Probing molecular interactions and mechanical properties of microbial cell surfaces by atomic force microscopy

Knowledge of the surface properties of microbial cells is a key to gain a detailed understanding of their functions in the natural environment and to efficiently exploit them in biotechnological processes. In this paper, we present force-distance curves recorded, by atomic force microscopy (AFM) in aqueous solutions, on various microbial samples: reconstituted S-layers, whole fungal spores and several bacterial strains. The approach and retraction curves exhibited important differences--depending on the type of microorganism, on the physiological state (dormancy versus germination) and on the environmental conditions (ionic strength)--which were shown to reflect differences in long-range surface forces, adhesion forces and mechanical properties. These data illustrate the great potential of AFM force measurements to elucidate the physical properties of microbial cells and to understand, at the molecular level, biointerfacial phenomena such as cell adhesion and cell aggregation.     

Adhesion and friction properties of hydrogenated amorphous carbon films measured by atomic force microscopy

Atomic force microscopy has been used to measure adhesion and friction forces at the interface between an oxidized metal probe tip and amorphous carbon films of varying hydrogen contents (12.3–39.0 atomic percent hydrogen). The interface of an oxide surface and a hard carbon coating models the unlubricated head-disk interface of current hard disk products. Adhesion forces normalized by the radius of curvature of the contacting tip range from 1.09 to 8.53 N/m. Coefficients of friction values, measured as the slope of the friction versus load plot, range from 0.33 to 0.87. A trend of increasing adhesion forces and coefficients of friction is observed for increasing hydrogen content in the films. We attribute the increase in adhesion and friction to increases in the surface free energy of the carbon films with the incorporation of hydrogen.     

Cross-sectional atomic force microscopy imaging of polycrystalline thin films

Atomic force microscopy (AFM) can be used to image cross-sections of thin-film samples. So far, however, it has mainly been used to study cross-sections of epitaxial systems or integrated circuits on crystalline substrates. In this paper, we show that AFM is a powerful tool to image fractured cross-sections of polycrystalline thin films deposited on crystalline and non-crystalline substrates, yielding unique information on the three-dimensional properties of the cross-sections, with a spatial resolution in the nm range. Original images of three different heterostructure systems are presented: Si(wafer)/SnO2/CdS/CdTe, glass/Mo/Cu(In,Ga)Se2,/CdS/ZnO, and glass/SnO2/WO3. We discuss the results by comparing AFM and scanning electron microscopy (SEM) images, and explain, for the different materials, why the AFM provides useful additional information.     

Frictional effects in atomic force microscopy of Langmuir-Blodgett films

Frictional effects in atomic force microscopy (AFM) of Langmuir-Blodgett films of 1, 2-dipalmitoyl-snglycero-phosphoglycerol were examined. Height measurements of the Langmuir layers are strongly influenced by the orientation of the cantilevers used in AFM relative to the sample. A simple model is used to describe the frictional effects and to calculate the real height of the monolayers.     

A symmetrical stretching stage for electrical atomic force microscopy

We present a remotely-controlled device for sample stretching, designed for use with atomic force microscopy (AFM) and providing electrical connection to the sample. Such a device enables nanoscale investigation of electrical properties of thin gold films deposited on polydimethylsiloxane (PDMS) substrate as a function of the elongation of the structure. Stretching and releasing is remotely controlled with use of a dc actuator. Moreover, the sample is stretched symmetrically, which gives an opportunity to perform AFM scans in the same site without a time-consuming finding procedure. Electrical connections to the sample are also provided, enabling Kelvin probe force microscopy (KPFM) investigations. Additionally, we present results of AFM imaging using the stretching stage.     

Probing local surface conductance using current sensing atomic force microscopy

We have analyzed correlations between surface morphology and current sensing images obtained using a current sensing atomic force microscope (CSAFM) and the implication of surface conductivity derived from the current sensing images. We found that in cases where the diameter of a CSAFM probe tip is much smaller than the correlation length of the surface morphological features, the current detected using the probe should have little correlation with the surface features imaged by the same probe. If the sample thickness is much larger than the tip size, the surface conductivity distribution of a sample can be derived from a current sensing image using the Holm resistance relation, and the current probed using a CSAFM reflects the conductance variations in a layer on the surface with the thickness comparable to the probe diameter. However, if the thickness of a sample is comparable to or smaller than the tip diameter, CSAFM measures the conductance across the entire portion of the sample sandwiched between the tip and the electrode.     

