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PubMed2001

From images to interactions: high-resolution phase imaging in tapping-mode atomic force microscopy.

In tapping-mode atomic force microscopy, the phase shift between excitation and response of the cantilever is used as a material-dependent signal complementary to topography. The localization of information in the phase signal is demonstrated with 1.4-nm lateral resolution on purple membrane of Halobacterium salinarum in buffer solution. In a first-order approximation, the phase signal is found to correlate with modulations of the tip oscillation amplitude, induced by topography. Extending the analysis to contributions of the tip-sample interaction area as a second-order approximation, a method is proposed to extract information about the interaction from the phase signal for surfaces with a roughness in the order of the tip radius.

Buffers↗
PubMed2004

An overview of the biophysical applications of atomic force microscopy.

The potentialities of the atomic force microscopy (AFM) make it a tool of undeniable value for the study of biologically relevant samples. AFM is progressively becoming a usual benchtop technique. In average, more than one paper is published every day on AFM biological applications. This figure overcomes materials science applications, showing that 17 years after its invention, AFM has completely crossed the limits of its traditional areas of application. Its potential to image the structure of biomolecules or bio-surfaces with molecular or even sub-molecular resolution, study samples under physiological conditions (which allows to follow in situ the real time dynamics of some biological events), measure local chemical, physical and mechanical properties of a sample and manipulate single molecules should be emphasized.

Animals↗
PubMed2005

Stepwise unfolding of ankyrin repeats in a single protein revealed by atomic force microscopy.

Using single-molecule atomic force microscopy, we find that a protein consisting of six identical ankyrin repeat units flanked by N- and C-terminal modules (N6C) unfolds in a stepwise, unit-by-unit fashion under a mechanical force. Stretching a N6C molecule results in a sawtooth pattern fingerprint, with as many as six peaks separated by approximately 10 nm and an average unfolding force of 50 +/- 20 pN. Our results demonstrate that a stretching force can unfold multiple repeat units individually in a single protein molecule, despite extensive hydrophobic interactions between adjacent units.

Ankyrin Repeat↗
PubMed1998

Mapping a protein-binding site on straightened DNA by atomic force microscopy.

We have developed an Atomic Force Microscopy (AFM)-based method for mapping protein-binding sites on individual, long DNA molecules (> 5 kb) at nanometer resolution. The protein is clearly detected at the apex of the bent DNA molecules. Randomly coiled DNA molecules or protein:DNA complexes were extended by a motor-controlled moving meniscus on an atomically flat surface. The immobilized molecules were detected by AFM. The straightened DNA displayed a sharp bend at the site of bound protein with the two DNA segments linearly extending from the protein-binding site. Using GAL4, a yeast transcription factor, we demonstrate good agreement of the position of the observed binding site on straightened DNA templates to the predicted binding site. The technique is expected to have significant implications in elucidating DNA and protein interactions in general, and specifically, for the measurement of promoter occupancy with unlabeled regulatory proteins at the single-molecule level.

Aluminum Silicates↗
PubMed1997

Probing single biomolecules with atomic force microscopy.

During the last years, atomic force microscopy (AFM) has developed from a microscopy tool for solid-state surface science toward a method employed in many scientific disciplines, such as biology, for investigating individual molecules on a nanometer scale. This article describes the current status of the imaging possibilities of AFM on RNA, IgG, and gold-labeled cell adhesion proteoglycans, as well as of measurements of intermolecular binding forces between biomolecules in order to investigate their molecular structure, function, and elasticity.

Animals↗
PubMed2002

Atomic force microscopy of Precambrian microscopic fossils.

Atomic force microscopy (AFM) is a technique used routinely in material science to image substances at a submicron (including nm) scale. We apply this technique to analysis of the fine structure of organic-walled Precambrian fossils, microscopic sphaeromorph acritarchs (cysts of planktonic unicellular protists) permineralized in approximately 650-million-year-old cherts of the Chichkan Formation of southern Kazakhstan. AFM images, backed by laser-Raman spectroscopic analysis of individual specimens, demonstrate that the walls of these petrified fossils are composed of stacked arrays of approximately 200-nm-sized angular platelets of polycyclic aromatic kerogen. Together, AFM and laser-Raman spectroscopy provide means by which to elucidate the submicron-scale structure of individual microscopic fossils, investigate the geochemical maturation of ancient organic matter, and, potentially, distinguish true fossils from pseudofossils and probe the mechanisms of fossil preservation by silica permineralization.

Animals↗
PubMed2001

Atomic force microscopy applications in macromolecular crystallography.

