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PubMed1993

Atomic force microscopy of DNA in aqueous solutions.

DNA on mica can be imaged in the atomic force microscope (AFM) in water or in some buffers if the sample has first been dehydrated thoroughly with propanol or by baking in vacuum and if the sample is imaged with a tip that has been deposited in the scanning electron microscope (SEM). Without adequate dehydration or with an unmodified tip, the DNA is scraped off the substrate by AFM-imaging in aqueous solutions. The measured heights and widths of DNA are larger in aqueous solutions than in propanol. The measured lengths of DNA molecules are the same in propanol and in aqueous solutions and correspond to the base spacing for B-DNA, the hydrated form of DNA; when the DNA is again imaged in propanol after buffer, however, it shortens to the length expected for dehydrated A-DNA. Other results include the imaging of E. coli RNA polymerase bound to DNA in a propanol-water mixture and the observation that washing samples in the AFM is an effective way of disaggregating salt-DNA complexes. The ability to image DNA in aqueous solutions has potential applications for observing processes involving DNA in the AFM.

1-Propanol↗

PubMed2004

Using the adhesive interaction between atomic force microscopy tips and polymer surfaces to measure the elastic modulus of compliant samples.

An atomic force microscope (AFM) method for measuring surface elasticity based on the adhesive interactions between an AFM tip and sample surfaces is introduced. The method is particularly useful when there is a large adhesion between the tip and soft samples, when the indentation method would be less accurate. For thin and soft samples, this method will have much less interference from the substrate than is found using the indentation method because there is only passive indentation induced by tip-sample adhesion; in contrast, a large indentation with a sharp tip in the sample may break its stress-strain linearity, or even make it fracture. For the case where it is difficult to accurately locate the tip-sample contact point, which is problematic for the indentation method, the method based on adhesive interactions is helpful because it does not require locating the tip-sample contact point when fitting the whole retraction force curve. The model is tested on PDMS polymers with different degrees of cross-linking.

Journal Article↗

PubMed2005

Probing the viscoelastic behavior of cultured airway smooth muscle cells with atomic force microscopy: stiffening induced by contractile agonist.

Complex rheology of airway smooth muscle cells and its dynamic response during contractile stimulation involves many molecular processes, foremost of which are actomyosin cross-bridge cycling and actin polymerization. With an atomic force microscope, we tracked the spatial and temporal variations of the viscoelastic properties of cultured airway smooth muscle cells. Elasticity mapping identified stiff structural elements of the cytoskeletal network. Using a precisely positioned microscale probe, picoNewton forces and nanometer level indentation modulations were applied to cell surfaces at frequencies ranging from 0.5 to 100 Hz. The resulting elastic storage modulus (G') and dissipative modulus (G'') increased dramatically, with hysteresivity (eta = G''/G') showing a definitive decrease after stimulation with the contractile agonist 5-hydroxytryptamine. Frequency-dependent assays showed weak power-law structural damping behavior and universal scaling in support of the soft-glassy material description of cellular biophysics. Additionally, a high-frequency component of the loss modulus (attributed to cellular Newtonian viscosity) increased fourfold during the contractile process. The complex shear modulus showed a strong sensitivity to the degree of actin polymerization. Inhibitors of myosin light chain kinase activity had little effect on the stiffening response to contractile stimulation. Thus, our measurements appear to be particularly well suited for characterization of dynamic actin rheology during airway smooth muscle contraction.

Actins↗

PubMed2003

Morphological study of the healthy human oculomotor nerve by atomic force microscopy.

This study concerns an investigation of oculomotor nerves via imaging semithin sections with an atomic force microscope (AFM). The study used 10 oculomotor nerves removed from individuals aged between 40 and 60 years old at time of death. We adopted semithin sections for our AFM studies to extract topographical data of the fibers of this nerve. Both light microscope and electron microscope images of the same samples were employed to augment our capabilities for orientation in the AFM analyses. Transverse sections of the oculomotor nerve, imaged via AFM, show clearly identifiable ring-like nerve fibers with the myelinated axons. The samples were simultaneously characterized by error signal mode and lateral force microscopy. These AFM-related techniques revealed no information concerning friction force and elasticity due to the presence of the embedding material (epoxy resin), but showed the myelinated nerve fibers with ring-like structures corresponding to the myelin sheaths. These are the first AFM images of physiological human oculomotor nerves, containing clear three-dimensional representations of these samples.

Adult↗

PubMedDate not supplied

Nerve collagens from diabetic and nondiabetic Sprague-Dawley and biobreeding rats: an atomic force microscopy study.

