Changes in the surface structure of purple membrane upon illumination measured by atomic force microscopy

Bacteriorhodopsin (BR) patches with a diameter of 1 to 3 μm were investigated in their native state by atomic force microscopy (AFM) in buffer solution. The patches were immobilized deposited and investigated on mica in 150 mM KCl and 10 mM Tris-buffer at pH 8. Under this buffer condition they adsorb preferred with their extracellular side to the solid support mica. The structure of the two-dimensional light adapted crystals was resolved with an imaging force of about 100 pN up to a resolution of 13 Å. The topography of the surface gets smoother if an imaging force of 1000 pN was applied indicating that protruding structures are compressed. Upon illumination with white light, during imaging with a force of 200 pN, the surface structure of the BR lattice changed. The force- and light-induced structural changes were reversible.     

Dielectrophoretic immobilization of proteins: Quantification by atomic force microscopy

The combination of alternating electric fields with nanometer‐sized electrodes allows the permanent immobilization of proteins by dielectrophoretic force. Here, atomic force microscopy is introduced as a quantification method, and results are compared with fluorescence microscopy. Experimental parameters, for example the applied voltage and duration of field application, are varied systematically, and the influence on the amount of immobilized proteins is investigated. A linear correlation to the duration of field application was found by atomic force microscopy, and both microscopical methods yield a square dependence of the amount of immobilized proteins on the applied voltage. While fluorescence microscopy allows real‐time imaging, atomic force microscopy reveals immobilized proteins obscured in fluorescence images due to low S/N. Furthermore, the higher spatial resolution of the atomic force microscope enables the visualization of the protein distribution on single nanoelectrodes. The electric field distribution is calculated and compared to experimental results with very good agreement to atomic force microscopy measurements.     

Dynamic imaging of single DNA-protein interactions using atomic force microscopy

Atomic force microscopy (AFM) imaging of static DNA-protein complexes, in air and in liquid, can be used to directly obtain quantitative and qualitative information on the structure of different complexes. For example, DNA length, the location of preferential binding sites for proteins and bending of DNA as a result of the complexation can all be measured. Recording consecutive AFM images of DNA and protein molecules under conditions that they are still able to move and interact, or dynamic AFM imaging, however, can reveal information on the dynamic aspects of the interactions between these molecules. Here, an overview is given of the technical challenges that need to be considered for successful dynamic AFM imaging studies of individual DNA-protein interactions. Necessary technical improvements to the AFM set-up and the development of new sample preparation methods are described in this paper.     

Interaction between solid surfaces in a polymer melt studied by atomic force microscopy

Using an atomic force microscope (AFM) the interaction between an AFM tip and a planar silicon oxide surface has been measured across poly(dimethylsiloxane) (PDMS, M_W = 18 000). Due to the small radius of curvature of the AFM tip the hydrodynamic repulsion of the tip was negligible and forces could be measured in equilibrium. This is confirmed by the fact that force-versus-distance curves measured at different approaching velocities were indistinguishable. In equilibrium a repulsive force was observed which could best be described by a power law, F ∝ 1/d^2.5 where d is the distance.     

Label-free quantification of Tacrolimus in biological samples by atomic force microscopy

In the present paper we describe an atomic force microscopy (AFM)-based method for the quantitative analysis of FK506 (Tacrolimus) in whole blood (WB) samples. Current reference methods used to quantify this immunosuppressive drug are based on mass spectrometry. In addition, an immunoenzymatic assay (ELISA) has been developed and is widely used in clinic, even though it shows a small but consistent overestimation of the actual drug concentration when compared with the mass spectrometry method. The AFM biosensor presented herein utilises the endogen drug receptor, FKBP12, to quantify Tacrolimus levels. The biosensor was first assayed to detect the free drug in solution, and subsequently used for the detection of Tacrolimus in blood samples. The sensor was suitable to generate a dose–response curve in the full range of clinical drug monitoring. A comparison with the clinically tested ELISA assay is also reported.     

