's geometry, the imaging parameters, the surface topography, and the viscoelastic and physicochemical properties of the cell surface. Additional problems arise from friction and from lateral displacement of the organism under study, which makes immobilization strategies critical.Beyond being an imaging device, the AFM has evolved as an instrument for measuring molecular interaction forces (21,22). Bio...

Surface Viscoelasticity of Individual Gram-Negative Bacterial Cells Measured Using Atomic Force Microscopy

“…An important requirement for AFM investigations is that the sample must be immobilized on a surface. For this purpose, an aliquot of the bacterial suspension of ϳ10 5 cells per ml was allowed to adhere through electrostatic interaction to a poly-L-lysine-coated glass substrate that was prepared as previously described (29). After 15 min, the bacterium-coated glass substrate was rinsed with deionized water to remove loosely attached bacteria and then transferred to the AFM for immediate measurement.…”

Force measurements with the atomic force microscope: Technique, interpretation and applications

“…Vadillo-Rodriguez et al [973] found an increase of adhesion on the 100 s timescale for Streptococcus thermophilus and Boonaert et al [974] observed an increase in adhesion with load for L. lactis bacteria. Another complication can arise from physicochemical and mechanical changes of the cell surface by cell immobilization [975].…”

Characteristic features of Bacillus cereus cell surfaces with biosorption of Pb(II) ions by AFM and FT-IR

“…It can provide three-dimensional images of the surface ultrastructure with molecular resolution, in real time, with minimal sample preparation and experiments can be executed under physiological conditions. Mild treatment of biological samples minimizes artifacts in images or force measurements [31,[35][36][37]. More importantly, imaging and force measurements can be carried out in buffer solutions representative of natural biological environments [35], thus providing new insights into the structure-functional relationship of microbial surfaces.…”