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PubMed1997

Imaging Plasmodium falciparum-infected ghost and parasite by atomic force microscopy.

Atomic force microscopy was used to image the membrane cytoskeleton network of normal and P. falciparum-infected ghosts. The membrane cytoskeleton network was examined in air-dried ghost preparations from normal and infected cells. We found that the spectrin network was changed in infected ghosts. The thickness of the normal red cell membrane was about 15.05 +/- 2.27 nm, while the thickness of the P. falciparum-infected membrane was found to be 22.97 +/- 3.84 nm. The ghost containing ring stage parasites exhibited areas of particle-like protrusions ranging in size from 0.2 to 0.7 micron. The surface of the P. falciparum parasite was also imaged in air-dried samples, showing the existence of a large protrusion extending from the parasite surface.

Animals↗
PubMedDate not supplied

Interferon alpha-2a interactions on glass vial surfaces measured by atomic force microscopy.

Atomic force microscopy was used to study adsorption and adhesion peculiarities of interferon alpha-2a on glass and mica surfaces. The specific protein adsorption behavior as a function of the pH value was illustrated on mica by single molecule imaging, while adhesion forces between interferon molecules and inner surfaces of borosilicate glass vials were measured directly under aqueous buffer conditions by force microscopy. We found that the adhesion force on Schott FIOLAX Type I plus was reduced by 40% of the total adhesion force measured on Schott FIOLAX, a standard type I borosilicate glass quality. These results reflect the anticipated superiority of the special "Type I plus" coating over undesired protein adsorption to glass. In addition, this study gives insight into a new method to predict unintended protein adsorption to glass container walls and to characterize the adsorption process by force measurement.

Adsorption↗
PubMed1997

Molecular resolution imaging of macromolecular crystals by atomic force microscopy.

Atomic force microscopy (AFM) images at the molecular level have been obtained for a number of different protein and virus crystals. They can be utilized in some special cases to obtain information useful to crystal structure analyses by x-ray diffraction. In particular, questions of space group enantiomer, the packing of molecules within a unit cell, the number of molecules per asymmetric unit, and the dispositions of multiple molecules within the asymmetric unit may be resolved. In addition, because of the increasing sensitivity and resolution of the AFM technique, some molecular features of very large asymmetric units may be within reach. We describe here high-resolution studies, using AFM, to visualize individual molecules and viruses in their crystal lattices. These investigations included fungal lipase, lysozyme, thaumatin, canavalin, and satellite tobacco mosaic virus (STMV).

Computer Simulation↗
PubMedDate not supplied

Determination of a translocation chromosome by atomic force microscopy.

Atomic force microscopy (AFM) has been used to study the translocation involving chromosomes 11 and 13. An amniocentesis procedure was performed at 18 weeks of pregnancy on a familial balanced translocation carrier mother whose karyotype was 46,XX,t(11;13) (q23;q34). After harvesting the tissue cultures, light microscopy studies (LM) have indicated that the fetus had the same translocation. A 0.3 microm gap region on the derivative chromosome 13 was determined by AFM; it was equivalent to a mid-sized G-band. The enhanced resolution of AFM with respect to its line measure analysis and three-dimensional image capture capability has allowed an extension and reconsideration of conclusions about chromosomal aberrations based on the study of LM preparations. In this manner, chromosomal disorders will be studied at nanoscale to help in the planning of new therapy strategies.

Adult↗
PubMed2002

Influence of metal ion sorption on colloidal surface forces measured by atomic force microscopy.

Atomic force microscopy (AFM) is employed to directly measure colloidal surface forces between a silica particle and a smooth glass plate in an aqueous solution with or without the presence of copper ions. Without the presence of copper ions, results show that the force between these two surfaces is repulsive and that its magnitude decreases with increasing ionic strength and decreasing pH. The surface forces are calculated based on the Derjaguin-Landau-Verwey-Overbeek (DLVO) theory for constant surface charge and are then compared with AFM force measurements. A good agreement between theory and experimental data is reported except at very small separation distances (<3 nm) between the silica particle and the glass plate. This behavior may be attributed to non-DLVO forces, such as the hydration effect that results from the bounded water molecules on the surface of the silica particle, or to surface roughness. When copper ions are present in acidic aqueous solutions, the magnitude of the force is found to be the same as that without the presence of copper ions, which indicates that no sorption of copper ions by the silica particle occurs under these conditions. Near neutral pH, sorption of copper ions causes charge reversal for the silica particle from negative to positive. Therefore, the force between the silica particle and the glass plate changes from repulsive to attractive. The transient zeta-potential of the silica particle during sorption of copper ions is determined by representing the experimental data with the DLVO theory. In alkaline solutions, where removal of copper ions is known to occur mainly by bulk precipitation, the measured force is similar to that without the presence of copper ions, which suggests that sorption does not occur under such conditions.