Microwave atomic force microscopy imaging for nanometer-scale electrical property characterization

We introduce a new type of microscopy which is capable of investigating surface topography and electrical property of conductive and dielectric materials simultaneously on a nanometer scale. The microwave atomic force microscopy is a combination of the principles of the scanning probe microscope and the microwave-measurement technique. As a result, under the noncontact AFM working conditions, we successfully generated a microwave image of a 200-nm Au film coating on a glass wafer substrate with a spatial resolution of 120 nm and a measured voltage difference of 19.2 mV between the two materials.     

Lift-off patterning of thin Au films on Si surfaces with atomic force microscopy

We studied a new lift-off process of thin Au film on silicon surfaces in nanometer-scale, combining anodic oxidation patterning with AFM, deposition of Au thin film on the patterned substrate and chemical etching processes of the Si oxide underneath the Au film. For Au films of thickness of 2-5 nm, the Au films on the Si oxide patterns were left unbroken and bent down to stick to Si surface after the removal of the oxide by the chemical etching. For an Au film of 1 nm in thickness, it was possible to lift-off the Au film on oxide patterns of the lines and dots in nanometer-scale using Si oxide as a sacrificial mask.     

Investigations into local ferroelectric properties by atomic force microscopy

In this article, we describe nanometer scale characterization of piezoelectric thin films of Lead-Zirconate-Titanate (PZT). Using the electric field from a biased conducting atomic-force microscopy (AFM) tip, we show that it is possible to form and subsequently image ferroelectric domains. Using a sphere-plane model for the tip-sample system we calculate the distribution of electric potential, field and polarization charge, and find good agreement with the experimental values. We also discuss the effects of surface contaminants on domain formation.     

Adhesion mode atomic force microscopy study of dual component protein films

Molecular recognition imaging by AFM was extended to dual component protein films adsorbed on mica. AFM probes were functionalized by covalently linking polyclonal antibodies against fibrinogen. Adhesion mapping mode of AFM was used to generate both topographic images and adhesion images. The efficacy of the functionalized probes was first established by performing adhesion mapping on patterned dual component protein films formed by microcontact printing bovine serum albumin on a mica surface and then backfilling with fibrinogen. Next, adhesion mapping was done on randomly distributed two-component protein monolayers generated by sequential adsorption of submonolayer amounts of fibrinogen followed by backfilling with bovine serum albumin. The adhesion maps were used to generate binary recognition images where the specific and non-specific interactions were differentiated based on a statistically derived cut-off value. The surface coverage of fibrinogen obtained from the recognition image over the complete dual protein monolayer was similar to that obtained prior to backfilling with bovine serum albumin. The number of recognition events that were observed decreased by >80% after blocking the surface with anti-fibrinogen antibodies. This result demonstrated that the positive events in the recognition image were indeed specific antibody-fibrinogen interactions.     

Low-voltage electron microscopy of polymer and organic molecular thin films

We have demonstrated the capabilities of a novel low-voltage electron microscope (LVEM) for imaging polymer and organic molecular thin films. The LVEM can operate in transmission electron microscopy, scanning transmission electron microscopy, scanning electron microscopy, and electron diffraction modes. The microscope operates at a nominal accelerating voltage of 5 kV and fits on a tabletop. A detailed discussion of the electron-sample interaction processes is presented, and the mean free path for total electron scattering was calculated to be 15 nm for organic samples at 5 kV. The total end point dose for the destruction of crystallinity at 5 kV was estimated at 5 x 10(-4) and 3.5 x 10(-2) C/cm2 for polyethylene and pentacene, respectively. These values are significantly lower than those measured at voltages greater than 100 kV. A defocus series of colloidal gold particles allowed us to estimate the experimental contrast transfer function of the microscope. Images taken of several organic materials have shown high contrast for low atomic number elements and a resolution of 2.5 nm. The materials studied here include thin films of the organic semiconductor pentacene, triblock copolymer films, single-molecule dendrimers, electrospun polymer fibers and gold nanoparticles.     

