Dielectrophoretic force microscopy of aqueous interfaces

A novel scanning probe microscopy technique has allowed dielectrophoretic force imaging with nanoscale spatial resolution. Dielectrophoresis (DEP) traditionally describes the mobility of polarizable particles in inhomogeneous alternating current (ac) electric fields. Integrating DEP with atomic force microscopy allows for noncontact imaging with the image contrast related to the local electric polarizability. By tuning the ac frequency, dielectric spectroscopy can be performed at solid/liquid interfaces with high spatial resolution. In studies of cells, the frequency-dependent dielectrophoretic force is sensitive to biologically relevant electrical properties, including local membrane capacitance and ion mobility. Consequently, dielectrophoretic force microscopy is well suited for in vitro noncontact scanning probe microscopy of biological systems.     

Atomic force microscopy for the characterization of immobilized enzyme molecules on biosensor surfaces

The development of biosensors has been one of the key areas in biotechnology and biomedical studies. Often it is difficult to investigate the immobilized biomolecules on the surfaces for biosensor optimization. Atomic force microscopy (AFM) should provide an ideal means for the visualization of biosensor surface and for the investigation of biomolecule activities. Therefore, AFM has been employed to study the surface topography of immobilized glutamate dehydrogenase (GDH) on two-dimensional glutamate biosensor surfaces. Correlation between the surface topography and the activity of the biosensor was investigated. Surface analysis has revealed that the enzymatic activity of the immobilized GDH molecules on the biosensor surface is linked to surface roughness, as measured by the peak-to-valley distance. Fractal dimension of the immobilization sensor surface was found to be a good parameter for judging the quality of the immobilized biosensors. As enzyme immobilization time increases, the biosensor has its maximum activity with around 18 h of immobilization in 10^–6 M GDH solution. Various biosensors prepared under different experimental conditions have been studied by AFM. This technique is shown to be an effective tool to characterize biosensor surfaces.     

Atomic force microscopy for the characterization of immobilized enzyme molecules on biosensor surfaces

The development of biosensors has been one of the key areas in biotechnology and biomedical studies. Often it is difficult to investigate the immobilized biomolecules on the surfaces for biosensor optimization. Atomic force microscopy (AFM) should provide an ideal means for the visualization of biosensor surface and for the investigation of biomolecule activities. Therefore, AFM has been employed to study the surface topography of immobilized glutamate dehydrogenase (GDH) on two-dimensional glutamate biosensor surfaces. Correlation between the surface topography and the activity of the biosensor was investigated. Surface analysis has revealed that the enzymatic activity of the immobilized GDH molecules on the biosensor surface is linked to surface roughness, as measured by the peak-to-valley distance. Fractal dimension of the immobilization sensor surface was found to be a good parameter for judging the quality of the immobilized biosensors. As enzyme immobilization time increases, the biosensor has its maximum activity with around 18 h of immobilization in 10(-6) M GDH solution. Various biosensors prepared under different experimental conditions have been studied by AFM. This technique is shown to be an effective tool to characterize biosensor surfaces.     

Atomic force microscopy and magnetic force microscopy study of model colloids

Atomic force microscopy (AFM) is used to study the size, shape, and polydispersity of a variety of magnetic and nonmagnetic model colloids, previously imaged by transmission electron microscopy (TEM) only. Both height and phase images are analyzed and special attention is given to 3D morphology and softness of particles, as well as structures and presence of secondary components in the colloid, difficult to investigate with TEM. Several methods of tip characterization followed by deconvolution were applied in order to improve the accuracy of lateral diameter determination. In the case of magnetite particles dispersed in conventional ferrofluids, we explore both experimentally and theoretically the possibility of using magnetic force microscopy (MFM). We propose and discuss several models which allow to estimate the magnetic moment of a single domain superparamagnetic sphere using MFM, which cannot be done with other techniques; alternatively the tip magnetization can be determined.     