BACKGROUND: Alterations in rat's nerve collagens due to diabetes may be related to the permanence of damage due to diabetic neuropathy. We (1) provide a methodology for determining the diameters of collagen fibers accounting for atomic force microscope (AFM) imaging artifacts, (2) present data on structural differences in sciatic nerve endoneurial, epineurial and tail tendon collagens of control and diabetic Sprague-Dawley and BioBreeding rats, and (3) compare results with literature values. METHODS: We measured collagen diameters and band spacing on endoneurial and epineurial sciatic nerve tissue, and tail tendon, in control and diabetic rats (STZ-induced 12-week diabetic SD and 16-week spontaneously diabetic BB rats). We also developed a model to interpret the raw AFM data. RESULTS: All types of fibrillar collagen diameters studied became larger for diabetic versus control animals. Values for diabetic and control collagen fiber diameters in SD rats were 78 nm and 72 nm for SN epineurium, and 49 nm and 43 nm for SN endoneurium. For diabetic and control BB rats, these values were 83 nm and 77 nm (SN epineurium) and 49 nm and 43 nm (SN endoneurium). Values of 161 nm and 125 nm were found for diabetic and control tail tendon of BB rats. No significant changes were observed in any of the five comparisons made in D-band spacings that ranged from 63 to 69 nm. CONCLUSIONS: The best means we have found to reduce raw AFM data is to measure several diameters with a single scan, using valley-to-valley measurements. Structural, fibrillar collagens of the nerve and tendon become larger in rats exposed to prolonged diabetes.

Animals↗

PubMed1998

Atomic force microscopy studies of crystal surface topology during enamel development.

During the secretory stage of enamel development, the hydroxyapatite crystals appear as thin ribbons which grow substantially in width and thickness during the later maturation stage. In this study, the atomic force microscope (AFM) was used to investigate developmentally-related changes in deproteinized enamel crystal surface topography in normal animals and in those receiving daily doses of fluoride. The AFM revealed previously undescribed surfaces features, some of which may represent growth sites or different crystalline phases. Secretory stage crystals had greater surface rugosity and were more irregular, with spherical sub-structures of 20-30 nm diameter arranged along the "c"-axis. Maturation stage crystals were smoother and larger but revealed both subnanometer steps and lateral grooves running parallel to the "c"-axis. Crystals from fluorotic tissue showed similar features but were more irregular with a higher degree of surface roughness, suggesting abnormal growth. The AFM may prove an important adjunct in determination of the mechanisms controlling crystal size and morphology in skeletal tissues.

Animals↗

PubMed1994

Atomic force microscopy produces faithful high-resolution images of protein surfaces in an aqueous environment.

The atomic force microscope has the potential to monitor structural changes of a biological system in its native environment. To correlate them with the biological function at a molecular level, high lateral and vertical resolution are required. Here we demonstrate that the atomic force microscope is capable of imaging the surface of the hexagonally packed intermediate layer of Deinococcus radiodurans in buffer solution with a lateral resolution of 1 nm and a vertical resolution of 0.1 nm. On average, these topographs differ from those determined by electron microscopy by <0.5 nm.

Journal Article↗

PubMedDate not supplied

[New approaches to the study of viruses using scanning probe microscopy].

Atomic force microscopy was applied for studying individual virus particles. The micromechanism of rotaviruses was studied using the force curves of an atomic force microscope, with the Young's modulus of an individual particle being 1.0 x10(8) +/- 0.3 x 10(8) Pa. The distribution of the mechanical and adhesive properties over the sample surface was analyzed. The application of substrates modified by the corresponding antibodies made it possible to increase the self-descriptiveness of analysis. Some details of applying these substrates in studies of adenoviruses by atomic force microscopy are described. The approaches proposed enable one to extend the set of characteristics describing the virus particle.

Adenoviridae↗

PubMed2005

The study of 3-dimensional structures of IgG with atomic force microscopy.

OBJECTIVE: To detect 3-dimensional images of anti-N-methyl-D-aspartate receptor Nr1 (NMDAr1) polycolonal IgG affixed on mica in physiological environment. METHODS: The images and data were obtained from a contact mode and commercial Si3N4 probed tip by using atomic force microscope (AFM). RESULTS: The anti-NMDAr1 polycolonal IgG has a characteristic structure described as an ellipse spherical shape of 136.4 A x 62.8 A x 26.1 A. On the section of the ellipse edge there were two peaks about 13 nm in width. CONCLUSIONS: Using AFM to investigate biomacromolecule can make us deeply understand the structure of IgG, which will instruct us to detect the membrane receptor protein as a labelling agent.

Adsorption↗

PubMed2005

Probing the viscoelastic response of glassy polymer films using atomic force microscopy.