Mechanical properties of asphalt binders evaluated by atomic force microscopy

Atomic Force Microscopy was employed in order to relate the features observed on the surface of a 50/70 asphalt binder according to its local stiffness and elastic recovery. Indentations were performed in different points of the surface and a significant variation of elasticity was observed between the points on the so-called bee structure and the matrix. Also, indentations varying the maximum force were performed on similar white spots in the bee structure and the recovery was followed up to 1 h after indentation. It was observed that the elastic recovery is very much dependent on the colloidal structure of the bee. The final surface state of the binder, close to the bee for usual bees is not the same as the initial one indicating severe plastic deformation. Also, permanent phase change could be observed for bright spots presented in not well-structured bee arrangements. A surface hardening was observed in the bee region.     

Drift-free atomic force microscopy measurements of cell height and mechanical properties

Atomic force microscopy (AFM) is used to study the morphological and mechanical properties of living cells. However, experiments performed over minutes to hours are subject to significant instrumental drift. The main sources of drift are the cantilever's geometrical asymmetry and bimorphic construction. We developed a simple software Stick-and-Move (SaM) routine for AFM that eliminates drift by continuously referencing the sample position to the substrate while acquiring force-distance curves. Control experiments show no drift over 15 min at an acquisition rate of 0.1 Hz. As a proof of concept, we applied the SaM to study the response of rat astrocytes to osmotic stress, observing dimensional and constitutive changes during volume regulation.     

Development of eddy current microscopy for high resolution electrical conductivity imaging using atomic force microscopy

We present a high resolution electrical conductivity imaging technique based on the principles of eddy current and atomic force microscopy (AFM). An electromagnetic coil is used to generate eddy currents in an electrically conducting material. The eddy currents generated in the conducting sample are detected and measured with a magnetic tip attached to a flexible cantilever of an AFM. The eddy current generation and its interaction with the magnetic tip cantilever are theoretically modeled using monopole approximation. The model is used to estimate the eddy current force between the magnetic tip and the electrically conducting sample. The theoretical model is also used to choose a magnetic tip-cantilever system with appropriate magnetic field and spring constant to facilitate the design of a high resolution electrical conductivity imaging system. The force between the tip and the sample due to eddy currents is measured as a function of the separation distance and compared to the model in a single crystal copper. Images of electrical conductivity variations in a polycrystalline dual phase titanium alloy (Ti-6Al-4V) sample are obtained by scanning the magnetic tip-cantilever held at a standoff distance from the sample surface. The contrast in the image is explained based on the electrical conductivity and eddy current force between the magnetic tip and the sample. The spatial resolution of the eddy current imaging system is determined by imaging carbon nanofibers in a polymer matrix. The advantages, limitations, and applications of the technique are discussed.     

Phase imaging with intermodulation atomic force microscopy

Intermodulation atomic force microscopy (IMAFM) is a dynamic mode of atomic force microscopy (AFM) with two-tone excitation. The oscillating AFM cantilever in close proximity to a surface experiences the nonlinear tip-sample force which mixes the drive tones and generates new frequency components in the cantilever response known as intermodulation products (IMPs). We present a procedure for extracting the phase at each IMP and demonstrate phase images made by recording this phase while scanning. Amplitude and phase images at intermodulation frequencies exhibit enhanced topographic and material contrast.     

A complementary-metal-oxide-semiconductor-field-effect-transistor-compatible atomic force microscopy tip fabrication process and integrated atomic force microscopy cantilevers fabricated with this process

A complementary-metal-oxide-semiconductor-field-effect-transistor-compatible process for the fabrication of atomic force microscopy cantilevers with integrated tips has been developed. For the first time, the tips are fabricated after the completion of the regular complementary metal-oxide-semiconductor-field-effect-transistor fabrication process sequence. On-chip circuit components, such as piezoresistive deflection sensors, deflection actuators, and amplifiers, are fabricated on the mirror-polished surface of the wafer, ensuring optimal performance. The tip fabrication process is based on anisotropic silicon etching at low temperature using a tetramethylammonium hydroxide solution. The anisotropic etching process has been optimized to ensure process controllability. Using the described process, complementary-metal-oxide-semiconductor-field-effect-transistor-based cantilevers with piezoresistive deflection sensors and integrated tips have been successfully fabricated. Force-distance curves and scanning images in constant-force mode have been recorded.     