Atomic force microscopy (AFM) can be applied both in situ and ex situ to study the growth of crystals from solution. The method is particularly useful for investigating the crystallization of proteins, nucleic acids and viruses because it can be carried out in the mother liquor and in a non-perturbing fashion. Interactions and transformations between various growth mechanisms can be directly visualized as a function of supersaturation, as can the incorporation of diverse impurities and the formation and propagation of defects. Because the crystals can be observed over long periods, it is also possible to obtain precise quantitative measures of the kinetic parameters for nucleation and growth. Finally, AFM has allowed us to identify a number of previously unsuspected phenomena that influence nucleation, rate of growth and the ultimate perfection of macromolecular crystals. These are all features which are important in determining the ultimate resolution and quality of a crystal's diffraction pattern.

Crystallization↗
PubMed2000

Atomic force microscopy of native purple membrane.

Atomic force microscopy (AFM) allows the observation of surface structures of purple membrane (PM) in buffer solution with subnanometer resolution. This offers the possibility to classify the major conformations of the native bacteriorhodopsin (BR) surfaces and to map the variability of individual polypeptide loops connecting transmembrane alpha-helices of BR. The position, the variability and the flexibility of these loops depend on the packing arrangement of BR molecules in the lipid bilayer with significant differences observed between the trigonal and orthorhombic crystal forms. Cleavage of the Schiff base bond leads to a disassembly of the trigonal PM crystal, which is restored by regenerating the bleached PM. The combination of single molecule AFM imaging and single molecule force-spectroscopy provides an unique insight into the interactions between individual BR molecules and the PM, and between secondary structure elements within BR.

Bacteriorhodopsins↗
PubMed1998

Visualization of trp repressor and its complexes with DNA by atomic force microscopy.

We used tapping mode atomic force microscopy to visualize the protein/protein and the protein/DNA complexes involved in transcriptional regulation by the trp repressor (TR). Plasmid fragments bearing the natural operators trp EDCBA and trp R, as well as nonspecific fragments, were deposited onto mica in the presence of varying concentrations of TR and imaged. In the presence of L-tryptophan, both specific and nonspecific complexes of TR with DNA are apparent, as well as free TR assemblies directly deposited onto the mica surface. We observed the expected decrease in specificity of TR for its operators with increasing protein concentration (1-5 nM). This loss of DNA-binding specificity is accompanied by the formation of large protein assemblies of varying sizes on the mica surface, consistent with the known tendency of the repressor to oligomerize in solution. When the co-repressor is omitted, no repressor molecules are seen, either on the plasmid fragments or free on the mica surface, probably because of the formation of larger aggregates that are removed from the surface upon washing. All these findings support a role for protein/protein interactions as an additional mechanism of transcriptional regulation by the trp repressor.

Bacterial Proteins↗
PubMed2000

Structure and dynamics of three-way DNA junctions: atomic force microscopy studies.

We have used atomic force microscopy (AFM) to study the conformation of three-way DNA junctions, intermediates of DNA replication and recombination. Immobile three-way junctions with one hairpin arm (50, 27, 18 and 7 bp long) and two relatively long linear arms were obtained by annealing two partially homologous restriction fragments. Fragments containing inverted repeats of specific length formed hairpins after denaturation. Three-way junctions were obtained by annealing one strand of a fragment from a parental plasmid with one strand of an inverted repeat-containing fragment, purified from gels, and examined by AFM. The molecules are clearly seen as three-armed molecules with one short arm and two flexible long arms. The AFM analysis revealed two important features of three-way DNA junctions. First, three-way junctions are very dynamic structures. This conclusion is supported by a high variability of the inter-arm angle detected on dried samples. The mobility of the junctions was observed directly by imaging the samples in liquid (AFM in situ). Second, measurements of the angle between the arms led to the conclusion that three-way junctions are not flat, but rather pyramid-like. Non-flatness of the junction should be taken into account in analysis of the AFM data.

DNA↗
PubMed1999

Determining the molecular-growth mechanisms of protein crystal faces by atomic force microscopy.