Intercalation-induced changes in DNA supercoiling observed in real-time by atomic force microscopy

Atomic force microscopy (AFM) has been employed to observe in real-time and in an aqueous environment the process of ethidium bromide induced supercoiling in individual DNA plasmid molecules. Image data reveal both the onset and the progressive presence of plectonemic DNA supercoiling. In addition, significant molecular motion of the surface adsorbed DNA is observed. These data illustrate the potential of AFM in the time-resolved study of biomolecular processes, and hence, provide new insights into biomolecular structure and function.     

Visualisation of an ultrafiltration membrane by non-contact atomic force microscopy at single pore resolution

Non-contact atomic force microscopy (AFM) has been used to investigate the furface pore structure of a polyethersulfone ultrafitration membrane of specified molecular weight cut off (MWCO) 25 000 (ES625, PCI Membrane Systems). Excellent images at up to single pore resolution were obtained. This is the first time that AFM images of a membrane at such high resolution have been presented. Analysis of the images gave a mean pore size of 5.1 nm with a standard deviation of 1.1 nm. The results have been compared to previously published studies of membranes of comparable MWCO using contact AFM and electron microscopy. Non-contact AFM is a powerful means of studying the surface pore characteristics of ultrafiltration membranes.     

Characterization of human cells exposed to deltamethrin by means of Raman microspectroscopy and atomic force microscopy

Raman spectroscopy is a powerful technique for studying cellular biochemistry. In fact, each toxic chemical induces biochemical changes related to the own action mechanism. In this investigation Raman microspectroscopy has been used, in correlation with atomic force microscopy images, to detect biochemical and structural damages occurring in cultured human cells as a consequence of deltamethrin exposure. Cultured human keratinocyte cells have been exposed at increasing concentrations of deltamethrin from 10⁻³ M to 10⁻⁶ M for 24 h. A viability test indicated that the cytotoxic dose corresponds to exposure at deltamethrin solution for 24 h with the chemical concentration between 10⁻⁴ M and 2.5 10⁻⁴ M. The compared analysis of Raman spectra and AFM images allows to state that an evident damage occurs in the plasmatic membrane and it is already detectable after exposure of keratinocytes at the lowest investigated deltamethrin concentration (10⁻⁶ M). The most important modifications are related to the breakdown of CH₂ bonds of lipidic chains, whereas proteineous bonds are less involved in the deltamethrin action. On the whole, cellular damage starts after exposure to deltamethrin doses well lower than that established as cytotoxic.     

Surface properties of Aspergillus oryzae spores investigated by atomic force microscopy

Spores of the filamentous fungus Aspergillus oryzae have a great biotechnological potential for the production of highly active proteins. To date, little is known about the molecular mechanisms of spore aggregation, a phenomenon observed during germination in liquid medium. Here, atomic force microscopy (AFM) imaging and force measurements were used to characterize, under aqueous conditions, the surface morphology and macromolecular interactions of A. oryzae spores in relation to their aggregation behavior. Dormant spores were covered with a discontinuous layer of about 35 nm thickness, as revealed by height images. High-resolution deflection images showed that this layer consisted of rodlets, 10±1 nm in diameter, that were assembled in parallel to form fascicles interlaced with different orientations. The germinating spore surface was much rougher and showed streaks oriented in the scanning direction, indicating that the probe was interacting with soft material. Retraction force curves were strikingly different depending on the spore physiological state: while dormant spores exhibited non-adhesive properties, germinating spores showed single or multiple attractive forces of 400±100 pN magnitude, along with characteristic elongation forces and rupture lengths ranging from 20 to 500 nm. These elongation forces are attributed to the stretching of long, flexible cell surface macromolecules and suggested to play a role in the aggregation process by promoting bridging interactions.     

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.     

Molecular imaging of membrane proteins and microfilaments using atomic force microscopy

Atomic force microscopy (AFM) is an emerging technique for a variety of uses involving the analysis of cells. AFM is widely applied to obtain information about both cellular structural and subcellular events. In particular, a variety of investigations into membrane proteins and microfilaments were performed with AFM. Here, we introduce applications of AFM to molecular imaging of membrane proteins, and various approaches for observation and identification of intracellular microfilaments at the molecular level. These approaches can contribute to many applications of AFM in cell imaging.     

