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Rapid visualization at high resolution of pathogens by atomic force microscopy: structural studies of herpes simplex virus-1.

A relatively crude preparation of herpes simplex virus was rapidly visualized by atomic force microscopy after exposure to conditions that produced gradual degradation of the virions. Images were obtained of 1) the intact, enveloped virus; 2) the underlying capsid with associated tegument proteins along with fragments of the membrane; 3) the capsomeres composing the capsid and their surface arrangement; 4) damaged and partially degraded capsids with missing capsomeres; and 5) the DNA extruded from damaged virions. These images provide a unique perspective on the structures of individual virus particles. Atomic force microscopy can thus be used as a diagnostic tool to provide a rapid way to obtain high-resolution images of human pathogens from crude preparations. It is a useful technique that complements X-ray-based structure determination, cryo-electron microscopy techniques, and optical microscopies in the field of molecular pathogenesis.

DNA, Viral↗

[Nonchemical methods of modifying substrates for studying biopolymers with atomic force microscopy].

Two simple procedures of DNA molecule fixation on mica for following imaging by the atomic force microscopy were developed. The distinctive features of the procedures are their simplicity, absence of chemical modification stages, and the possibility to obtain the images in air under relative high humidity. Comparison of the features of the images obtained indicated that the procedures developed were competitive with the procedures earlier suggested.

Aluminum Silicates↗

Adhesion forces between protein layers studied by means of atomic force microscopy.

Adhesion forces between different protein layers adsorbed on different substrates in aqueous media have been measured by means of an atomic force microscope using the colloid probe technique. The effects of the loading force, the salt concentration and pH of the medium, and the electrolyte type on the strength, the pull-off distance, and the separation energy of such adhesion forces have been analyzed in depth. Two very different proteins (bovine serum albumin and apoferritin) and two dissimilar substrates (silica and polystyrene) were used in the experiments. The results clearly point out a very important contribution of the electrostatic interactions in the adhesion between protein layers.

Adhesiveness↗

Pretransition and progressive softening of bovine carbonic anhydrase II as probed by single molecule atomic force microscopy.

To develop a simple method for probing the physical state of surface adsorbed proteins, we adopted the force curve mode of an atomic force microscope (AFM) to extract information on the mechanical properties of surface immobilized bovine carbonic anhydrase II under native conditions and in the course of guanidinium chloride-induced denaturation. A progressive increase in the population of individually softened molecules was probed under mildly to fully denaturing conditions. The use of the approach regime of force curves gave information regarding the height and rigidity of the molecule under compressive stress, whereas use of the retracting regime of the curves gave information about the tensile characteristics of the protein. The results showed that protein molecules at the beginning of the transition region possessed slightly more flattened and significantly more softened conformations compared with that of native molecules, but were still not fully denatured, in agreement with results based on solution studies. Thus the force curve mode of an AFM was shown to be sensitive enough to provide information concerning the different physical states of single molecules of globular proteins.

Animals↗

GTG banding pattern on human metaphase chromosomes revealed by high resolution atomic-force microscopy.

Surface topography of human metaphase chromosomes following GTG banding was examined using high resolution atomic force microscopy (AFM). Although using a completely different imaging mechanism, which is based on the mechanical interaction of a probe tip with the chromosome, the observed banding pattern is comparable to results from light microscopy and a karyotype of the AFM imaged metaphase spread can be generated. The AFM imaging process was performed on a normal 2n = 46, XX karyotype and on a 2n = 46, XY, t(2;15)(q23;q15) karyotype as an example of a translocation of chromosomal bands.

Azure Stains↗

Atomic force microscopy and other scanning probe microscopies.

The highlight of the past year is the unfolding and refolding of the muscle protein titin in the atomic force microscope. A related highlight in the intersection between experiment and theory is a recent review of the effects of molecular forces on biochemical kinetics. Other advances in scanning probe microscopy include entropic brushes, molecular sandwiches and applications of atomic force microscopy to gene therapy.

Annexin A5↗

Atomic force microscopy analysis of enveloped and non-enveloped viral entry into, and egress from, cultured cells.