Colloids↗
PubMed2002

Characterization of the adhesive mucilages secreted by live diatom cells using atomic force microscopy.

Atomic Force Microscopy (AFM) resolved the topography and mechanical properties of two distinct adhesive mucilages secreted by the marine, fouling diatom Craspedostauros australis. Tapping mode images of live cells revealed a soft and cohesive outer mucilage layer that encased most of the diatom's siliceous wall, and force curves revealed an adhesive force of 3.58 nN. High loading force, contact mode imaging resulted in cantilever 'cleaned' cell walls, which enabled the first direct observation of the active secretion of soft mucilage via pore openings. A second adhesive mucilage consisted of strands secreted at the raphe, a distinct slit in the silica wall involved in cell-substratum attachment and motility. Force measurements revealed a raphe adhesive strand(s) resistant to breaking forces up to 60 nN, and these strands could only be detached from the AFM cantilever probe using the manual stepper motor.

Adhesiveness↗
PubMed2002

Direct observation of polyhydroxyalkanoate chains by atomic force microscopy.

Atomic force microscopy in the tapping mode was used to investigate aqueous acetone-treated polyhydroxyalkanoate (PHA) inclusions freshly isolated from a recombinant bacterium. The PHA is a copolymer containing about 95 mol% 3-hydroxybutyrate units while the rests are units of 3-hydroxyhexanoate, 3-hydroxyoctanoate, 3-hydroxydecanoate, and 3-hydroxydodecanoate. Polymer chains extending to several micrometers in length were observed on glass cover slips upon the evaporation of the aqueous acetone. The polymer chains seem to exist in the form of fibrillar aggregates. The height of the microfibrils was about 1 nm. Upon prolonged standing at ambient conditions, the microfibrils dissociated into finer strands of about 0.5 nm in height. The results suggest that biosynthesized PHA are stored in the inclusions in an amorphous state but with minimal chain entanglement. This is possible because the PHA chains exist in the form of fibrillar aggregates that may be the product of a special biosynthesis mechanism.

Cupriavidus necator↗
PubMed2003

Real-time imaging of the surface topography of living yeast cells by atomic force microscopy.

Atomic force microscopy (AFM) was used to image the surface topography of living Saccharomyces cerevisiae cells at high resolution and to monitor enzyme digestion of the cell wall in real time. Apart from the presence of bud scars, the surface of native cells imaged in aqueous solution was homogeneous and smooth. Topographic images of the surface were recorded to a lateral resolution of 2 nm without significant modification of the surface morphology. Successive images of single cells were collected at fixed time intervals following addition of protease and amyloglucosidase solutions. Protease caused a progressive increase of surface roughness. Large depressions surrounded by protruding edges, approximately 50 nm in height, were formed and attributed to the erosion of the mannoprotein outer layer. By contrast, no modification of the cell surface was noted upon addition of amyloglucosidase, which was consistent with the cell wall biochemical composition. These results indicate that AFM is a complementary tool to electron microscopy in that it allows the surface of living cells to be explored directly in real time.

Cell Wall↗
PubMed1997

Topography of ribosomes and initiation complexes from rat liver as revealed by atomic force microscopy.

Atomic force microscopy (AFM) was used to image ribosomes and ribosomal subunits (60S, 40S and native 40S ribosomal subunits) isolated from rat liver. A variety of topographic images were obtained directly and found to be consistent with models established by other biophysical methods. In addition, the ternary complex of eIF-2 x GTP x Met-tRNA(i) and the 43S preinitiation complex have been discerned by AFM directly. Detailed information about the binding sites for eIF-1A, eIF-2, eIF-3, and Met-tRNA(i) on the 40S ribosomal subunit was derived from the AFM images. Finally, factors which may give rise to artifactual images, namely, convolution of the AFM tip on ribosomes, surface tension collapse effect and dehydration, are discussed. This work demonstrates that AFM is useful for imaging ribosomes and translational complexes and provides valuable information that can be used to complement other well-established techniques.

Animals↗
PubMed2003

Characterisation of heterogeneous arabinoxylans by direct imaging of individual molecules by atomic force microscopy.

