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PubMed2001

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.

Animals↗

PubMed2003

Observing the growth of individual actin filaments in cell extracts by time-lapse atomic force microscopy.

High-resolution atomic force microscopy (AFM) was applied to directly observe the dynamic assembly of single actin filaments in HeLa cell extracts in vitro. The F-actin filaments established a dynamic network and formed different types of junctions and branches. The connections of this network were X-, Y- or T-shaped. It was found that the actin filaments were densely covered by endosomes and vesicles from the cell extract, which are thought to stabilize their structures. Using time-lapse AFM, the growth, shrinkage, branching and the interaction of actin filaments with endosomes could be characterized. Our results indicate that the majority of F-actin filaments are static in HeLa extract and that only a minor fraction of filaments undergo dynamic changes. Furthermore, the AFM imaging approach not only provides unique insights into the assembly and dynamics of actin networks; it also builds an avenue to study in vitro assays of complex biological systems.

Actin Cytoskeleton↗

PubMed2006

Stiffness of normal and pathological erythrocytes studied by means of atomic force microscopy.

During recent years, atomic force microscopy has become a powerful technique for studying the mechanical properties (such as stiffness, viscoelasticity, hardness and adhesion) of various biological materials. The unique combination of high-resolution imaging and operation in physiological environment made it useful in investigations of cell properties. In this work, the microscope was applied to measure the stiffness of human red blood cells (erythrocytes). Erythrocytes were attached to the poly-L-lysine-coated glass surface by fixation using 0.5% glutaraldehyde for 1 min. Different erythrocyte samples were studied: erythrocytes from patients with hemolytic anemias such as hereditary spherocytosis and glucose-6-phosphate-dehydrogenase deficiency patients with thalassemia, and patients with anisocytosis of various causes. The determined Young's modulus was compared with that obtained from measurements of erythrocytes from healthy subjects. The results showed that the Young's modulus of pathological erythrocytes was higher than in normal cells. Observed differences indicate possible changes in the organization of cell cytoskeleton associated with various diseases.

Cell Shape↗

PubMedDate not supplied

Solid-state microstructure of poly(l-lactide) and l-lactide/meso-lactide random copolymers by atomic force microscopy (AFM).

Tapping mode atomic force microscopy was used to investigate the lamellar morphology of poly(l-lactide) and two poly(l-lactide-co-meso-lactide) random copolymers containing 3% and 6% meso-lactide. Samples were isothermally crystallized at selected temperatures, and qualitative and quantitative analyses of lamellar structure were performed using height and phase images. This is the first study of the morphology of polylactide stereocopolymers using a real-space probe, and the important effects of scanning parameters on the acquired images are described. More open spherulites with an abundance of screw dislocations between edge-on lamellar stacks were observed in samples crystallized at higher temperatures. Mean lamellar thicknesses are lower for the random copolymers compared to PLLA, particularly at lower DeltaT, in agreement with the results of our previous small-angle X-ray scattering (SAXS) experiments. Mean lamellar thicknesses derived from the current real-space examination are in good agreement with those determined previously from SAXS. Internal surfaces-from microtomed specimens-were also studied to investigate the bulk crystal morphology. Although quantitative analysis was not feasible (for reasons discussed in the text), lamellar organization similar to that seen in the surface experiments is observed at high magnifications.

Biocompatible Materials↗

PubMed2003

Atomic force microscopy of actin.

Atomic force microscope (AFM) is a powerful and novel tool to investigate the surface and submembranous structures of living cells under physiological conditions at high resolution, and has force measure at nano-Newton level. AFM can image and manipulate samples (single macromolecule, cells, and so on) at very high, sometimes atomic resolution by scanning a fine tip over the surface of interest and detecting physical interactions between the tip and sample. Actin is one of the most important proteins, involved extensively in various cell physiological activities and functions in eukaryotes. The present paper clarified the crucial significance and the enormous applicable prospects of AFM in modern biological sciences by reviewing the usage of AFM in actin study, which can be taken as a good example for expounding almost all functions that AFM owns, and can be utilized in life sciences exploration.

Actins↗

PubMed2005

Multiple membrane tethers probed by atomic force microscopy.