The mechanical properties of glassy films and glass surfaces have been studied using an atomic force microscope (AFM) through various imaging modes and measuring methods. In this paper, we discuss the viscoelastic response of a glassy surface probed using an AFM. We analyzed the force-distance curves measured on a glassy film or a glassy surface at temperatures near the glass transition temperature, Tg, using a Burgers model. We found that the material's characteristics of reversible anelastic response and viscous creep can be extracted from a force-distance curve. Anelastic response shifts the repulsive force-distance curve while viscous creep strongly affects the slope of the repulsive force-distance curve. When coupled with capillary force, due to the condensation of a thin layer of liquid film at the tip-surface joint, the anelasticity and viscous creep can alter the curve significantly in the attractive region.

Journal Article↗

PubMed2002

Study of the modifications caused by cisplatin, transplatin, and Pd(II) and Pt(II) mepirizole derivatives on pBR322 DNA by atomic force microscopy.

Modifications of the structure of pBR322 DNA caused by interaction with cisplatin, transplatin and Pd(II) and Pt(II) mepirizole derivatives were studied. The compounds were incubated with the plasmid DNA for 24 h at 37 degrees C and then observed with an atomic force microscope. Circular DNA was used because the small tertiary structural changes are easy to monitor. Likewise, its superhelical nature mimics DNA better than certain forms of intracellular DNA such as chromatin. AFM images clearly reveal that the complexes induce changes in the topological forms of fully relaxed pBR322 DNA. Most of the compounds produce a more compact DNA structure with modified writhing number. Analysis of gel migration of the relaxed pBR322 DNA incubated with the platinum complexes provides complementary information, which is in good agreement with AFM results.

Antineoplastic Agents↗

PubMed2006

In-situ thickness measurement of porous alumina by atomic force microscopy and the reflectance wavelength measurement from 400-1000 nm.

This article presents a novel method to measure the in situ thickness of porous alumina (PA) films. The PA films were prepared in oxalic acid at 30, 40, and 60 V direct current. Based on the atomic force microscope measurements, PA film porosities and refractive indexes measurements were acquired. With the observation of the reflectance spectra of PA films over the wavelength range 400-1000 nm, the nondestructive thickness measurement of the PA films were accurate and were found to be 3.66, 7.76, and 11.38 mum, respectively. Experiments showed that when the applied voltage increased, the pores diameters and interpore distances were enlarged, and the interference pattern was stronger and exhibited a greater number of oscillations over the given wavelength range, which indicated that the PA film's thickness increased. Our results match with the theoretical predictions and analysis quite well.

Aluminum Oxide↗

PubMed2003

Combined nanomanipulation by atomic force microscopy and UV-laser ablation for chromosomal dissection.

Nanomanipulation and nanoextraction on a scale close to and beyond the resolution limit of light microscopy is needed for many modern applications in biological research. For the manipulation of biological specimens a combined microscope allowing for ultraviolet (UV) microbeam laser manipulation together with manipulation by an atomic force microscope (AFM) was used. In a one-step procedure, human metaphase chromosomes were dissected optically by the UV-laser ablation and mechanically by AFM manipulation. With both methods, sub-400-nm cuts could be achieved routinely. Thus, the AFM is an indispensable tool for in situ quality control of nanomanipulation. However, already on this scale the dilation of the topographic AFM image due to the tip geometry can become significant. Therefore the AFM images were restored using a tip geometry obtained by a blind tip-reconstruction algorithm. Cross-sectional analysis of the restored image reveals a 380-nm-wide UV-laser cut and AFM cuts between 70 nm and 280 nm.

Chromosomes↗

PubMed2006

Atomic force microscopy study of the specific adhesion between a colloid particle and a living melanoma cell: Effect of the charge and the hydrophobicity of the particle surface.

We investigated the effect of the charge and the hydrophobicity of drug delivery system (DDS) carriers on their specificity to living malignant melanoma B16F10 cells with the atomic force microscope. To model various nanoparticle DDS carriers, we used silica particles that were modified with silane coupling agents. We then measured the compression and decompression forces between the modified colloid probes and the living B16F10 cell in a physiological buffer as a function of their separation distances. The maximum adhesive force on decompression was related to the strength of the specificity of the DDS to the malignant cell. A comparison of the average maximum adhesive force of each functionality group surprisingly showed that negatively charged surfaces and hydrophobic modified surfaces all had similar low values. Additionally, we saw the unexpected result that there was no observable dependence on the degree of hydrophobicity of the probe surface to a B16F10 cell. Only the positively charged particle gave a strong adhesive force with the B16F10 cell. This indicated that DDS carriers with positive charges appeared to have the highest affinity for malignant melanoma cells and that the use of hydrophobic materials unexpectedly did not improve their affinity.

Adhesiveness↗

PubMedDate not supplied

Detection of rokitamycin-induced morphostructural alterations in Helicobacter pylori by atomic force microscopy.

