Towards artefact-free AFM image presentation and interpretation.

Atomic force microscopy (AFM) is based upon a simple operational principle. However, the presentation and interpretation of AFM images can easily suffer from consequential artefacts that are easily overlooked. Here we discuss results from AFM and its companion variations AFM-IR (AFM combined with infrared spectroscopy) and PF-QNM (an AFM mode called peak-force quantitative nano-mechanical mapping) by imaging 'bee' structures in asphalt binder (bitumen) as examples. We show how common problems manifest themselves and provide solutions, with the intent that authors can present their results clearly and avoid interpreting artefacts as true physical properties, thereby raising the quality of AFM research.

Relating the physical properties of Pseudomonas aeruginosa lipopolysaccharides to virulence by atomic force microscopy.

Lipopolysaccharides (LPS) are an important class of macromolecules that are components of the outer membrane of Gram-negative bacteria such as Pseudomonas aeruginosa. P. aeruginosa contains two different sugar chains, the homopolymer common antigen (A band) and the heteropolymer O antigen (B band), which impart serospecificity. The characteristics of LPS are generally assessed after isolation rather than in the context of whole bacteria. Here we used atomic force microscopy (AFM) to probe the physical properties of the LPS of P. aeruginosa strain PA103 (serogroup O11) in situ. This strain contains a mixture of long and very long polymers of O antigen, regulated by two different genes. For this analysis, we studied the wild-type strain and four mutants, ΔWzz1 (producing only very long LPS), ΔWzz2 (producing only long LPS), DΔM (with both the wzz1 and wzz2 genes deleted), and Wzy::GM (producing an LPS core oligosaccharide plus one unit of O antigen). Forces of adhesion between the LPS on these strains and the silicon nitride AFM tip were measured, and the Alexander and de Gennes model of steric repulsion between a flat surface and a polymer brush was used to calculate the LPS layer thickness (which we refer to as length), compressibility, and spacing between the individual molecules. LPS chains were longest for the wild-type strain and ΔWzz1, at 170.6 and 212.4 nm, respectively, and these values were not statistically significantly different from one another. Wzy::GM and DΔM have reduced LPS lengths, at 34.6 and 37.7 nm, respectively. Adhesion forces were not correlated with LPS length, but a relationship between adhesion force and bacterial pathogenicity was found in a mouse acute pneumonia model of infection. The adhesion forces with the AFM probe were lower for strains with LPS mutations, suggesting that the wild-type strain is optimized for maximal adhesion. Our research contributes to further understanding of the role of LPS in the adhesion and virulence of P. aeruginosa.

Atomic force microscopy study of the role of LPS O-antigen on adhesion of E. coli.

The O-antigen is a highly variable component of the lipopolysaccharide (LPS) among Escherichia coli strains and is useful for strain identification and assessing virulence. While the O-antigen has been chemically well characterized in terms of sugar composition, physical properties such as O-antigen length of E. coli LPS have not been well studied, even though LPS length is important for determining binding of bacteria to biomolecules and epithelial cells. Atomic force microscopy (AFM) was used to characterize the physicochemical properties of the LPS of eight E. coli strains. Steric repulsion between the AFM tip (silicon nitride) and the E. coli cells was measured and modeled, to determine LPS lengths for three O157 and two O113 E. coli strains, and three control (K12) strains that do not express the O-antigen. For strains with an O-antigen, the LPS lengths ranged from 17 +/- 10 to 37 +/- 9 nm, and LPS length was positively correlated with the force of adhesion (F(adh)). Longer lengths of LPS may have allowed for more hydrogen bonding between the O-antigen and silanol groups of the AFM silicon nitride tip, which controlled the magnitude of F(adh). For control strains, LPS lengths ranged from 3 +/- 2 to 5 +/- 3 nm, and there was no relationship between LPS length and adhesion force between the bacterium and the silicon nitride tip. In the absence of the O-antigen, we attributed F(adh) to electrostatic interactions with lipids in the bacterial membrane.

Understanding Calcium-Mediated Adhesion of Nanomaterials in Reservoir Fluids by Insights from Molecular Dynamics Simulations.