A high-resolution atomic force microscopy (AFM) study has shown that the molecular packing on the tetragonal lysozyme (110) face corresponds to only one of two possible packing arrangements, suggesting that growth layers on this face are of bimolecular height [Li et al. (1999). Acta Cryst. D55, 1023-1035]. Theoretical analyses of the packing also indicated that growth of this face should proceed by the addition of growth units of at least tetramer size, corresponding to the 43 helices in the crystal. In this study, an AFM linescan technique was used to measure the dimensions of individual growth units on protein crystal faces as they were being incorporated into the lattice. Images of individual growth events on the (110) face of tetragonal lysozyme crystals were observed, shown by jump discontinuities in the growth step in the linescan images. The growth-unit dimension in the scanned direction was obtained from these images. A large number of scans in two directions on the (110) face were performed and the distribution of lysozyme growth-unit sizes were obtained. A variety of unit sizes corresponding to 43 helices were shown to participate in the growth process, with the 43 tetramer being the minimum observed size. This technique represents a new application for AFM, allowing time-resolved studies of molecular processes to be carried out.

Animals↗
PubMed1997

Escherichia coli RNA polymerase activity observed using atomic force microscopy.

Fluid tapping-mode atomic force microscopy (AFM) was used to observe Escherichia coli RNA polymerase (RNAP) transcribing two different linear double-stranded (ds) DNA templates. The transcription process was detected by observing the translocation of the DNA template by RNAP on addition of ribonucleoside 5'-triphosphates (NTPs) in sequential AFM images. Stalled ternary complexes of RNAP, dsDNA and nascent RNA were adsorbed onto a mica surface and imaged under continuously flowing buffer. On introduction of all four NTPs, we observed some DNA molecules being pulled through the RNAP, some dissociating from the RNAP and others which did not move relative to the RNAP. The transcription rates were observed to be approximately 0.5-2 bases/s at our NTP concentrations, approximately 5 microM. The RNA transcripts were not unambiguously imaged in fluid. However, in experiments using a small single-stranded (ss) circular DNA template, known as a rolling circle, transcripts up to 1 or 2 microns long could be observed with tapping mode AFM once the samples were dried and imaged in air. This confirmed our observations of the transcriptional activity of RNAP adsorbed onto mica. This work illustrates that the development of tapping-mode in fluid has made it possible to use AFM to follow biological processes at the molecular level and get new insights about the variability of activity of individual molecules bound to a surface.

Aluminum Silicates↗
PubMedDate not supplied

Research on double-probe, double- and triple-tip effects during atomic force microscopy scanning.

Information obtained by atomic force microscopy (AFM) depends strongly on the kind of probe or tip used; therefore, probe and tip effects have to be taken into account when verifying or interpreting the data acquired. In many papers, double-tip effects have been mentioned while other research was done; however, there are only a few special reports on double- or triple-tip effects, especially double-probe effects. In our paper, metaphase chromosomes of Chinese hamster ovary (CHO) cells, aggregates of pectin molecules, membrane surface of mouse embryonic stem cells, and R-phycoerythrin-conjugated immunoglobulin G complexes were imaged by AFM with high-quality probes, double-probe cantilever, and double-tip and triple-tip probes, respectively, in order to determine double-probe, double-tip, and triple-tip effects during AFM scanning. We found that the double-probe, double-tip, and triple-tip effects share the same principle, and that these effects correlate with distance and height differences between probes of double-probe cantilever or tips of double-tip or multiple-tip probes. Since many other factors influence double-probe or double-tip effects, more in-depth studies must be undertaken. However, this initial research will make all users of AFM techniques aware of double-probe and double-tip or triple-tip effects during AFM scanning and aid in verifying or interpreting the data acquired.

Animals↗
PubMed1995

Applications for atomic force microscopy of DNA.

Tapping mode atomic force microscopy (AFM) of DNA in propanol, dry helium, and aqueous buffer each have specific applications. Resolution is best in propanol, which precipitates and immobilizes the DNA and provides a fluid imaging environment where adhesive forces are minimized. Resolution on exceptional images of DNA appears to be approximately 2 nm, sufficient to see helix turns in detail, but the smallest substructures typically seen on DNA in propanol are approximately 6-10 nm in size. Tapping AFM in dry helium provides a convenient way of imaging such things as conformations of DNA molecules and positions of proteins on DNA. Images of single-stranded DNA and RecA-DNA complexes are presented. In aqueous buffer DNA molecules as small as 300 bp have been imaged even when in motion. Images are presented of the changes in shape and position of circular plasmid DNA molecules.

Bacteriophage lambda↗
PubMed2004

Hierarchical networks of casein proteins: an elasticity study based on atomic force microscopy.