Since its invention, the atomic force microscope has been used to image a wide variety of biological samples, including viruses. Viral entry into, and egress from, cultured cells has been extensively studied using numerous scientific techniques and to a limited extent using atomic force microscopy. One of the main structural differences that can exist between viruses is the absence, or presence, of an envelope and this factor has consequences for the mode of viral entry and egress. In this study, the entry into, and egress from, cultured cells of enveloped and non-enveloped viruses were investigated using atomic force microscopy. No significant cell surface changes were observed following infection with enveloped or non-enveloped viruses. Although roughness analysis of viral entry revealed cell smoothing post-infection, no differences between the roughness values of enveloped and non-enveloped viral entry were observed. Line analysis of viral entry revealed minor differences between cells infected with an enveloped rather than a non-enveloped virus. These differences may represent a distinction between the uptake processes of enveloped and non-enveloped viruses. Studies of viral egress revealed that infected cells were undergoing cytopathic changes. Whilst topographic, height and roughness differences clearly occurred between virally- and mock-infected cells, no significant differences were elucidated between enveloped and non-enveloped viral egress.

Animals↗

Application of atomic force microscopy to microbial surfaces: from reconstituted cell surface layers to living cells.

The application of atomic force microscopy (AFM) to probe the ultrastructure and physical properties of microbial cell surfaces is reviewed. The unique capabilities of AFM can be summarized as follows: imaging surface topography with (sub)nanometer lateral resolution; examining biological specimens under physiological conditions; measuring local properties and interaction forces. AFM is being used increasingly for: (i) visualizing the surface ultrastructure of microbial cell surface layers, including bacterial S-layers, purple membranes, porin OmpF crystals and fungal rodlet layers; (ii) monitoring conformational changes of individual membrane proteins; (iii) examining the morphology of bacterial biofilms, (iv) revealing the nanoscale structure of living microbial cells, including fungi, yeasts and bacteria, (v) mapping interaction forces at microbial surfaces, such as van der Waals and electrostatic forces, solvation forces, and steric/bridging forces; and (vi) probing the local mechanical properties of cell surface layers and of single cells.

Bacteria↗

Atomic force microscopy studies on DNA structural changes induced by vincristine sulfate and aspirin.

We report that atomic force microscopy (AFM) studies on structural variations of a linear plasmid DNA interact with various concentrations of vincristine sulfate and aspirin. The different binding images show that vincrinstine sulfate binding DNA chains caused some loops and cleavages of the DNA fragments, whereas aspirin interaction caused the width changes and conformational transition of the DNA fragments. Two different DNA structural alternations could be explained by the different mechanisms of the interactions with these two components. Our work indicates that the AFM is a powerful tool in studying the interaction between DNA and small molecules.

Aspirin↗

Atomic force microscopy measurements. Measurements of binding strength between a single pair of molecules in physiological solutions.

Atomic force microscopy (AFM) measurements of intermolecular binding strength between a single pair of complementary cell adhesion molecules in physiological solutions provided the first quantitative evidence for their cohesive function. This novel AFM based nanobiotechnology opens a molecular mechanic approach for studying structure to function related properties of any type of individual biological macromolecules. The presented example of Porifera cell adhesion glyconectin proteoglycans showed that homotypic carbohydrate to carbohydrate interactions between two primordial proteoglycans can hold the weight of 1600 cells. Thus, glyconectin type carbohydrates, as the most peripheral cell surface molecules of sponges (today's simplest living Metazoa), are proposed to be the primary cell adhesive molecules essential for the evolution of the multicellularity.

Animals↗

Penetration of bovine serum albumin into dipalmitoylphosphatidylglycerol monolayers: direct observation by atomic force microscopy.

The penetration of bovine serum albumin (BSA) into dipalmitoylphosphatidylglycerol (DPPG) monolayers was observed using atomic force microscopy (AFM) and surface pressure measurements. The effects of surface pressure, amount of BSA and the addition of ganglioside GM1 (GM1) were investigated. The surface pressure of the DPPG monolayer was increased by the penetration of BSA, and the increase in surface pressure was greater in the liquid-expanded film than that in the liquid-condensed film. The AFM images indicated that BSA penetrated into the DPPG monolayer. The amount of BSA that penetrated into the DPPG monolayer increased with time and with the amount of BSA added. On the contrary, the AFM image showed that BSA penetration into the mixed DPPG/GM1 (9 : 1) monolayer scarcely occurred. GM1 inhibited the penetration of BSA into the DPPG monolayer.

Animals↗

Transverse stiffness of myofibrils of skeletal and cardiac muscles studied by atomic force microscopy.