Using the atomic force microscope to locally probe the cell membrane, we observed the formation of multiple tethers (thin nanotubes, each requiring a similar pulling force) as reproducible features within force profiles recorded on individual cells. Forces obtained with Chinese hamster ovary cells, a malignant human brain tumor cell line, and human endothelial cells (EA hy926) were found to be 28 +/- 10 pN, 29 +/- 9 pN, and 29 +/- 10 pN, respectively, independent of the nature of attachment to the cantilever. The rather large variation of the tether pulling forces measured at several locations on individual cells points to the existence of heterogeneity in the membrane properties of a morphologically homogeneous cell. Measurement of the summary lengths of the simultaneously extracted tethers provides a measure of the size of the available membrane reservoir through which co-existing tethers are associated. As expected, partial disruption of the actin cytoskeleton and removal of the hyaluronan backbone of the glycocalyx were observed to result in a marked decrease (30-50%) in the magnitude and a significant sharpening of the force distribution indicating reduced heterogeneity of membrane properties. Taken together, our results demonstrate the ability of the plasma membrane to locally produce multiple interdependent tethers-a process that could play an important role in the mechanical association of cells with their environment.

Animals↗

PubMed1998

Elastic properties of living fibroblasts as imaged using force modulation mode in atomic force microscopy.

Using the force modulation mode in atomic force microscopy, we measured elastic properties of living mouse fibroblasts (NIH3T3) in a culture medium. The topographic images of the cellular surface and the corresponding elastic images of the cellular surface were able to be captured simultaneously with high spatial resolution. The consecutive images were useful for examining time-dependent changes in the cellular surface. We observed that some cells continued to shrink and change their softness for 2 hours. Then the force modulation mode in atomic force microscopy shows potential use in analyzing time-dependent regional elastic properties of living cells with high spatial resolution.

3T3 Cells↗

PubMed2004

Purification of the recombinant hepatitis B virus core antigen (rHBcAg) produced in the yeast Saccharomyces cerevisiae and comparative observation of its particles by transmission electron microscopy (TEM) and atomic force microscopy (AFM).

Hepatitis B virus core antigen (HBcAg) gene (C gene) was expressed in Saccharomyces cerevisiae and the products (rHBcAg or core particles) were purified from a crude lysate of the yeast by three steps: Sephrose CL-4B chromatography, Sucrose step-gradient ultracentrifugation and CsCl-isopycnic ultracentrifugation. It has been observed that HBcAg was synthesized in yeast cells as a particle consisting of polypeptides with a molecular weight of 21.5 kDa (p21.5). Results of ELISA test and density analysis of CsCl-isopycnic ultracentrifugation indicated that the purified products (rHBcAg particles) with HBcAg antigenicity mainly located at the densities of 1.27 and 1.40 g ml(-1), respectively. Observation and analysis of the purified rHBcAg products by TEM indicated that rHBcAg peptides could mainly self-assemble into two size classes of core particles. The larger particles were approximately 30.1 nm and the smaller were approximately 21.5 nm in mean diameter. Further observation and analysis of the same rHBcAg (core) particles by AFM also indicated that rHBcAg (core) particles were similar to the native HBcAg (core) particles from infected human hepatocytes and mainly composed of two size classes of partides core. The larger particles were approximately 31.3 nm and the smaller were approximately 22.5 nm in mean diameter which was similar to the results obtained by TEM. All results from both TEM and AFM suggested that core particles (capsids) produced in S. cerevisiae possessed dimorphism.

Genes, Viral↗

PubMed2000

Quantitative determination of contact stiffness using atomic force acoustic microscopy

Atomic force acoustic microscopy is a near-field technique which combines the ability of ultrasonics to image elastic properties with the high lateral resolution of scanning probe microscopes. We present a technique to measure the contact stiffness and the Young's modulus of sample surfaces quantitatively, with a resolution of approximately 20 nm, exploiting the contact resonance frequencies of standard cantilevers used in atomic force microscopy. The Young's modulus of nanocrystalline ferrite films has been measured as a function of oxidation temperature. Furthermore, images showing the domain structure of piezoelectric lead zirconate titanate ceramics have been taken.

Journal Article↗

PubMed2000

New insight into cellulose structure by atomic force microscopy shows the i(alpha) crystal phase at near-atomic resolution.

The organization of the surface of cellulose is important in cell structure, as well as in industrial processing and modification. Using atomic force microscopy, we show that the I(alpha) phase of native cellulose first proposed in 1984 and subsequently characterized by a triclinic unit cell exists over large areas of the surface of microcrystals from Valonia, one of the most highly crystalline celluloses. There is startling agreement between the observed structure and crystal models, and it is possible to identify the specific crystal face being imaged. The near-atomic resolution images also offer an insight into structural reconstructions at the surface compared to the interior. We are able to assign features in the images to particular side groups attached to the glucose ring and find indications of subtle modifications of the position of surface hydroxyls due to changes in hydrogen bonding.