Interest in nanomaterials for subsurface applications has grown markedly due to their successful application in a variety of disciplines, such as biotechnology and medicine. Nevertheless, nanotechnology application in the petroleum industry presents greater challenges to implementation because of the harsh conditions (i.e. high temperature, high pressure, and high salinity) that exist in the subsurface that far exceed those present in biological applications. The most common subsurface nanomaterial failures include colloidal instability (aggregation) and sticking to mineral surfaces (irreversible retention). We previously reported an atomic force microscopy (AFM) study on the calcium-mediated adhesion of nanomaterials in reservoir fluids (S. L. Eichmann and N. A. Burnham, Sci. Rep. 7, 11613, 2017), where we discovered that the functionalized and bare AFM tips showed mitigated adhesion forces in calcium ion rich fluids. Herein, molecular dynamics reveal the molecular-level details in the AFM experiments. Special attention was given to the carboxylate-functionalized AFM tips because of their prominent ion-specific effects. The simulation results unambiguously demonstrated that in calcium ion rich fluids, the strong carboxylate-calcium ion complexes prevented direct carboxylate-calcite interactions, thus lowering the AFM adhesion forces. We performed the force measurement simulations on five representative calcite crystallographic surfaces and observed that the adhesion forces were about two to three fold higher in the calcium ion deficient fluids compared to the calcium ion rich fluids for all calcite surfaces. Moreover, in calcium ion deficient fluids, the adhesion forces were significantly stronger on the calcite surfaces with higher calcium ion exposures. This indicated that the interactions between the functionalized AFM tips and the calcite surfaces were mainly through carboxylate interactions with the calcium ions on calcite surfaces. Finally, when analyzing the order parameters of the tethered functional groups, we observed significantly different behavior of the alkanethiols depending on the absence or presence of calcium ions. These observations agreed well with AFM experiments and provided new insights for the competing carboxylate/calcite/calcium ion interactions.

Effect of electrode roughness on the capacitive behavior of self-assembled monolayers.

Analytical gold electrodes were polished mechanically and electrochemically and the true area of the electrode surface was measured by quantitative oxidative/reductive cycling of the electrode. A roughness factor for each electrode was determined from the ratio of the true area to the geometric area. The roughness is fully described by a combination of microscopic roughness (up to tens of nanometers) and macroscopic roughness (on the order of hundreds of nanometers) terms. The electrodes were then derivatized with a self-assembled monolayer (SAM) of dodecanethiol or a thioalkane azacrown and characterized by impedance spectroscopy. The behavior of the electrodes was modeled with either a Helmholtz or Randles equivalent circuit (depending on the SAM used) in which the capacitance was replaced with a constant phase element. From the model, an effective capacitance and an alpha factor that quantifies the nonideality of the SAM capacitance was obtained. The effective capacitance divided by the roughness factor yields the capacitance per unit true area, which is only a function of microscopic roughness. The relationship between this capacitance and the alpha factor indicates that microscopic roughness predominantly affects the nonideality of the film while macroscopic roughness predominantly affects the magnitude of the film's capacitance. Understanding the contribution of the electrode topography to the magnitude and ideality of the SAM capacitance is important in the construction of SAM-based capacitive sensors because it predicts the importance of electrode-electrode variations.

Calcium-Mediated Adhesion of Nanomaterials in Reservoir Fluids.

Globally, a small percentage of oil is recovered from reservoirs using primary and secondary recovery mechanisms, and thus a major focus of the oil industry is toward developing new technologies to increase recovery. Many new technologies utilize surfactants, macromolecules, and even nanoparticles, which are difficult to deploy in harsh reservoir conditions and where failures cause material aggregation and sticking to rock surfaces. To combat these issues, typically material properties are adjusted, but recent studies show that adjusting the dispersing fluid chemistry could have significant impact on material survivability. Herein, the effect of injection fluid salinity and composition on nanomaterial fate is explored using atomic force microscopy (AFM). The results show that the calcium content in reservoir fluids affects the interactions of an AFM tip with a calcite surface, as surrogates for nanomaterials interacting with carbonate reservoir rock. The extreme force sensitivity of AFM provides the ability to elucidate small differences in adhesion at the pico-Newton (pN) level and provides direct information about material survivability. Increasing the calcium content mitigates adhesion at the pN-scale, a possible means to increase nanomaterial survivability in oil reservoirs or to control nanomaterial fate in other aqueous environments.

Surface microstructure of bitumen characterized by atomic force microscopy.