Monitoring of glass derivatization with pulsed force mode atomic force microscopy

Non-specific adsorption of proteins at solid/liquid interfaces is a major problem in the use of synthetic biomaterials and in ultrasensitive detection methods. Grafting surfaces with a dense layer of poly(ethylene glycol) (PEG) or other polymers is a most widely used strategy to solve this task. While such modified surfaces have been characterized by their ability to resist protein adsorption, the polymer layers themselves have rarely been studied in fine detail. Atomic force microscopy (AFM) using the pulsed force mode (PFM), is an ideal technique to investigate structural features and physiochemical properties of surfaces because topology and adhesion are simultaneously detected with high lateral resolution. In the present study, PFM-AFM was applied to thoroughly characterize different stages of glass derivatization, up to the formation of a dense PEG layer. Lateral inhomogeneities in topology and/or adhesion were observed at all stages before PEG attachment. The covalently bound PEG, however, was seen to form a densely packed monolayer with maximal thickness, smooth surface, and weak adhesion. Thus, PFM-AFM appears to be a valuable tool for the characterization of protein-repelling surfaces in solution.     

Spectroscopy and atomic force microscopy of biomass

Scanning probe microscopy has emerged as a powerful approach to a broader understanding of the molecular architecture of cell walls, which may shed light on the challenge of efficient cellulosic ethanol production. We have obtained preliminary images of both Populus and switchgrass samples using atomic force microscopy (AFM). The results show distinctive features that are shared by switchgrass and Populus. These features may be attributable to the lignocellulosic cell wall composition, as the collected images exhibit the characteristic macromolecular globule structures attributable to the lignocellulosic systems. Using both AFM and a single case of mode synthesizing atomic force microscopy (MSAFM) to characterize Populus, we obtained images that clearly show the cell wall structure. The results are of importance in providing a better understanding of the characteristic features of both mature cells as well as developing plant cells. In addition, we present spectroscopic investigation of the same samples.     

Analysis of protein crystal growth at molecular resolution by atomic force microscopy

High-resolution atomic force microscopy (AFM) studies have been performed to analyze the molecularity of growth steps of the (1 1 0) face of tetragonal lysozyme crystals. Besides a major population of step heights of about 5.5 nm also step heights of about half this size were observed. The latter steps always appeared pairwise. Both surfaces the 1 1 0) face and the (1 0 1) face could be imaged at molecular level. Comparison of the height pattern of the corresponding surface structure indicates that the (1 1 0) face is relatively smooth of less than 0.2 nm compared to the (1 0 1) face of about 1.5 nm. AFM linescan images of the (1 0 1) face indicate the insertion of lysozyme aggregates in solution to the crystal surface rather than lysozyme monomers. This study suggests that insertion of lysozyme aggregates in the solution yields growth steps of the (1 1 0) face of monomolecular as well as of bimolecular unit height.     

Chemical force microscopy of microcontact-printed self-assembled monolayers by pulsed-force-mode atomic force microscopy

A novel chemically sensitive imaging mode based on adhesive force detection by previously developed pulsed-force-mode atomic force microscopy (PFM-AFM) is presented. PFM-AFM enables simultaneous imaging of surface topography and adhesive force between tip and sample surfaces. Since the adhesive forces are directly related to interaction between chemical functional groups on tip and sample surfaces, we combined the adhesive force mapping by PFM-AFM with chemically modified tips to accomplish imaging of a sample surface with chemical sensitivity. The adhesive force mapping by PFM-AFM both in air and pure water with CH3- and COOH-modified tips clearly discriminated the chemical functional groups on the patterned self-assembled monolayers (SAMs) consisting of COOH- and CH3-terminated regions prepared by microcontact printing (microCP). These results indicate that the adhesive force mapping by PFM-AFM can be used to image distribution of different chemical functional groups on a sample surface. The discrimination mechanism based upon adhesive forces measured by PFM-AFM was compared with that based upon friction forces measured by friction force microscopy. The former is related to observed difference in interactions between tip and sample surfaces when the different interfaces are detached, while the latter depends on difference in periodic corrugated interfacial potentials due to Pauli repulsive forces between the outermost functional groups facing each other and also difference in shear moduli of elasticities between different SAMs.