Atomic force microscopy of a hydrated bacterial surface protein

The protein surface layer of the bacterium Deinococcus radiodurans (HPI layer) was examined with an atomic force microscope (AFM). The measurements on the air-dried, but still hydrated layer were performed in the attractive imaging mode in which the forces between tip and sample are much smaller than in AFM in the repulsive mode or in scanning tunnelling microscopy (STM). The results are compared with STM and transmission electron microscopy (TEM) data.     

Atomic force microscopy observations of in situ deformed materials: application to single crystals and thin films on substrates

An experimental apparatus which consists of a compression machine interfaced with an atomic force microscope has been realized and allows the in situ observation of a sample surface under compressive stress. Taking advantage of the high resolution offered by this microscopy, the equipment is particularly suited both to analysing the fine slip line structure of deformed single crystals, providing interesting complementary information about plastic mechanisms taking place in the bulk, and to characterizing the mechanical behaviour of thin films on substrates with the investigation of the buckling phenomenon.     

Atomic force microscopy to characterize the molecular size of prion protein

The conformational transition of α‐helix‐rich cellular prion protein (PrP^C) to an isomer with high β‐sheet content is associated with transmissible spongiform encephalopathies. With the ultimate long‐term goal of using imaging techniques to study PrP aggregation, we report the results of initial experiments to determine whether PrP molecules could be visualized as single molecules, and if the observed size corresponded to the calculated size for PrP. The investigation of single molecules, and not those embedded into larger aggregates, was the key in our experimental approach. Using atomic force microscopy (AFM) as an imaging method, the immobilization of recombinant histidine (His)₁₀‐tagged PrP on mica was performed in the presence of different heavy metal ions. The addition of Cu²⁺ resulted in an enhanced PrP immobilization, whereas Ni²⁺ reduced coverage of the surface by PrP. High‐resolution data from dried PrP preparations provided a first approximation to geometrical parameters of PrP precipitates, which indicated that the volume of a single PrP molecule was 30 nm³. Molecular dynamics simulations performed to complement the structural aspects of the AFM investigation yielded a calculated molecular volume of 33 nm³ for PrP. These experimentally observed and theoretically expected values provide basic knowledge for further studies on the size and composition of larger amyloidal PrP aggregates, PrP isoforms or mutants such as PrP molecules without octarepeats.     

Atomic force microscopy of purple membranes

The surface structure of purple membranes was imaged using an atomic force probe mounted in a scanning tunnelling microscope. One of the two different membrane surfaces showed protruding, disc-shaped features forming an hexagonal lattice with about 6 nm centre to centre spacing. These are identified as the cytoplasmic surfaces of trimers of bacteriorhodopsin molecules and are correlated with the structural information on bacteriorhodopsin obtained from numerous earlier electron microscope and diffraction studies.     

Atomic force microscopy of human hair

The atomic force microscope (AFM) was used to investigate the surface architecture of the entire lengths of cleaned human head hairs. Many features previously seen with the scanning electron microscope (SEM) were identified. However, the AFM has provided much greater detail and, in particular, the hair's cuticular surfaces appear not to be as smooth as had been previously supposed. A consistent feature was of step discontinuities or "ghosts" on the scale surfaces. These delineated the original location of each overlying scale before its edge had been chipped away. There was a change in the longitudinal angular presentation of the surfaces about each ghost. This means the distal ends of each cuticle cell have been synthesised in the follicle to be thicker than where that same cuticle cell is bounded on both sides by other cuticle cells. The undamaged outer cuticular surfaces at the root end of each hair were covered everywhere by longitudinal ridges (striations). Where the hair surface was worn, the striations terminated at a scale edge ghost. The ridges were approximately 9 nm high and were in parallel array with a lateral repeat spacing of about 350 nm. The striations are evidently formed on the outer surface of each cuticle cell following earlier contact in the hair follicle with the inner root sheath. The study of stained transverse sections of hairs in the transmission electron microscope (TEM) is suggested as a means for throwing some light on the underlying structure and chemistry of the striations. Finally, our AFM studies have revealed that the surface of the freshly emergent hair gradually changes over a distance of about 20 mm and that the surface of the hair for most of its length is quite different from that near the root. This is likely to be of import to those engaged in the hair toiletries industry.     