Atomic force microscopy of coated glasses

Atomic force microscopy has been used to investigate the topology of alkoxide gel dip coatings on different substrates. Results of SiO(2) - TiO(2) - ZrO(2) (STZ) coatings are presented on float glass, on polished fused silica, on commercially coated insulating flat glass, and on PtRh. Consolidated STZ coatings display the so-called glass pattern with ripples equal or less than 2 nm high. The same pattern is seen on partially dense STZ coatings, as soon as the surface is stiff enough for scanning, and also on the bottom of a 50 nm deep sputtering crater in the consolidated coating. The vitreous STZ coating on the fire side of the float glass is as flat as the float glass itself. It has the same tendency to contamination. 100 nm wide and 50 nm deep polishing grooves on fused silica have been filled up with the 80 nm thick coating, only dips of a few nm remain. The trenches between the SnO(2) crystallites on the insulating flat glass were filled up and the roughness of the substrate was partially reduced. PtRh sheet remained rough even after the coating. On the partially densified STZ coating, sputtering generates a grained surface.     

Atomic force microscopy of coated glasses

Atomic force microscopy has been used to investigate the topology of alkoxide gel dip coatings on different substrates. Results of SiO₂ – TiO₂ – ZrO₂ (STZ) coatings are presented on float glass, on polished fused silica, on commercially coated insulating flat glass, and on PtRh.· Consolidated STZ coatings display the so-called glass pattern with ripples equal or less than 2 nm high. The same pattern is seen on partially dense STZ coatings, as soon as the surface is stiff enough for scanning, and also on the bottom of a 50 nm deep sputtering crater in the consolidated coating.· The vitreous STZ coating on the fire side of the float glass is as flat as the float glass itself. It has the same tendency to contamination. 100 nm wide and 50 nm deep polishing grooves on fused silica have been filled up with the 80 nm thick coating, only dips of a few nm remain. The trenches between the SnO₂ crystallites on the insulating flat glass were filled up and the roughness of the substrate was partially reduced. PtRh sheet remained rough even after the coating.· On the partially densified STZ coating, sputtering generates a grained surface.     

Atomic force microscopy of potato virus A

Potato virus A is studied by atomic force microscopy. Topographic images of virus particles deposited onto mica are obtained. Geometric characteristics of potato virus A are determined by computer-aided analysis of the images obtained.     

Atomic force microscopy of model lipid membranes

Supported lipid bilayers (SLBs) are biomimetic model systems that are now widely used to address the biophysical and biochemical properties of biological membranes. Two main methods are usually employed to form SLBs: the transfer of two successive monolayers by Langmuir–Blodgett or Langmuir–Schaefer techniques, and the fusion of preformed lipid vesicles. The transfer of lipid films on flat solid substrates offers the possibility to apply a wide range of surface analytical techniques that are very sensitive. Among them, atomic force microscopy (AFM) has opened new opportunities for determining the nanoscale organization of SLBs under physiological conditions. In this review, we first focus on the different protocols generally employed to prepare SLBs. Then, we describe AFM studies on the nanoscale lateral organization and mechanical properties of SLBs. Lastly, we survey recent developments in the AFM monitoring of bilayer alteration, remodeling, or digestion, by incubation with exogenous agents such as drugs, proteins, peptides, and nanoparticles.

Atomic force microscopy in viscous ionic liquids

Extracting quantitative information from amplitude-modulation atomic force microscopy (AM-AFM) in viscous ionic liquids is difficult because existing theory requires knowledge of the cantilever natural frequency, which cannot be measured in the absence of a resonance peak. We present a new model that describes cantilever dynamics in an overdamped medium (Q < 0.5) and derive the theory necessary to extract the stiffness and damping in highly viscous liquids. The proposed methodology is used to measure the solvation layers of an ionic liquid at a gold electrode.     

Atomic force microscopy of extended-chain crystals of polyethylene

Extended-chain crystals of polyethylene grown at elevated pressure and temperature were analyzed for the first time by atomic force microscopy. It was possible to compare the typical fracture surface striation features with those obtained earlier by electron microscopy. High resolution atomic force microscopy on flat surfaces enabled the recording of an atomic scale regularity that could not be fully indentified. © 1993 John Wiley & Sons, Inc.     

