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Results for “Microscopy, Scanning Tunneling”

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At least 19 recordsLinked to original sources
PubMed1996

Microscopic analysis of DNA and DNA-protein assembly by transmission electron microscopy, scanning tunneling microscopy and scanning force microscopy.

To investigate DNA and DNA-protein assembly, nucleic acids were adsorbed to freshly cleaved mica in the presence of magnesium ions. The efficiency of DNA adhesion and the distribution of the molecules on the mica surface were checked by transmission electron microscopy. In addition, various kinds of DNA-protein interactions including DNA wrapping and DNA supercoiling were analyzed using electron microscopy. In parallel, this Mg2+/mica method can be applied (1) to analyze embedded DNA by scanning tunneling microscopy, (2) to visualize freeze-dried, metal coated DNA-protein complexes by tunneling microscopy, and (3) to image DNA or DNA-protein interaction in air or in liquid by scanning force microscopy. An advantage of such a correlative approach is that parallel imaging can reveal complementary information. The benefit of such a combined approach in analysis of protein-induced DNA bending is discussed.

Aluminum Silicates↗
PubMed1989

Structure of the globular protein vicilin revealed by scanning tunnelling microscopy.

Scanning tunnelling microscopy (s.t.m.) has been used to study the structure of the non-crystalline globular protein vicilin. Molecules were deposited on amorphous carbon substrates and imaged both in air and in vacuo without additional sample preparation. Current-voltage plots of an individual protein molecule are also reported. The s.t.m. images are compared with conventional transmission electron micrographs and with a model of vicilin based on small-angle synchrotron X-ray scattering data.

Electric Conductivity↗
PubMed1992

Observation of the inverted hexagonal phase of lipids by scanning tunneling microscopy.

Scanning tunneling microscopy (STM) has been used in direct observations of the inverted hexagonal phase of several lipids, such as cardiolipin (CL) in the presence of Ca2+, soybean PE, DOPE and a binary system of CL and DMPC as well. Single tube-like structures, bundles of tubes and the cross-sectional view of the HII phase can clearly be seen at room temperature in a near natural state of these lipids. The success of this study shed some light on the applicability of STM in the investigation of lipid polymorphism and its role in the function of biological membranes.

Cardiolipins↗
PubMed1992

Visualization of the algal polysaccharide carrageenan by scanning tunnelling microscopy.

Scanning tunnelling microscopy has been used to obtain images in the constant-current mode in air and moist conditions at molecular resolution for the kappa- and iota-carrageenan algal polysaccharides. The molecules were deposited from an aqueous solution onto a graphite substrate. The samples formed aligned nematic-like arrays and were also found as individual molecules. The molecular dimensions of width, height and repeat distance along the molecule were found to be close to the values previously determined by X-ray diffraction. The results support a molecular model based on a double-helix structure for carrageenan.

Carbohydrate Sequence↗
PubMed1994

Use of molybdenum telluride as a substrate for the imaging of biological molecules during scanning tunnelling microscopy.

Scanning tunnelling microscopy was used to image biological molecules including supercoiled deoxyribonacetic acid and specific retrovirus enzymes, the reverse transcriptases of the avian myeloblastosis virus, the moloney murine leukaemia virus and the human immunodeficiency virus. Measurements were carried out on graphite and Group VI transition metal dichalcogenide layered crystals. Images obtained with graphite could not be unequivocally interpreted and attachment appears to occur solely at surface defect sites. The layered crystal MoTe2 shows different imaging properties. The bimolecules are clearly visible, distributed over the semiconductor surface, and the molecular shapes and dimensions show good correlation with structure predictions.

DNA, Superhelical↗
PubMed1991

Structure of immunoglobulin G by scanning tunnelling microscopy.

Scanning tunnelling microscopy (STM) has been used to examine the shape of individual immunoglobulin G (IgG) molecules deposited onto a graphite surface. IgG was chosen for this study as it has a well-characterized and distinctive three-dimensional structure. The micrographs clearly reveal the IgG molecule as trilobed, corresponding with the known structural organization of IgG. Comparison of these images with the structure of IgG determined by X-ray crystallography shows that the STM images are consistent with the crystal structure. This illustrates that STM is a valuable technique for examining protein structure, allowing rapid determination of the overall molecular shape that is consistent with more established techniques.

Immunoglobulin G↗
PubMed1992

Imaging of proteins by scanning tunnelling microscopy.

Scanning tunnelling microscopy has been used to examine the structure of proteins deposited on a graphite surface. Three molecules have been studied; immunoglobulin G (IgG), Complement component 1q (C1q) and ATP-citrate lyase (ACL). The images show IgG as a tri-lobed molecule, consistent with the known 3D structure as determined by X-ray crystallography. The C1q images differ from the well known "tulip bunch" model derived by electron microscopy, but are consistent with the model if it is assumed that the six globular heads have aggregated. Molecules of ACL are visible as discrete units, with some hints of substructure. These results highlight the potential of STM in studying protein structures, but also illustrate the difficulties of interpreting micrographs of proteins whose structure is currently unknown.