人类染色体的原子力显微术研究

The atomic force microscope(AFM),invented by Binnig et al.in 1986,can obtain topographic images at high spatial resolution in various(vacuum,air and liquid)environments,and has been applied to studies for imaging various biological samples from DNA to living cells [1,2].We have been especially interested in AFM imaging of human chromosomes for analyzing their three-dimensional structure.In this paper,we briefly reviewed our AFM studies of human metaphase chromosomes,and introduced our results on the high-order structure of the chromosomes.     

Atomic force microscopy study of chromosome surface structure changed by protein extraction

We applied atomic force microscopy (AFM) to investigate the surface structure of barley chromosome in combination with a chemical treatment method. As a result, we have obtained high-resolution topographic images of granular structures with a diameter of ca. 50 nm on the surface of critical-point dried metaphase chromosomes. Treatment with 2M NaCl significantly modified the chromosome surface structure: surface roughness was increased and chromosome thickness was decreased. The NaCl treatment extracted two major proteins with molecular weights of 4000 and 20,000 Da. These proteins might be belonging to non-histone protein families that do not contain any aromatic amino acid. The results demonstrate the advantage of the combined method of high-resolution AFM imaging and chemical treatments for understanding nano-scale surface structures of the chromosome.     

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.     

Atomic force microscopy for ultrafiltration membrane imaging

Atomic force microscopy (AFM) can reveal nanometer-scale structure of samples without the sample preparation techniques that involve dehydration. This is particularly important for hydrophilic organic materials. An asymmetric polysulfone ultrafiltration membrane (molecular weight cutoff rated at 10 kg/mol) was imaged by AFM. Sample mounting methods tried include cyanoacrylate, double-sided tape, and paraffin. Wax and tape bonding did not lead to usable images. Cyanoacrylate bonding resulted in images that appear to show 2.8° 10⁹ pores/m² approximately 3 nm in diameter, creating a porosity of 2%. This is consistent with estimates of molecular sizes for 10 kg/mol proteins, but not with the results of other AFM studies of similar membranes. The discrepancies can be explained largely by differences in sample preparation techniques.     

Atomic force microscopy imaging of polycrystalline CuInSe2 thin films

In this study, atomic force microscopy (AFM) imaging has been used to study the structural properties of polycrystalline CuInSe₂ films, which are widely used as absorber materials in thin film solar cell devices. This technique demonstrated an excellent capability for the reproducible imaging of these rough polycrystalline materials. AFM imaging in combination with statistical analysis revealed distinct differences in the structural properties (i.e. grain width and height distributions, root‐mean‐square (RMS) and peak to valley (R_(p–v)) roughness values) as a function of the specific growth technique used and the bulk composition of the films. In the case of Cu‐rich films, prepared by the H₂Se/Ar treatment of Cu/In/Cu alloys, rough surface structures were in general observed. Statistical analysis revealed two distinct distribution of grains in these samples (1.0–2.5 μm and 3–5.5 μm) with large RMS and R_(p–v) roughness values of 380 nm and 2.6 μm, respectively. In‐rich films were characterized by the presence of much smaller, roughly circular clusters with a significant reduction in both the width and height distributions as well as RMS and R_(p–v) roughness values. The most successful growth techniques, in terms of producing homogeneous and dense films, were in the cases of H₂Se/Ar treated metallic InSe/Cu/InSe alloys and the coevaporation of all materials to form CuInSe₂. Both these techniques produced absorber films with very narrow grain width and height distributions as well as small roughness values. It was possible to establish that high efficiency devices are associated with the use of absorber films with narrow width distributions between 0.5 and 2 μm and small RMS (> 300 nm) roughness values. These values are used as a figure of merit in our laboratories to evaluate the structural properties of our CuInSe₂ thin films.     

Atomic force and scanning electron microscopy of atmospheric particles

Atomic force microscopy (AFM) and scanning electron microscopy with energy dispersive spectroscopy (SEM-EDS) have been used for both morphological and elemental mass analysis study of atmospheric particles. As part of the geometrical particle analysis, and in addition to the traditional height profile measurement of individual particles, AFM was used to measure the volume relative to the projection area for each particle separately, providing a particle shape model. The element identification was done by the EDS analysis, and the element mass content was calculated based on laboratory calibration with particles of known composition. The SEM-EDS mass measurements from two samples collected at 150 and 500 m above the surface of the Mediterranean Sea were found to be similar to mass calculations derived from the AFM volume measurements. The AFM results show that the volume of most of the aerosols that were identified as soluble marine sulfate and nitrate aerosol particles can be better estimated using cylindrical shapes than spherical or conical geometry.     