Bitumen, also called asphalt binder, plays important roles in many industrial applications. It is used as the primary binding agent in asphalt concrete, as a key component in damping systems such as rubber, and as an indispensable additive in paint and ink. Consisting of a large number of hydrocarbons of different sizes and polarities, together with heteroatoms and traces of metals, bitumen displays rich surface microstructures that affect its rheological properties. This paper reviews the current understanding of bitumen's surface microstructures characterized by Atomic Force Microscopy (AFM). Microstructures of bitumen develop to different forms depending on crude oil source, thermal history, and sample preparation method. While some bitumens display surface microstructures with fine domains, flake-like domains, and dendrite structuring, 'bee-structures' with wavy patterns several micrometers in diameter and tens of nanometers in height are commonly seen in other binders. Controversy exists regarding the chemical origin of the 'bee-structures', which has been related to the asphaltene fraction, the metal content, or the crystallizing waxes in bitumen. The rich chemistry of bitumen can result in complicated intermolecular associations such as coprecipitation of wax and metalloporphyrins in asphaltenes. Therefore, it is the molecular interactions among the different chemical components in bitumen, rather than a single chemical fraction, that are responsible for the evolution of bitumen's diverse microstructures, including the 'bee-structures'. Mechanisms such as curvature elasticity and surface wrinkling that explain the rippled structures observed in polymer crystals might be responsible for the formation of 'bee-structures' in bitumen. Despite the progress made on morphological characterization of bitumen using AFM, the fundamental question whether the microstructures observed on bitumen surfaces represent its bulk structure remains to be addressed. In addition, critical technical challenges associated with AFM characterization of bitumen surface structures are discussed, with possible solutions recommended. For future work, combining AFM with other chemical analysis tools that can generate comparable high resolution to AFM would provide an avenue to linking bitumen's chemistry to its microscopic morphological and mechanical properties and consequently benefit the efforts of developing structure-related models for bituminous materials across the different length scales.

A high throughput MATLAB program for automated force-curve processing using the AdG polymer model.

Research in understanding biofilm formation is dependent on accurate and representative measurements of the steric forces related to brush on bacterial surfaces. A MATLAB program to analyze force curves from an AFM efficiently, accurately, and with minimal user bias has been developed. The analysis is based on a modified version of the Alexander and de Gennes (AdG) polymer model, which is a function of equilibrium polymer brush length, probe radius, temperature, separation distance, and a density variable. Automating the analysis reduces the amount of time required to process 100 force curves from several days to less than 2min. The use of this program to crop and fit force curves to the AdG model will allow researchers to ensure proper processing of large amounts of experimental data and reduce the time required for analysis and comparison of data, thereby enabling higher quality results in a shorter period of time.

Measuring the mechanical properties of living cells using atomic force microscopy.

Mechanical properties of cells and extracellular matrix (ECM) play important roles in many biological processes including stem cell differentiation, tumor formation, and wound healing. Changes in stiffness of cells and ECM are often signs of changes in cell physiology or diseases in tissues. Hence, cell stiffness is an index to evaluate the status of cell cultures. Among the multitude of methods applied to measure the stiffness of cells and tissues, micro-indentation using an Atomic Force Microscope (AFM) provides a way to reliably measure the stiffness of living cells. This method has been widely applied to characterize the micro-scale stiffness for a variety of materials ranging from metal surfaces to soft biological tissues and cells. The basic principle of this method is to indent a cell with an AFM tip of selected geometry and measure the applied force from the bending of the AFM cantilever. Fitting the force-indentation curve to the Hertz model for the corresponding tip geometry can give quantitative measurements of material stiffness. This paper demonstrates the procedure to characterize the stiffness of living cells using AFM. Key steps including the process of AFM calibration, force-curve acquisition, and data analysis using a MATLAB routine are demonstrated. Limitations of this method are also discussed.

Shape-independent lateral force calibration.

Current methods for lateral force calibration are often time-consuming, expensive, or cause significant wear of the tip. A quick and simple alternative is presented in which the linear relationship between force and voltage is exploited. The technique is independent of the shapes of the sample and cantilever and eliminates common problems, while maintaining better than 10% precision. This advance will facilitate quantitative comparisons between experiments.

Substrate rigidity regulates the formation and maintenance of tissues.

The ability of cells to form tissues represents one of the most fundamental issues in biology. However, it is unclear what triggers cells to adhere to one another in tissues and to migrate once a piece of tissue is planted on culture surfaces. Using substrates of identical chemical composition but different flexibility, we show that this process is controlled by substrate rigidity: on stiff substrates, cells migrate away from one another and spread on surfaces, whereas on soft substrates they merge to form tissue-like structures. Similar behavior was observed not only with fibroblastic and epithelial cell lines but also explants from neonatal rat hearts. Cell compaction on soft substrates involves a combination of weakened adhesions to the substrate and myosin II-dependent contractile forces that drive cells toward one another. Our results suggest that tissue formation and maintenance is regulated by differential mechanical signals between cell-cell and cell-substrate interactions, which in turn elicit differential contractile forces and adhesions to determine the preferred direction of cell migration and association.