Atomic force microscopy study of polypropylene-based self-reinforced composites

Self-reinforced composites are polymeric materials formed by a reinforcement core and a low-melting point skin, which acts as a matrix after the consolidation step. These materials are widely exploited in industrial applications for their mechanical resistance and durability, which are themselves influenced by processing conditions and polymer composition. In the present work, two similar polypropylene-based commercial fabrics were used to evaluate the surface modifications after laminate compaction and after artificial aging using atomic force microscopy. The results were correlated with the chemical and physical-chemical interactions obtained from scanning electron microscopy, transmission electron microscopy, raman and thermal analysis experiments. Single tape consolidated laminate before and after aging displayed different superficial features that can explain the differences in the macroscopic behavior of the two products.     

Atomic force microscopy images of lyotropic lamellar phases

For the very first time, atomic force microscope images of lamellar phases were observed combined with a freeze fracture technique that does not involve the use of replicas. Samples are rapidly frozen, fractured, and scanned directly with atomic force microscopy, at liquid nitrogen temperature and in high vacuum. This procedure can be used to investigate micro-structured liquids. The lamellar phases in Sodium bis(2-ethylhexyl) sulfosuccinate (AOT)/water and in C12E5/water systems were used to asses this new technique. Our observations were compared with x-ray diffraction measurements and with other freeze fracture methods reported in the literature. Our results show that this technique is useful to image lyotropic lamellar phases and the estimated repeat distances for lamellar periodicity are consistent with those obtained by x-ray diffraction.     

Atomic force microscopy assisted immobilization of lipid vesicles

We report on a new approach to direct the immobilization of unilamellar lipid vesicles on substrate-supported lipid bilayers in a spatially confined manner. The adsorption of vesicles from solution is limited to areas of disorder in the bilayers, which is induced by scanning a pattern in situ with an atomic force microscopy (AFM) tip using high imaging forces. Lines of vesicles with a length exceeding 25 microm and a width corresponding to that of a single surface-immobilized vesicle have been fabricated. The adsorbed vesicles are effectively immobilized and do not desorb spontaneously. However, AFM with forces of several nanoNewtons allows one to displace vesicles selectively. The novel methodology described, which may serve as a platform for research on proteins incorporated in the lipid bilayers comprising the vesicles, does not require chemical labeling of the vesicles to guide their deposition.     

Atomic force microscopy characterization of an electrochemical DNA-biosensor

Electrode surface characteristics represent an important aspect on the construction of sensitive DNA electrochemical biosensors for rapid detection of DNA interaction and damage. Two different immobilization procedures of double-stranded DNA (dsDNA) at the surface of a HOPG electrode were evaluated by MAC mode AFM performed in air. A thin dsDNA adsorbed film forming a network structure with holes exposing the electrode surface and a thick dsDNA film completely covering the electrode surface, presenting a much rougher structure, were investigated. The DNA surface characteristics and structure are discussed with respect to the degree of surface coverage.     

Atomic force microscopy investigation of phage infection of bacteria

Atomic force microscopy (AFM) was used to study the process of infection of bacterial cells by bacteriophages, for which purpose experimental protocols were elaborated. Three types of bacteriophages were characterized with AFM and transmission electron microscopy (TEM). Bacteriophage interaction with cells was studied for three bacterial hosts: Gram-negative Escherichia coli 057 and Salmonella enteritidis 89 and Gram-positive Bacillus thuringiensis 393. Depending on the phase of lytic cycle, different cell surface changes are observed in AFM images of infected cells in comparison with intact cells: from phage adsorption on the cells and flagella to complete lysis of the cells, accompanied by the release of a large number of newly formed phages. Control experiments (cells without phages and cells with nonspecific phages) did not reveal any surface changes. Penetration of phages inside obligate aerobe Bacillus thuringiensis was shown to be oxygen-dependent and required aeration in laboratory conditions. Our results show great potential of using AFM for numerous fundamental and applied tasks connected with pathogen-host interaction.     