Complement C1q↗
PubMed1993

Analysis of ordered arrays of adsorbed lysozyme by scanning tunneling microscopy.

Scanning tunneling microscopy (STM) has been used to observe lysozyme at a graphite surface directly in order to gain mechanistic information about the molecular events involved in protein adsorption. The experiments were performed using an insulated tip in an aqueous protein solution, allowing the time course of the adsorption process to be followed, including the evolution of ordered arrays. Ordered arrays of protein molecules were observed, with lattice spacings that varied with bulk protein concentration and salt strength. Fourier analysis was used to determine the average cell dimensions of an array. From the observed lattice spacings, it was possible to estimate the surface coverage of the protein, and thus, by varying the conditions, adsorption isotherms could be obtained. These isotherms compare well with adsorption isotherms measured using total internal reflectance fluorescence (TIRF) spectroscopy on a hydrophobic surface. Since the protein is charged and the electrolyte has an effect on the isotherms, electrostatics are a likely controlling factor. Molecular electrostatics computations were thus used to investigate the possible origins of the lattice structure, and they suggest that favorable intermolecular interactions among adsorbed molecules are consistent with hydrophobically dominated protein-surface interactions.

Adsorption↗
PubMed1996

Investigation of ascorbate-Cu (II) induced cleavage of DNA by scanning tunneling microscopy.

Scanning Tunneling Microscopy (STM) was used for the investigation of oxidative DNA damage. A PCR amplified fragment of human beta-globin gene was used as a model for time dependent cleavage reaction by ascorbate and copper. Cleavage reactions were carried out in a medium containing 0.5 microgram/20 microliters DNA, 20 nM Tris-HC1 pH, 7.8 and ascorbate-Cu (II) in the final concentrations of 1 mM and 30 microM, respectively. The mixtures were incubated at 37 degrees C for 5, 15 and 30 min. For STM studies, 3 pg/5 microliters DNA samples were deposited on the gold coated mica and dried in a water flow vacuum drier. The STM was operated in air at atmospheric pressure with a tip-to-substrate bias of 100 mV and tunneling currents of < 10 pA. Etched tips of Pt/Ir wires were used in a constant current mode. The degradated DNA structure can be distinguished from the intact DNA and the sizes of the degradation products can be identified in the STM micrographs. The size of fragments decreased from approximately 3000 A to 34 A in ascorbate-Cu (II) medium, after 30 min of incubation.

Ascorbic Acid↗
PubMed1993

Identification of DNA--cisplatin adducts in a blind trial of in situ scanning tunneling microscopy.

Scanning tunneling microscopy (STM) reveals nanometer scale details of hydrated DNA but the interpretation of the images is controversial because of substrate artifacts and the lack of a theory for image contrast. We demonstrate that we have overcome these problems by identifying five DNA samples by their STM images alone in a blinded trial. The samples were single-stranded and double-stranded DNA with and without covalent modification by the anti-tumor drug cisplatin. The cisplatin adducts were distinguished by substantial kinking at the drug binding site. The oligomers were 20 bases in length, which was too short to permit the kinking angle to be determined with precision. However, models with a 45 degree kink gave a better fit to the images of the duplex adducts than models with a 90 degrees kink. A variety of structures was observed for the single-stranded adducts.

Base Sequence↗
PubMed1993

Transmission electron microscopy, scanning tunneling microscopy, and atomic force microscopy of the cell envelope layers of the archaeobacterium Methanospirillum hungatei GP1.

Methanospirillum hungatei GP1 possesses paracrystalline cell envelope components including end plugs and a sheath formed from stacked hoops. Both negative-stain transmission electron microscopy (TEM) and scanning tunneling microscopy (STM) distinguished the 2.8-nm repeat on the outer surface of the sheath, while negative-stain TEM alone demonstrated this repeat around the outer circumference of individual hoops. Thin sections revealed a wave-like outer sheath surface, while STM showed the presence of deep grooves that precisely defined the hoop-to-hoop boundaries at the waveform nodes. Atomic force microscopy of sheath tubes containing entrapped end plugs emphasized the end plug structure, suggesting that the sheath was malleable enough to collapse over the end plugs and deform to mimic the shape of the underlying structure. High-resolution atomic force microscopy has revised the former idea of end plug structure so that we believe each plug consists of at least four discs, each of which is approximately 3.5 nm thick. PT shadow TEM and STM both demonstrated the 14-nm hexagonal, particulate surface of an end plug, and STM showed the constituent particles to be lobed structures with numerous smaller projections, presumably corresponding to the molecular folding of the particle.

Cell Membrane↗
PubMed1989

Imaging of single uncoated DNA molecules by scanning tunneling microscopy.