Probing buried carbon nanotubes within polymer-nanotube composite matrices by atomic force microscopy

Multi-walled carbon nanotubes (MW-CNT) inside a polyamide-6 (PA6)-MW-CNT composite were visualized by atomic force microscopy (i) in a field-assisted intermittent contact and (ii) in the tunneling (TUNA) mode. Individual buried MW-CNTs were clearly discerned within the PA6 matrix. An average diameter of 33 ± 5 nm of the MW-CNTs was determined based on field-assisted intermittent contact mode AFM images, which is consistent with the expected size of PA6-coated MW-CNTs. Single well dispersed MW-CNTs that are located in the sub-surface region of the composite were also observed in the TUNA mode. These new AFM approaches circumvent the tedious sample preparation based on ultramicrotoming required for high resolution electron microscopy studies to obtain “in-depth” morphological information and hence are expected to facilitate the analysis of CNT-based and other nanocomposites in the future.     

Characterization of elastomeric blends by atomic force microscopy

The bulk mechanical properties of a blend of elastomers are found to depend on the micro and nano scale morphology of the phases of the materials in the blend. In this study, we examine the phase morphology of blends of incompatible elastomers using Atomic Force Microscopy (AFM). Specifically, nanoindentation and Tapping Mode AFM (TMAFM) imaging techniques are used as experimental tools for mapping the composition of unfilled elastomeric blends. Depending on the composition of the blend, either co‐continuous or discontinuous domain/matrix morphology is observed. To identify the different components in bromobutyl (BIIR)/natural rubber (NR) blends, nanoscale indentation measurements were made on the observed phase‐separated regions. Results from force mode AFM and mechanical measurements of bulk NR and BIIR are used to assist in the interpretation of the TMAFM results for the BIIR/NR blends. © 2005 Wiley Periodicals, Inc. J Polym Sci Part B: Polym Phys 44: 492–503, 2006     

Melting behavior of lamellae of isotactic polypropylene studied using hot-stage atomic force microscopy

The α- and β-form lamellae of isotactic polypropylene were developed at different temperatures. The melting behaviors of the lamellae were observed in real time at elevated temperatures using a hot-stage atomic force microscopy. The melting behavior of the α-form lamellae was determined by the lamellar defects. For the α-form lamellae developed at different undercoolings, the larger the undercoolings, the relatively higher amount of defect in the lamellae was observed. The lamellae with defects were melted into lamellar segments, and recrystallization took place during the heating process. The β-form lamellae had lower thermal stability, and they melted firstly and separately from that of α-form.     

Current-sensing atomic force microscopy for analyzing conformations and properties of polymer membranes for fuel cells

The three-dimensional structures of polymer membranes are different at surfaces and inside bulks, and thus, in general, physical/chemical properties are also different. Morphologies and properties of membrane surfaces are now visualized by current-sensing atomic force microscopy. The increase in performances of a single cell is discussed based on the three-dimensional structures of the polymer membrane, anion-exchange membrane as an example, used for fuel cells. Other reports on Nafion®, proton-exchange membrane, are also introduced to show the importance of this microscopic method.     

Molecular positional order in a dipalmitoylphosphatidic acid Langmuir–Blodgett monolayer by atomic force microscopy

Langmuir–Blodgett monolayers of dipalmitoylphosphatidic acid were studied by using atomic force microscopy on the large- and nano-scale. A molecularly resolved image was achieved at high surface pressure. The monolayer shows a dramatic long-range orientational and positional ordering of molecular organization of aliphatic tails. The ordered molecular arrangement of aliphatic tails may result from the strong intermolecular hydrogen bonding interactions between adjacent phosphate groups in the polar region.     