Atomic force microscopy force mapping in the study of supported lipid bilayers

Investigating the structural and mechanical properties of lipid bilayer membrane systems is vital in elucidating their biological function. One route to directly correlate the morphology of phase-segregated membranes with their indentation and rupture mechanics is the collection of atomic force microscopy (AFM) force maps. These force maps, while containing rich mechanical information, require lengthy processing time due to the large number of force curves needed to attain a high spatial resolution. A force curve analysis toolset was created to perform data extraction, calculation and reporting specifically in studying lipid membrane morphology and mechanical stability. The procedure was automated to allow for high-throughput processing of force maps with greatly reduced processing time. The resulting program was successfully used in systematically analyzing a number of supported lipid membrane systems in the investigation of their structure and nanomechanics.     

Atomic force microscopy of parallel DNA branched junction arrays

BACKGROUND: The four arms of the Holliday junction are known to stack in pairs forming two helical domains whose orientations are antiparallel, but twisted positively by about 60 degrees, based on electrophoretic, FRET and AFM measurements. Recent gel retardation studies suggest that a bowtie junction (containing 5',5' and 3',3' linkages in its crossover strands) may adopt a parallel conformation. RESULTS: An AFM study of two-dimensional arrays produced by parallelograms of bowtie junctions shows that the angle between helical domains is in the range of -68+/-2 degrees. We demonstrate by AFM that the domains are parallel by constructing V-shaped structures whose arms are separated by approximately 68 degrees and approximately 112 degrees. CONCLUSIONS: The arms of the bowtie junction are parallel rather than antiparallel. The parallel or antiparallel nature of the junction apparently is determined by the local structure of the junction, but the sign of the angle appears to be a consequence of interarm electrostatic interactions.     

Atomic force microscopy of soil and stream fulvic acids

Atomic force microscopy (AFM) was used to image fulvic acid (FA) deposited from aqueous solution on to the basal-plane surfaces of freshly cleaved muscovite, and allowed to air dry. Two fulvic acid samples were used: a soil fulvic acid (SFA) prepared by NaOH extraction from a muck soil underlying a freshwater fen in the New Jersey Pinelands and the IHSS standard Suwannee River fulvic acid (SRFA). The use of tapping-mode AFM (TMAFM), a relatively new technique which reduces the lateral frictional forces generally associated with contact-mode AFM, allowed excellent images of delicate FA structures to be obtained with minimal sample disturbance. Four main structures were observed on SFA. At low concentrations, sponge-like structures consisting of rings ( 15 nm in diameter) appeared, along with small spheres (10–50 nm). At higher concentrations, aggregates of spheres formed branches and chain-like assemblies. At very high surface coverage, perforated sheets were observed. On some samples, all of these structures were apparent, perhaps owing to concentration gradients on drying. SRFA samples were only imagined at higher concentrations. Spheres, aggregated branches, and perforated sheets were apparent. The results agree with previous work by Stevenson and Schnitzer [Soil Sci., 133(1992) 179], who applied TEM to soil FAs freeze-dried on muscovite. However, the TEM images did not detect the smaller spheres and sponge-like structures observed by AFM at low concentrations. The relevance of imaging dried samples remains questionable; hence, it is hoped in the future to use new in situ TMAFM to image FAs sorbed to surfaces in solution. Although TMAFM provided excellent images, a variety of artifacts and potential problems were encountered, as discussed.     

Atomic force microscopy of mechanically trapped bacterial cells.

This article presents a study on the influence of the protocol used for immobilization of bacterial cells onto surfaces by mechanically trapping them into a filter. In this sense, the surface and structure of trapped cells are analyzed. Bacteria can be present solely or with extracellular polymeric substances (EPS). To test the behavior of the EPS layer duing the filtering process, different strains of a well-known EPS-producer bacteria (Staphylococcus epidermidis), which produce an extracellular matrix clearly visible in AFM images, have been used. Results show that this immobilization method can cause severe structural and mechanical deformation to the cell membrane. This altered mechanical state may possibly influence the parameters derived from AFM force curves (which are micro/nano-mechanical tests). Also, our results suggest that the EPS layer might move during the filtering process and could accumulate at the upper part of the cell, thus favoring distorted data of adhesion/pull-off forces as measured by an AFM tip, especially in the case of submicron-sized microbial cells such as bacteria.