Carbohydrate Conformation↗

PubMed2006

Surface characterization by atomic force microscopy of sterilized PLGA microspheres.

Atomic force microscopy (AFM) is recognized a suitable and powerful technique for surface and morphological analysis. Even if until now this technique has not been frequently used in the pharmaceutical field, it can contribute to an accurate morphologic characterization of microspheres and nanospheres. In this work, atomic force microscopy has been used to perform the surface characterization of sterilized microspheres. The aim is to investigate the morphologic modifications induced by gamma irradiation on poly(lactide-co-glycolide) microspheres loaded with ovalbumin and to compare the results obtained by AFM to those obtained by scanning electron microscopy (SEM). The results obtained show that, with respect to SEM, AFM can give some additional information regarding the modifications induced by gamma-irradiation on microspheres surface morphology. The significant changes in surface roughness after irradiation are indicative of damage due to gamma-irradiation. The unchanged surface roughness values calculated for microspheres containing PEG in their matrix, suggest that this polymer exerts a protective effect towards gamma-irradiation.

Drug Carriers↗

PubMed1995

Recent advances in biological atomic force microscopy.

Recent developments in biological atomic force microscopy are reviewed. In addition to the advances in methodology, new structural information of different biological systems revealed by the atomic force microscopy is also presented. A discussion regarding the contrast, resolution and specimen deformation is provided based on a theoretical model.

Bacterial Toxins↗

PubMed2006

An integrated instrumental setup for the combination of atomic force microscopy with optical spectroscopy.

In recent years, the study of single biomolecules using fluorescence microscopy and atomic force microscopy (AFM) techniques has resulted in a plethora of new information regarding the physics underlying these complex biological systems. It is especially advantageous to be able to measure the optical, topographical, and mechanical properties of single molecules simultaneously. Here an AFM is used that is especially designed for integration with an inverted optical microscope and that has a near-infrared light source (850 nm) to eliminate interference between the optical experiment and the AFM operation. The Tip Assisted Optics (TAO) system consists of an additional 100 x 100-microm(2) X-Y scanner for the sample, which can be independently and simultaneously used with the AFM scanner. This allows the offset to be removed between the confocal optical image obtained with the sample scanner and the simultaneously acquired AFM topography image. The tip can be positioned exactly into the optical focus while the user can still navigate within the AFM image for imaging or manipulation of the sample. Thus the tip-enhancement effect can be maximized and it becomes possible to perform single molecule manipulation experiments within the focus of a confocal optical image. Here this is applied to simultaneous measurement of single quantum dot fluorescence and topography with high spatial resolution.

Image Processing, Computer-Assisted↗

PubMed1997

Simultaneous imaging of the surface and the submembraneous cytoskeleton in living cells by tapping mode atomic force microscopy.

Contact and tapping mode atomic force microscopy have been used to visualize the surface of cultured CV-1 kidney cells in aqueous medium. The height images obtained from living cells were comparable when using contact and tapping modes. In contrast, the corresponding, and simultaneously acquired, deflection images differed markedly. Whereas, as expected, deflection images enhanced the surface features in the contact mode, they revealed the presence of a filamentous network when using the tapping mode. This network became disorganized upon addition of cytochalasin, which strongly suggests that it corresponded to the submembraneous cytoskeleton. Examination of fixed cells further supported this assumption. These data show that, in addition to the structural information on the cell surface, the use of the tapping mode in liquid can also provide a good visualization of the membrane cytoskeleton. Tapping mode atomic force microscopy appears to be a promising technique for studying interactions between cell surface and subsurface structures, a critical step in many biological processes.

Animals↗

PubMed1999

Atomic force microscopy study of tooth surfaces.

Atomic force microscopy (AFM) was used to study tooth surfaces in order to compare the pattern of particle distribution in the outermost layer of the tooth surfaces. Human teeth and teeth from a rodent (Golden hamster), from a fish (piranha), and from a grazing mollusk (chiton) with distinct feeding habits were analyzed in terms of particle arrangement, packing, and size distribution. Scanning electron microscopy and transmission electron microscopy were used for comparison. It was found that AFM gives high-contrast, high-resolution images and is an important tool as a source of complementary and/or new structural information. All teeth were cleaned and some were etched with acidic solutions before analysis. It was observed that human enamel (permanent teeth) presents particles tightly packed in the outer surface, whereas enamel from the hamster (continuously growing teeth) shows particles of less dense packing. The piranha teeth have a thin cuticle covering the long apatite crystals of the underlying enameloid. This cuticle has a rough surface of particles that have a globular appearance after the brief acidic treatment. The similar appearance of the in vivo naturally etched tooth surface suggests that the pattern of globule distribution may be due to the presence of an organic material. Elemental analysis of this cuticle indicated that calcium, phosphorus, and iron are the main components of the structure while electron microdiffraction of pulverized cuticle particles showed a pattern consistent with hydroxyapatite. The chiton mineralized tooth cusp had a smooth surface in an unabraded region and a very rough structure with the magnetite crystals (already known to make part of the structure) protruding from the surface. It was concluded that the structures analyzed are optimized for efficiency in feeding mechanism and life span of the teeth.