Atomic force microscopy has been used to characterise populations of extracted water-soluble wheat endosperm arabinoxylans. The adsorbed molecules are extended structures with an estimated Kuhn statistical segment length of 128 nm, suggesting that they adopt an ordered helical structure. However, estimates of the molecular weight distribution, coupled with size exclusion data, suggest that, in solution, the polysaccharides behave as semi-flexible coils, with a Kuhn length of 16 nm. These data imply that adsorption of the arabinoxylan structures onto mica promotes formation of the helical structure. Adoption of this ordered structure is fortunate because it has permitted characterisation of branching observed in a small proportion (approximately 15%) of the population of otherwise linear molecules. The degree of branching has been found to increase with the contour length of the molecules. Degradation of the polysaccharides with xylanase has been used to confirm that both the backbone and branches are based on beta-(1-->4) linked D-xylopyranosyl residues.

Adsorption↗
PubMed1997

Localization of amiloride-sensitive sodium channels in A6 cells by atomic force microscopy.

Atomic force microscopy (AFM) was used for high-resolution imaging of the apical distribution of epithelial Na+ channels in A6 renal epithelial cells. A6 cells grown on coverslips were labeled with antibodies generated against an amiloride-sensitive epithelial Na+ channel complex purified from bovine renal medulla that had been conjugated to 8-nm colloidal gold particles before preparation for AFM. AFM revealed that there was a marked increase in the height of the microvilli in cells labeled with the anti-epithelial Na+ channel antibodies compared with unlabeled cells or cells labeled with rabbit nonimmune immunoglobulin G conjugated to colloidal gold particles. We interpret this apparent increase in microvillar height to be due to anti-epithelial Na+ channel antibody binding to the apical microvilli. These data demonstrate that epithelial Na+ channels are restricted to the apical microvilli in Na+-transporting renal epithelial cells. Furthermore, they demonstrate the applicability of using AFM for high-resolution imaging of the cell surface distribution of epithelial ion channels.

Amiloride↗
PubMed2003

Analysis of the intrinsic bend in the M13 origin of replication by atomic force microscopy.

Atomic force microscopy (AFM) has been used to image a 471-bp bent DNA restriction fragment derived from the M13 origin of replication in plasmid LITMUS 28, and a 476-bp normal, unbent fragment from plasmid pUC19. The most probable angle of curvature of the 471-bp DNA fragment is 40-50 degrees, in reasonably good agreement with the bend angle determined by transient electric birefringence, 38 degrees +/- 7 degrees. The normal 476-bp DNA fragment exhibited a Gaussian distribution of bend angles centered at 0 degrees, indicating that this fragment does not contain an intrinsic bend. The persistence length, P, was estimated to be 60 +/- 8 and 62 +/- 8 nm for the 471- and 476-bp fragments, respectively, from the observed mean-square end-to-end distances in the AFM images. Since the P-values of the normal and bent fragments are close to each other, the overall flexibility of DNA fragments of this size is only marginally affected by the presence of a stable bend. The close agreement of AFM and transient electric birefringence results validates the suitability of both methods for characterizing DNA bending and flexibility.

Crystallography↗
PubMed2003

Preliminary analysis of polyhydroxyalkanoate inclusions using atomic force microscopy.

Atomic force microscopy analysis of polyhydroxyalkanoate (PHA) inclusions isolated from sonicated Ralstonia eutropha cells revealed that they exhibit two types of surface structure and shape; rough and ovoid, or smooth and spherical. Smooth inclusions possessed linear surface structures that were in parallel arrays with 7-nm spacing. Occasionally, cracks or fissures could be seen on the surface of the rough inclusions, which allowed a measurement of approximately 4 nm for the thickness of the boundary layer. When the rough inclusions were imaged at higher resolution, globular structures, 35 nm in diameter, having a central pore could be seen. These globular structures were connected by a network of 4-nm-wide linear structures. When the inclusions were treated with sodium lauryl sulfate, the boundary layer of the inclusion deteriorated in a manner that would be consistent with a lipid envelope. When the boundary layer was largely gone, 35-nm globular disks could be imaged laying on the surface of the filter beside the inclusions. These data have facilitated the development of a preliminary model for PHA inclusion structure that is more advanced than previous models.

Cupriavidus necator↗
PubMed2002

Structural perturbations in DNA caused by bis-intercalation of ditercalinium visualised by atomic force microscopy.

Atomic force microscopy (AFM) has been used to examine perturbations in the tertiary structure of DNA induced by the binding of ditercalinium, a DNA bis-intercalator with strong anti-tumour properties. We report AFM images of plasmid DNA of both circular and linearised forms showing a difference in the formation of supercoils and plectonemic coils caused at least in part by alterations in the superhelical stress upon bis-intercalation. A further investigation of the effects of drug binding performed with 292 bp mixed-sequence DNA fragments, and using increment in contour length as a reliable measure of intercalation, revealed saturation occurring at a point where sufficient drug was present to interact with every other available binding site. Moment analysis based on the distribution of angles between segments along single DNA molecules showed that at this level of bis-intercalation, the apparent persistence length of the molecules was 91.7 +/- 5.7 nm, approximately twice as long as that of naked DNA. We conclude that images of single molecules generated using AFM provide a valuable supplement to solution-based techniques for evaluation of physical properties of biological macromolecules.