Thickness and morphology of polyelectrolyte coatings on silica surfaces before and after protein exposure studied by atomic force microscopy

Analyte–wall interaction is a significant problem in capillary electrophoresis (CE) as it may compromise separation efficiencies and migration time repeatability. In CE, self-assembled polyelectrolyte multilayer films of Polybrene (PB) and dextran sulfate (DS) or poly(vinylsulfonic acid) (PVS) have been used to coat the capillary inner wall and thereby prevent analyte adsorption. In this study, atomic force microscopy (AFM) was employed to investigate the layer thickness and surface morphology of monolayer (PB), bilayer, (PB-DS and PB-PVS), and trilayer (PB-DS-PB and PB-PVS-PB) coatings on glass surfaces. AFM nanoshaving experiments providing height distributions demonstrated that the coating procedures led to average layer thicknesses between 1 nm (PB) and 5 nm (PB-DS-PB), suggesting the individual polyelectrolytes adhere flat on the silica surface. Investigation of the surface morphology of the different coatings by AFM revealed that the PB coating does not completely cover the silica surface, whereas full coverage was observed for the trilayer coatings. The DS-containing coatings appeared on average 1 nm thicker than the corresponding PVS-containing coatings, which could be attributed to the molecular structure of the anionic polymers applied. Upon exposure to the basic protein cytochrome c, AFM measurements showed an increase of the layer thickness for bare (3.1 nm) and PB-DS-coated (4.6 nm) silica, indicating substantial protein adsorption. In contrast, a very small or no increase of the layer thickness was observed for the PB and PB-DS-PB coatings, demonstrating their effectiveness against protein adsorption. The AFM results are consistent with earlier obtained CE data obtained for proteins using the same polyelectrolyte coatings.     

Estimating the thickness of hydrated ultrathin poly(o-phenylenediamine) film by atomic force microscopy

A novel method to measure ultrathin poly(o-phenylenediamine) (PPD) film electropolymerized on gold electrode in liquid was developed. It is based on the force versus distance curve (force curve) of atomic force microscopy (AFM). When 1-0.25 μm/s was chosen as the rising rate of the scanner, and 50% of the confidence interval (CI) as the qualifying threshold value, the thickness of the hydrated polymer film could be calculated. This result was compared with one obtained from an AFM image. A step-like electrode fabricated by a photolithographic process was used. The height difference of the electrode before and after the PPD coating was imaged in liquid, and then the real thickness, 19.6±5.2 nm, was obtained. The sample was also measured by estimating the transition range of the force curve of hydrated PPD film, and the thickness of the hydrated PPD film was determined to be 19.3±8.2 nm. However, the results calculated by integrating the electropolymerized charge for the oxidation process of o-phenylenediamine (o-PD) was only one-third as large as it was when using the two previously described methods. This indicated that the structure of hydrated PPD film might have been swollen.     

Size fractionation of aquatic colloids and particles by cross-flow filtration: analysis by scanning electron and atomic force microscopy

The suitability of the combined application of environmental scanning electron microscopy (ESEM), scanning electron microscopy (SEM) and atomic force microscopy (AFM) for the evaluation of the ability of cross-flow filtration (CFF) to perform adequate size fractionation of freshwater colloids and particles was examined. ESEM and SEM imaging provided reference images of the CFF-generated fractions and, in estimating the experimental cut-off diameter of the membrane, provided evidence that separation was not consistent with nominal pore sizes of the membranes. However, analysis of the images showed that size distribution of CFF-generated fractions and the estimated cut-off diameter of the membranes were dependent on the advantages and limitations of the two imaging techniques. With both ESEM and SEM, best estimates of size cut-offs were lower than the nominal pore size of the membrane in the case of 0.45 μm membranes, but roughly accurate in the case of 0.1 μm pore size membranes. The results also suggested that the effectiveness of CFF may benefit from a pre-separation step using a minimally perturbing technique such as split thin-flow fractionation. AFM demonstrated the presence of colloids smaller than 50 nm in all fractions including the retentates, showing that CFF fractionation is not fully quantitative and not based on size alone. The results indicate that previous studies investigating trace element partitioning using CFF may need re-evaluation as the importance of particles and large colloids may be over-estimated.     

Atomic force microscope studies of membranes: Surface pore structures of Cyclopore and Anopore membranes

Non-contact atomic force microscopy has been used to investigate the surface pore structure of Cyclopore and Anopore microfiltration membranes in air. Three Cyclopore membranes and three Anopore membranes of different pore sizes were studied. Excellent high resolution images were obtained. Analysis of the images gave quantitative information on the surface pore structure, in particular the pore size distribution. Non-contact AFM is an excellent means of obtaining such information for microfiltration membranes.