The subinhibitory and suprainhibitory concentrations of many antibiotics are capable of interfering with the morphology of bacteria, thus disrupting the integrity of their structures and reducing bacterial virulence. Studies of this type have so far been performed using optical or scanning electron microscopes, but a new type of lens-free microscope, the atomic force microscope (AFM), has recently been introduced which is capable of investigating fine three-dimensional surface topography. The present study made use of this new technique to investigate the morphological alterations in Helicobacter pylori as a bacterial specimen exposed or not to different concentrations of rokitamycin. The produced images clearly show that AFM is a very useful tool for obtaining fine-quality three-dimensional images of bacterial morphology. The breakthrough in applying this new type of microscopy is also due to the fact that the images can be digitally collected at very high resolution in the z-axis, without the need to adopt the critical point-drying method or vacuum conditions, and without the need to coat the surface of the sample with a metal layer, a result not otherwise attainable with other microscopy techniques.

Anti-Bacterial Agents↗

PubMed2005

Atomic force microscopy shows that vaccinia topoisomerase IB generates filaments on DNA in a cooperative fashion.

Type IB DNA topoisomerases cleave and rejoin one strand of the DNA duplex, allowing for the removal of supercoils generated during replication and transcription. In addition, electron microscopy of cellular and viral TopIB-DNA complexes has suggested that the enzyme promotes long-range DNA-DNA crossovers and synapses. Here, we have used the atomic force microscope to visualize and quantify the interaction between vaccinia topoisomerase IB (vTopIB) and DNA. vTopIB was found to form filaments on nicked-circular DNA by intramolecular synapsis of two segments of a single DNA molecule. Measuring the filament length as a function of protein concentration showed that synapsis is a highly cooperative process. At high protein:DNA ratios, synapses between distinct DNA molecules were observed, which led to the formation of large vTopIB-induced DNA clusters. These clusters were observed in the presence of Mg2+, Ca2+ or Mn2+, suggesting that the formation of intermolecular vTopIB-mediated DNA synapsis is favored by screening of the DNA charge.

DNA↗

PubMed1997

Gi regulation of secretory vesicle swelling examined by atomic force microscopy.

In the last decade, several monomeric and heterotrimeric guanine nucleotide binding proteins have been identified to associate with secretory vesicles and to be implicated in exocytosis. Vesicle volume also has been proposed to play a regulatory role in secretory vesicle fusion at the plasma membrane. However, the molecular mechanism of function of the guanine nucleotide binding proteins and of the regulation of secretory vesicle volume in the exocytotic process remains unclear. In this study, we report association of the secretory vesicle membrane with the alpha subunit of a heterotrimeric GTP binding protein G(alpha i3) and implicate its involvement in vesicle swelling. Using an atomic force microscope in combination with confocal microscopy, we were able to study the dynamics of isolated zymogen granules, the secretory vesicles in exocrine pancreas. Exposure of zymogen granules to GTP resulted in a 15-25% increase in vesicle height as measured by the atomic force microscope and a similar increase in vesicle diameter as determined by confocal microscopy. Mas7, an active mastoparan analog known to stimulate Gi proteins, was found to stimulate the GTPase activity of isolated zymogen granules and cause swelling. Increase in vesicle size in the presence of GTP, NaF, and Mas7 were irreversible and KCl-sensitive. Ca2+ had no effect on zymogen granule size. Taken together, the results indicate that G(alpha i3) protein localized in the secretory vesicle membrane mediates vesicle swelling, a potentially important prerequisite for vesicle fusion at the cell plasma membrane.

Animals↗

PubMed2006

Fast-scanning atomic force microscopy reveals the ATP/ADP-dependent conformational changes of GroEL.

In order to fold non-native proteins, chaperonin GroEL undergoes numerous conformational changes and GroES binding in the ATP-dependent reaction cycle. We constructed the real-time three-dimensional-observation system at high resolution using a newly developed fast-scanning atomic force microscope. Using this system, we visualized the GroES binding to and dissociation from individual GroEL with a lifetime of 6 s (k=0.17 s(-1)). We also caught ATP/ADP-induced open-closed conformational changes of individual GroEL in the absence of qGroES and substrate proteins. Namely, the ATP/ADP-bound GroEL can change its conformation 'from closed to open' without additional ATP hydrolysis. Furthermore, the lifetime of open conformation in the presence of ADP ( approximately 1.0 s) was apparently lower than those of ATP and ATP-analogs (2-3 s), meaning that ADP-bound open-form is structurally less stable than ATP-bound open-form. These results indicate that GroEL has at least two distinct open-conformations in the presence of nucleotide; ATP-bound prehydrolysis open-form and ADP-bound open-form, and the ATP hydrolysis in open-form destabilizes its open-conformation and induces the 'from open to closed' conformational change of GroEL.

Adenosine Diphosphate↗