Scanning tunneling microscope images of DNA molecules absorbed onto highly oriented pyrolytic graphite have been obtained. Three methods of deposition and sample preparation have been utilized. In the first method, a highly concentrated solution of DNA is sonicated, and a drop is deposited on freshly cleaved graphite. Under these conditions, the molecules tend to align in a parallel fashion, forming liquid-crystalline phases. In the second method, a solution of DNA is deposited directly on the graphite surface without sonication. In this case, ammonium acetate, a volatile salt, is used to decrease the amount of the residual salt crystals left after drying. In the third method, a solution containing lysed phage particles and DNA is adsorbed onto a graphite surface. The molecules are seen either isolated or in small bundles. The values of height, periodicity, and thickness observed and the handedness of the molecules are consistent with those expected for DNA. In all cases, the molecules were identified by their characteristic periodic structure and because, at higher magnification, no graphite-like structure was detectable on the surface of the molecules. Often the DNA molecules appear to adsorb in areas of the graphite that have many steps and defects. A mechanism that explains the magnitude of the tunneling currents measured in DNA is proposed. This mechanism, in turn, suggests a general method by which large insulating molecules can be rendered conductive.

DNA↗
PubMed1989

Images of single-stranded nucleic acids by scanning tunnelling microscopy.

The scanning tunnelling microscope has the potential to resolve the structure of biological molecules with atomic detail. Progress has been made in the imaging of dried, unshadowed double helices of DNA4-7 and in recording images of DNA under water. Also, images of unshadowed complexes of DNA with the RecA protein from Escherichia coli indicate that this technique may not be restricted to thin biological samples. Here we present images of polydeoxyadenylate molecules aligned in parallel, with their bases lying flat on a surface of highly oriented pyrolytic graphite and with their charged phosphodiester backbones protruding upwards. Based on these images, a molecular model has been built which suggests the presence of a hydrogen bond that could stabilize the parallel alignment. Our micrographs demonstrate the potential application of scanning tunnelling microscopy in structural studies of nucleic acids and provide evidence that it could be used to sequence DNA.

Adsorption↗
PubMed1994

Atomic force microscopy and scanning tunnelling microscopy: refining techniques for studying biomolecules.

The scanning tunnelling microscope and the atomic force microscope offer the prospect of real-time, nanometre-scale imaging of biomolecules and biosurfaces under physiological environments. Much effort is therefore being made to establish these techniques as routine biophysical tools. The considerable recent progress that has been made in biotechnological applications is reviewed, highlighting specific examples of the applications of this new and exciting method of analysis.

Biological Products↗
PubMed1994

A study of lignin formation at the molecular level by scanning tunneling microscopy.

A scanning tunneling microscope (STM) was used to observe the temporal formation and organization of dehydrogenative polymer (DHP) synthesized from coniferyl alcohol. The images obtained elucidate this structure for the first time. The structure of DHP, as seen from STM images, shows long-range order. It appears that DHP consists of building units or modules assembled into larger assemblies called supermodules. Supermodules are interconnected into the overall lattice-like polymer structure with or without spherical regions. One module consists of about 20 monomers, while the supermodule contains about 500 monomers. Calculated molecular weights for modules and supermodules agree with DHP molecular weight distribution peaks. Samples prepared at two different pH values, 6.4 and 7.6, have the same characteristics. The results presented demonstrate that the process of lignification, even in in vitro conditions, is highly ordered, and as such contribute to our understanding of the structure of lignin, a significant constitutive and functional element of cell walls.

Hydrogen-Ion Concentration↗
PubMed1990

Atomic-scale imaging of DNA using scanning tunnelling microscopy.

The scanning tunnelling microscope (STM) has been used to visualize DNA under water, under oil and in air. Images of single-stranded DNA have shown that submolecular resolution is possible. Here we describe atomic-resolution imaging of duplex DNA. Topographic STM images of uncoated duplex DNA on a graphite substrate obtained in ultra-high vacuum are presented that show double-helical structure, base pairs, and atomic-scale substructure. Experimental STM profiles show excellent correlation with atomic contours of the van der Waals surface of A-form DNA derived from X-ray crystallography. A comparison of variations in the barrier to quantum mechanical tunnelling (barrier-height) with atomic-scale topography shows correlation over the phosphate-sugar backbone but anticorrelation over the base pairs. This relationship may be due to the different chemical characteristics of parts of the molecule. Further investigation of this phenomenon should lead to a better understanding of the physics of imaging adsorbates with the STM and may prove useful in sequencing DNA. The improved resolution compared with previously published STM images of DNA may be attributable to ultra-high vacuum, high data-pixel density, slow scan rate, a fortuitously clean and sharp tip and/or a relatively dilute and extremely clean sample solution. This work demonstrates the potential of the STM for characterization of large biomolecular structures, but additional development will be required to make such high resolution imaging of DNA and other large molecules routine.

Animals↗
PubMed1991

Application of scanning tunneling microscopy to structural biology.

Scanning tunneling microscopy offers the possibility of visualizing biological molecules in conditions similar to those in vivo with molecular resolution. Images of DNA and various proteins have been obtained, but insufficient conductivity through, and inhomogeneous and unstable adsorption of the biomolecules continue to prevent reliable imaging. Applying a metal coating to samples, to separate the conductivity and deposition problems has yielded satisfactory deposition procedures in various laboratories, but extension of this protocol to high resolution imaging of macromolecules has yet to be demonstrated. In this paper we present a review of the main results obtained in our laboratory, which illustrate the main problems encountered by investigators attempting to image metal-coated and uncoated biological specimens.

DNA↗