Carbazoles↗
PubMed2000

Morphology and transverse stiffness of Drosophila myofibrils measured by atomic force microscopy.

Atomic force microscopy was used to investigate the surface morphology and transverse stiffness of myofibrils from Drosophila indirect flight muscle exposed to different physiologic solutions. I- and A-bands were clearly observed, and thick filaments were resolved along the periphery of the myofibril. Interfilament spacings correlated well with estimates from previous x-ray diffraction studies. Transverse stiffness was measured by using a blunt tip to indent a small section of the myofibrillar surface in the region of myofilament overlap. At 10 nm indention, the effective transverse stiffness (K( perpendicular)) of myofibrils in rigor solution (ATP-free, pCa 4.5) was 10.3 +/- 5.0 pN nm(-1) (mean +/- SEM, n = 8); in activating solution (pCa 4.5), 5.9 +/- 3.1 pN nm(-1); and in relaxing solution (pCa 8), 4.4 +/- 2.0 pN nm(-1). The apparent transverse Young's modulus (E( perpendicular)) was 94 +/- 41 kPa in the rigor state and 40 +/- 17 kPa in the relaxed state. The value of E( perpendicular) for calcium-activated myofibrils (55 +/- 29 kPa) was approximately a tenth that of Young's modulus in the longitudinal direction, a difference that at least partly reflects the transverse flexibility of the myosin molecule.

Animals↗
PubMed2003

Size and shape of mineralites in young bovine bone measured by atomic force microscopy.

Atomic force microscopy (AFM) was used to obtain three-dimensional images of isolated mineralites extracted from young postnatal bovine bone. The mean mineralite size is 9 nm x 6 nm x 2.0 nm, significantly shorter and thicker than the mineralites of mature bovine bone measured by the same technique. Mineralites of the young postnatal bone can be accommodated within the hole zone regions of a quasi-hexagonally packed collagen fibril in the fashion described by Hodge [9] in which laterally adjacent hole zone regions form continuous "channels" across the diameter of a fibril for a distance of at least 10 nm. Deposition of mineralites of the size noted above in this void volume of the fibrils would result in little or no distortion of the collagen molecules or supramolecular structure of the collagen fibril. The new AFM data supporting this claim is consistent with findings obtained by electron microscopy and low-angle x-ray and neutron diffraction that mineralites formed within collagen fibrils during initial stages of calcification occur within the hole zone region. However, the deposition of additional mineralites in the intermolecular spaces between collagen molecules in the overlap region of the fibrils would significantly distort the fibrils since the space available between adjacent molecules is considerably less than even the smallest dimension of the mineralites.

Animals↗
PubMed2001

Morphology and current-voltage characteristics of nanostructured pentacene thin films probed by atomic force microscopy.

Atomic force microscopy was used to study the growth modes (on SiO2, MoS2, and Au substrates) and the current-voltage (I-V) characteristics of organic semiconductor pentacene. Pentacene films grow on SiO2 substrate in a layer-by-layer manner with full coverage at an average thickness of 20 A and have the highest degree of molecular ordering with large dendritic grains among the pentacene films deposited on the three different substrates. Films grown on MoS2 substrate reveal two different growth modes, snowflake-like growth and granular growth, both of which seem to compete with each other. On the other hand, films deposited on Au substrate show granular structure for thinner coverages (no crystal structure) and dendritic growth for higher coverages (crystal structure). I-V measurements were performed with a platinum tip on a pentacene film deposited on a Au substrate. The I-V curves on pentacene film reveal symmetric tunneling type character. The field dependence of the current indicates that the main transport mechanism at high field intensities is hopping (Poole-Frenkel effect). From these measurements, we have estimated a field lowering coefficient of 9.77 x 10(-6) V-1/2 m1/2 and an ideality factor of 18 for pentacene.

Crystallization↗
PubMed1998

Measuring elasticity of biological materials by atomic force microscopy.

Atomic force microscopy (AFM) has proved its value not only for resolving the topographical structure of biological samples, but also for probing inherent properties of biological structures, like local interaction forces, mechanical properties or dynamics in a natural (physiological) environment. This minireview focuses on the acquisition of elasticity data of biomaterials by AFM. A possible theoretical model is presented, followed by a practical 'how to do it with AFM', and, finally, a brief overview of publications in this field is given.

Animals↗