2D- and 3D-atomic force microscopy (AFM) experiments were performed on single casein micelles (CM) in native state, submerged in liquid, using a home-built AFM instrument. The micelles were immobilized via carbodiimide chemistry to a self-assembled monolayer supported on gold-coated slides. Off-line data analysis allowed the extraction of both surface topography and elastic properties. Relative Young moduli (E*) were derived from force-vs-indentation curves, using the Hertz theory. The obtained E* values were found to increase with CM diameter, following a straight line dependence. The data showed that temperature, via its influence on both the protein-protein interactions and the composition of the micelle, has a clear effect on the mechanical properties of the CMs: higher temperatures and lower serum casein concentrations result in stiffer micelles. For pH < or = 5.6, effecting calcium phosphate release from the micelles by decreasing the pH does not have a large effect on CM stiffness. On decrease of the pH below 5.0, particulate gels and multilayers were obtained. Their measured elasticity (expressed by an equivalent G'AFM) agrees remarkably well with the storage moduli as measured with a conventional rheometer. Compared to single micelles, gels from nonheated CM suspensions are about 3 orders of magnitude softer. The "softness" of these gels (measured under compression or shear) therefore must come from the microscopic and/or mesoscopic links rather than the micelles themselves.

Caseins↗
PubMed2005

The structure of C-banded human metaphase chromosomes as observed by atomic force microscopy.

The ultrastructure of C-banded human metaphase chromosomes was studied by the combined use of light microscopy and atomic force microscopy (AFM). Light microscopy of the C-banded chromosomes showed that the centromeric regions of all chromosomes except the Y chromosome were positively stained. AFM further revealed that the C-positive region was higher than the C-negative region. The area of the C-positive region was specific depending on each chromosome; it ranged from the centromere to the proximal end of the long arm in chromosome 1, while it was restricted to the centromere in chromosomes 2 and 3. At higher magnification, chromatin fibers about 50 nm thick were clearly shown in the entire length of the chromosomes. In the C-positive region, these chromatin fibers were densely packed, while chromatin fibers were loosely packed with gentle twisting in the C-negative region. These AFM findings suggest that certain factors related to the chromatin fiber compaction remain in the C-positive region even after successive C-banging treatment.

Centromere↗
PubMed2006

Therapeutic ultrasound-mediated DNA to cell and nucleus: bioeffects revealed by confocal and atomic force microscopy.

Therapeutic ultrasound (TUS) has the potential of becoming a powerful nonviral method for the delivery of genes into cells and tissues. Understanding the mechanism by which TUS delivers genes, its bioeffects on cells and the kinetic of gene entrances to the nucleus can improve transfection efficiency and allow better control of this modality when bringing it to clinical settings. In the present study, direct evidence for the role and possible mechanism of TUS (with or without Optison) in the in vitro gene-delivery process are presented. Appling a 1 MHz TUS, at 2 W/cm(2), 30%DC for 30 min was found to achieve the highest transfection level and efficiency while maintaining high cell viability (>80%). Adding Optison further increase transfection level and efficiency by 1.5 to three-fold. Confocal microscopy studies indicate that long-term TUS application localizes the DNA in cell and nucleus regardless of Optison addition. Thus, TUS significantly affects transfection efficiency and protein kinetic expression. Using innovative direct microscopy approaches: atomic force microscopy, we demonstrate that TUS exerts bioeffects, which differ from the ones obtained when Optison is used together with TUS. Our data suggest that TUS alone affect the cell membrane in a different mechanism than when Optison is used.

Albumins↗
PubMed1998

Force-mediated kinetics of single P-selectin/ligand complexes observed by atomic force microscopy.

Leukocytes roll along the endothelium of postcapillary venules in response to inflammatory signals. Rolling under the hydrodynamic drag forces of blood flow is mediated by the interaction between selectins and their ligands across the leukocyte and endothelial cell surfaces. Here we present force-spectroscopy experiments on single complexes of P-selectin and P-selectin glycoprotein ligand-1 by atomic force microscopy to determine the intrinsic molecular properties of this dynamic adhesion process. By modeling intermolecular and intramolecular forces as well as the adhesion probability in atomic force microscopy experiments we gain information on rupture forces, elasticity, and kinetics of the P-selectin/P-selectin glycoprotein ligand-1 interaction. The complexes are able to withstand forces up to 165 pN and show a chain-like elasticity with a molecular spring constant of 5.3 pN nm-1 and a persistence length of 0.35 nm. The dissociation constant (off-rate) varies over three orders of magnitude from 0.02 s-1 under zero force up to 15 s-1 under external applied forces. Rupture force and lifetime of the complexes are not constant, but directly depend on the applied force per unit time, which is a product of the intrinsic molecular elasticity and the external pulling velocity. The high strength of binding combined with force-dependent rate constants and high molecular elasticity are tailored to support physiological leukocyte rolling.

Base Sequence↗