The transverse stiffness of single myofibrils of skeletal and cardiac muscles was examined by atomic force microscopy. The microscopic images of both skeletal and cardiac myofibrils in a rigor state showed periodical striation patterns separated by Z-bands, which is characteristic of striated muscle fibers. However, sarcomere patterns were hardly distinguishable in the stiffness distributions of the relaxed myofibrils of skeletal and cardiac muscles. Myofibrils in a rigor state were significantly stiff compared with those in a relaxed state, and in each state, cardiac myofibrils were significantly stiffer compared with skeletal myofibrils. By proteolytic digestions of sarcomere components of myofibrils, it was suggested that cardiac myofibrils are laterally stiffer than skeletal myofibrils because Z-bands, connectin (titin) filament networks, and other components of sarcomere structures for the former myofibrils are stronger than those for the latter.

Animals↗

A new method of biosensing with 1 microl of Escherichia coli suspension using atomic force microscopy.

We developed a new method for detecting bacterial cells from 1-mul samples with atomic force microscopy (AFM). The use of a parafilm surface as a sample palette was effective for reacting small amounts of samples with an AFM probe. This was due to the parafilm's hydrophobic, semitransparent, and nonadhesive surface. In this way, all processes, such as the surface functionalization of a cantilever and the adhesion of Escherichia coli cells to a cantilever, were easily completed. In addition, we succeeded in detecting cell adsorption on the same AFM cantilever by both the drive mode and the thermal mode. The resonance frequency shift caused by cell adhesion was clearly detected by the two modes for the first time. Our data indicated the potential of applying AFM nanobiosensing to extremely small amounts of samples.

Bacterial Adhesion↗

Topographic and phase-contrast imaging in atomic force microscopy

Phase-contrast imaging in the tapping mode atomic force microscopy (AFM) is a powerful method in surface characterization. This method can provide fine details about rough surfaces, which are normally obscured in topographic imaging. To illustrate some of the capabilities of phase-contrast imaging, AFM studies of Pt/Ti/SiO2/Si and Pb(Zr0.52Ti0.48)O3 (PZT) films were carried out. Phase-contrast imaging revealed fine details of their microstructures, including grain boundaries, triple junctions and twinning, which could not be detected by topographic imaging. The studies showed that phase-contrast imaging is capable of providing superior information about surface characteristics when compared to the standard topographic imaging.

Journal Article↗

Atomic force microscopy of DNA on mica and chemically modified mica.

Atomic force microscopy (AFM) was used to image circular DNA adsorbed on freshly cleaved mica and mica chemically modified with Mg(II), Co(II), La(III), and Zr(IV). Images obtained on unmodified mica show coiling of DNA due to forces involved during the drying process. The coiling or super twisting appeared to be right handed and the extent of super twisting could be controlled by the drying conditions. Images of DNA observed on chemically modified surfaces show isolated open circular DNA that is free from super twisting, presumably due to strong binding of DNA on chemically modified surfaces.

Adsorption↗

Covalent and reversible short-range electrostatic imaging in noncontact atomic force microscopy.

We present a computational study of atomic-scale image formation in noncontact atomic force microscopy on metallic surfaces. We find two imaging scenarios: (1). atomic resolution arising due to very strong covalent tip-sample interaction exhibiting striking similarity with the imaging mechanism found on semiconductor surfaces, and (2). a completely new mechanism, reversible short-range electrostatic imaging, arising due to subtle charge-transfer interactions. Contrary to the strong covalent-bond imaging, the newly identified mechanism causes only negligible surface perturbation and can account for results recently observed experimentally.

Journal Article↗

TectoRNA and 'kissing-loop' RNA: atomic force microscopy of self-assembling RNA structures.

RNA molecules have been much less studied by atomic force microscopy (AFM) than have DNA molecules. In this paper, AFM imaging is presented for two different RNA molecules able to self-assemble into complex supramolecular architectures. The first one is a molecular dimer of a 230-nt RNA fragment coming from the RNA genome of a murine leukaemia virus. The monomeric RNA fragment, which appears by AFM as an elongated structure with a mean aspect ratio of 1.4, assembles into a dimer of elongated structures through the formation of a 'kissing-loop' RNA interaction. The second one is a large supramolecular fibre formed of artificial self-assembling RNA molecular units called tectoRNA. The fibre lengths by AFM suggest that there are 50-70 tectoRNA units per fibre. Some methods and limitations are presented for measuring molecular volumes from AFM images.

Animals↗