Atomic force microscopy of the erythrocyte membrane skeleton

The atomic force microscope was used to examine the cytoplasmic surface of untreated as well as fixed human erythrocyte membranes that had been continuously maintained under aqueous solutions. To assess the effects of drying, some membranes were examined in air. Erythrocytes attached to mica or glass were sheared open with a stream of isotonic buffer, which allowed access to the cytoplasmic membrane face without exposing cells to non‐physiological ionic strength solutions. Under these conditions of examination, the unfixed cytoplasmic membrane face revealed an irregular meshwork that appeared to be a mixture largely of triangular and rectilinear openings with mesh sizes that varied from 35 to 100 nm, although few were at the upper limit. Fixed ghosts were similar, but slightly more contracted. These features represent the membrane skeleton, as when the ghosts were treated to extract spectrin and actin, these meshworks were largely removed. Direct measurements of the thickness of the membrane skeleton and of the lateral dimensions of features in the images suggested that, especially when air dried, spectrin can cluster into large, quite regularly distributed aggregates. Aggregation of cytoskeletal components was also favoured when the cells were attached to a polylysine‐treated substrate. In contrast, the membrane skeletons of cells attached to substrates rendered positively charged by chemical derivatization with a cationic silane were much more resistant to aggregation. As steps were taken to reduce the possibility of change of the skeleton after opening the cells, the aggregates and voids were eliminated, and the observed structures became shorter and thinner. Ghosts treated with Triton X‐100 solutions to remove the bilayer revealed a meshwork having aggregated components resembling those seen in air. These findings support the proposition that the end‐to‐end distance of spectrin tetramers in the cell in the equilibrium state is much shorter than the contour length of the molecule and that substantial rearrangements of the spectrin‐actin network occur when it is expanded by low ionic strength extraction from the cell. This study demonstrates the applicability of AFM for imaging the erythrocyte membrane skeleton at a resolution that appears adequate to identify major components of the membrane skeleton under near‐physiological conditions.     

Atomic force microscopy in the photochemistry of chalcones

The application of atomic force microscopy (AFM) to photodimerization of crystalline chalcones provides new insights into the detailed mechanisms of solid-state reactions on the molecular level. Well-directed long-range transport phenomena are found which reach far beyond the crystal lattice distances. Reactions occur in the surface region where the light is absorbed. Characteristic features are built up that depend on crystal structure and crystal face. This could not be foreseen by previous theories based solely on a topochemical postulate/principle. There is now a much more intimate correlation of crystal structure with solid-state reactivity and this is directly studied and proven experimentally by AFM. Even solid-state reactions which are in opposition to topochemistry can be studied and understood on a molecular basis. The three-dimensional resolution of undisturbed insulating surfaces which is obtained by AFM is not available by any other technique.     

Atomic force microscopy of conventional and unconventional nucleic acid structures

Images of conventional (Watson–Crick base paired) and unconventional (G4 RNA) nucleic acid structures have been obtained by atomic force microscopy. The images are reproducibly generated from samples deposited on freshly cleaved mica. Periodic substructural features are evident in fibres observed in both cases. In the case of G4 RNA, tip-induced formation of large fibres is observed.     

Atomic and magnetic force microscopy imaging of thin-film recording heads

Investigations on the use of the scanning probe microscope (SPM) in the atomic force microscopy (AFM) mode for topography imaging and the magnetic force microscopy (MFM) mode for magnetic imaging are presented for a thin-film recording head. Results showed that the SPM is suitable for imaging the surface profile of the recording head, determining the width of the pole gap region, and mapping the magnetic field patterns of the recording head excited under current bias conditions of different polarity. For the cobalt sputter-coated tips used in MFM imaging, it was found that the magnetic field patterns obtained under different polarities of the current bias to the recording head were similar. This can be explained by the nature of the thin-film MFM tip, in which the direction of the tip magnetic moment can follow the stray magnetic field of the sample as the current bias to the recording head reverses in direction.     

Atomic force microscopy wear characterization of biomedical polymer coatings

Atomic force microscopy (AFM) has become established as a powerful and versatile tool for investigating local mechanical properties. In addition, through the AFM tip–sample interaction, it has become possible to study the effects of perturbations and modifications to the surface of soft samples, such as polymers. The accurate knowledge of their response to continuous AFM scanning could help to design new materials having desirable mechanical properties. In this paper, we present the results obtained by applying a new methodology to investigate wear properties on two different type of polymer, poly(methyl‐methacrylate) and poly(l ‐lactic acid). These polymers have been widely employed in biomedical applications and have recently been considered as good candidates for coronary metallic stent coatings. Copyright © 2006 John Wiley & Sons, Ltd.     