Acids↗

PubMed1997

Atomic force microscopy of arthropod gap junctions.

Atomic force microscopy has been used to characterize gap junctions isolated from the hepatopancreas of Nephrops norvegicus. The major polypeptide of these gap junctions is ductin, a highly conserved 16- to 18-kDa protein. The hydrated gap junctions, imaged in phosphate-buffered saline, appeared as membrane plaques with a thickness of 14 nm, consistent with their being a pair of apposing membranes. The upper membrane was removed by force dissection using an increased imaging force. The thickness of the lower membrane was 6 nm, giving a separation or gap between the two membranes of 2 nm. High-resolution images show fine details of the force-dissected extracellular surfaces, as previously reported for vertebrate and heart gap junctions. In addition high-resolution AFM images show for the first time detailed substructure on the cytoplasmic face of hydrated gap junctions of either vertebrate or invertebrate. The plaques had particles on their exposed and force-dissected faces. These particles were packed in a hexagonal lattice (a = b = 8.9 nm on both faces) and had a diameter of approximately 6.5 nm, with a central, pore-like depression. Fourier maps calculated from the AFM data suggested that each particle was composed of six subunits. These images show a marked similarity to the widely accepted structure of the connexon channel of vertebrate gap junctions.

Animals↗

PubMed2006

Investigation of sisal fibers by atomic force microscopy: morphological and adhesive characteristics.

Atomic force microscopy (AFM) was used to study the nanoscale surface chemistry and morphological changes caused by chemical treatment of sisal fibers. Scanning Electron Microscopy (SEM) micrographs indicated that sisal in natura (bundle of fibers) is formed by fibers with diameters of approximately 10 microm. AFM images showed that these fibers consist of microfibrils with diameters varying from 250 to 600 nm, which are made up of nanofibrils of ca. 20 nm in diameter. The adhesion force (pull-off force) between the AFM tip and the fibers surface increased after benzylation, pointing to a decrease in the polar groups on the sisal fiber. The adhesion map measured over a scan range of 3 microm was heterogeneous in samples treated with 40% NaOH and the low adhesion sites disappeared after benzylation. Using an established mathematical model, it was possible to evaluate the increase in adhesion work and consequently in the interaction between the AFM tip and sisal fibers. These results indicated that AFM can detect heterogeneity in the wettability of sisal fibers with nanometer resolution and can be applied in the study of fiber-matrix adhesion in polymer composites.

Adhesives↗

PubMed2004

A study of phase separation in peptide-loaded HPMC films using T(zero)-modulated temperature DSC, atomic force microscopy, and scanning electron microscopy.

Despite the widespread use of drug-loaded polymeric systems, there is still considerable uncertainty with regard to the nature of the distribution of the drug within the polymer matrix. The aim of this investigation was to develop thermal and microscopic techniques whereby the miscibility and spatial distribution of a model peptide, cyclosporin A (CyA), in hydroxypropyl methylcellulose (HPMC) films may be studied. The new technique of T(zero)-modulated temperature differential scanning calorimetry (T(zero) MTDSC), scanning electron microscopy (SEM), and pulse force mode atomic force microscopy (PFM-AFM) were used in conjunction to study films prepared using a solvent evaporation process, with a solvent extraction study performed to elucidate the nature of the observed phases. T(zero) MTDSC studies showed glass transitions for both the HPMC and CycA, with the T(g) for the HPMC and CycA seen for the mixed systems. SEM showed two spherical phases of differing electron density. PFM-AFM also showed spheres of differing adhesion that increased in size on addition of drug. Pixel intensity analysis indicated that the smaller spheres corresponded to CycA. Exposure of the films to dichloromethane, in which CycA is soluble but HPMC is not, resulted in the presence of voids that corresponded well to the spheres suggested to correspond to the drug. It was concluded that the system had undergone extensive or complete phase separation, and that the thermal and microscopic techniques outlined above are an effective means by which this issue may be studied.

Calorimetry, Differential Scanning↗