Atomic force microscopy observations of acyl chains in phospholipids

The potential use of atomic force microscopy (AFM) to image the mode of assembly and to measure the corresponding lattice parameters of model systems consisting of ordered aggregates of cardiolipin molecules has been investigated. An unprecedented resolution of about 0·2 nm has been achieved on suitably prepared specimens. This enables the orientational order and the positional correlations of the individual molecules in the lattice to be defined, and submolecular details, such as the acyl chains and the polar groups, to be imaged. The structural parameters derived from AFMhave been compared with those obtained by transmission electron diffraction of the same specimen and found to be in excellent agreement. AFM turns out to be a powerful and probably a unique tool to reveal local phase variations in systems, such as biological membranes, that have non-homogeneous composition and organization.     

Atomic force microscopy of freeze-fracture replicas of rat atrial tissue

Atomic force microscopy (AFM) has provided three-dimensional (3-D) surface images of many biological specimens at molecular resolution. In the absence of spectroscopic capability for AFM, it is often difficult to distinguish individual components if the specimen contains a population of mixed structures such as in a cellular membrane. In an effort to understand the AFM images better, a correlative study between AFM and the well-established technique of transmission electron microscopy (TEM) was performed. Freeze-fractured replicas of adult rat atrial tissue were examined by both TEM and AFM. The same replicas were analysed and the same details were identified, which allowed a critical comparison of surface topography by both techniques. AFM images of large-scale subcellular structures (nuclei, mitochondria, granules) correlated well with TEM images. AFM images of smaller features and surface textures appeared somewhat different from the TEM images. This presumably reflects the difference in the surface sensitivity of AFM versus TEM, as well as the nature of images in AFM (3-D surface contour) and TEM (2-D projection). AFM images also provided new information about the replica itself. Unlike TEM, it was possible to examine both sides of the replica with AFM; the resolution on one side was significantly greater compared with the other side. It was also possible to obtain quantitative height information which is not readily available with TEM.     

Atomic force microscopy characterization of Xenopus laevis oocyte plasma membrane

We used atomic force microscopy (AFM) to characterize the plasma membrane of Xenopus laevis oocytes. The samples were prepared according to novel protocols, which allowed the investigation of the extra- and intracellular sides of the membrane, both of which showed sparsely distributed spherical-like protrusions. Regions with comparably sized and densely packed structures arranged in an orderly manner were visualized and dimensionally characterized. In particular, two different arrangements, hexagonal and square packing, were recognizable in ordered regions. The lateral dimension of structures visualized on the external side had a normal distribution centered on 25.5 +/- 0.3 nm (mean value +/- SE), whereas that on the intracellular side showed a normal distribution centered on 30.2 +/- 0.8 nm. The height of the protrusions was 2-5 nm on the external side and 1-3 nm on the intracellular side. The mean number of structures on the external and intracellular sides of the plasma membrane was about 1000 microm(-2) and 850 microm(-2) respectively. Trypsin treatment greatly decreased the size of the membrane protrusions, thus confirming the proteic nature of the structures. These results show that AFM is a useful tool for structural characterization of proteins in a native eukaryotic membrane.     

Atomic force microscopy study of living diatoms in ambient conditions

We present the first in vivo study of diatoms using atomic force microscopy (AFM). Three chain‐forming, benthic freshwater species –Eunotia sudetica, Navicula seminulum and a yet unidentified species – are directly imaged while growing on glass slides. Using the AFM, we imaged the topography of the diatom frustules at the nanometre range scale and we determined the thickness of the organic case enveloping the siliceous skeleton of the cell (10 nm). Imaging proved to be stable for several hours, thereby offering the possibility to study long‐term dynamic changes, such as biomineralization or cell movement, as they occur. We also focused on the natural adhesives produced by these unicellular organisms to adhere to other cells or the substratum. Most man‐made adhesives fail in wet conditions, owing to chemical modification of the adhesive or its substrate. Diatoms produce adhesives that are extremely strong and robust both in fresh‐ and in seawater environments. Our phase‐imaging and force‐pulling experiments reveal the characteristics of these natural adhesives that might be of use in designing man‐made analogues that function in wet environments. Engineering stable underwater adhesives currently poses a major technical challenge.