Deoxyribonucleic acid and chromatin imaging of endometriosis and endometrial carcinoma using atomic force microscopy

Abstract

Introduction: Clinical differential diagnosis of endometrial carcinoma is challenging, as signs and symptoms may vary considerably and there is a lack of reliable diagnostic serum biomarkers.

Aim of the study: The aim of our work was to characterize the deoxyribonucleic acid and chromatin changes in the tissue of patients confirmed to be suffering from endometriosis and endometrial adenocarcinoma and compare it with a healthy control group.

Material and methods: All samples were collected during recommended surgical interventions, and after the DNA isolation, a sonification followed by crosslink of chromatin were done. Consequently, both DNA and chromatin were examined using atomic force microscopy.

Results: A chromatin immunoprecipitation was used for the in vivo observation of conformational chromatin changes. The width of ssDNA showed a significant difference, almost double the control value in the endometrial cancer sample versus control (by 73?±?5% wider, p?<?0.001). In contrast, the height of ssDNA was highest in the frozen pelvis patient sample (by 510?±?12% compared to control, p?<?0.01).

Conclusion: Our results suggest that the horizontal size of single-stranded deoxyribonucleic acid and nucleosomes can help to identify potential patients with endometrial adenocarcinoma, while the height of the same parameter is associated with endometriosis.     

Understanding tapping-mode atomic force microscopy data on the surface of soft block copolymers

In this paper, we focus on better understanding tapping-mode atomic force microscopy (AFM) data of soft block copolymer materials with regard to: (1) phase attribution; (2) the relationship between topography and inside structure; (3) contrast-reversal artifacts; (4) the influence of annealing treatment on topography. The experiments were performed on the surface of poly(styrene–ethylene/butylene–styrene) (SEBS) triblock copolymer acting as a model system. First, by coupling AFM with transmission electron microscopy (TEM) measurements, the phase attribution for AFM images was determined. Secondly, by imaging an atomically flat SEBS surface as well as an AFM tip-scratched SEBS surface, it was confirmed that the contrast in AFM height images of soft block copolymers is not necessarily the result of surface topography but the result of lateral differences in tip-indentation depth between soft and hard microdomains. It was also found that there is an enlarging effect in AFM images on the domain size of block copolymers due to the tip-indention mechanism. Thirdly, based on the tip-indention mechanism, tentative explanations in some detail for the observed AFM artifacts (a reversal in phase image followed by another reversal in height image) as function of imaging parameters were given. Last, it was demonstrated that the commonly used annealing treatments in AFM sample preparation of block copolymers may in some cases lead to a dramatic topography change due to the unexpected order-to-order structure transition.     

Binding studies of costunolide and dehydrocostuslactone with HSA by spectroscopy and atomic force microscopy

Human serum albumin (HSA), a major plasma protein and plasma-derived therapeutic, interacts with a wide variety of drugs and native plasma metabolites. In this study the interactions of costunolide (CE) and dehydrocostuslactone (DE) with HSA were investigated by molecule modeling, atomic force microscopy (AFM), and different optical techniques. In the mechanism discussion, it was proved that fluorescence quenching of HSA by both of the drugs is a result of the formation of drug-HSA complexes. Binding parameters for the reactions were determined according to the Stern-Volmer equation and static quenching. The results of thermodynamic parameters ΔG⁰, ΔH⁰, and ΔS⁰ at different temperatures indicated that hydrogen bonding interactions play a major role in the drug-HSA associations process. The binding properties were further studied by quantitative analysis of CD, FTIR, and Raman spectra. Furthermore, AFM results showed that the dimension of HSA molecules became more swollen after binding with the drugs.     

Atomistic simulations of fluid structure and solvation forces in atomic force microscopy

We describe results of atomistic molecular dynamics simulations modelling an atomic force microscope (AFM) tip immersed in a fluid. Both the tip and the surface are modelled by rigid arrays of atoms. The tip is pyramidal and the surface is the (100) face of a fcc crystal. The focus is on the solvation forces acting on the tip and on the surface and their relation to the structural and dynamic properties of the fluid. Fluid particles in the neighborhood of the tip-surface junction are found to be highly ordered compared to the bulk, as shown by localized variations in the average fluid density. The atomistic nature of the model gives rise to several effects related to the discrete sizes of the fluid, tip, and surface particles which are not observed in continuum-based theories. A number of simulated force-distance curves are presented, along with an analysis of the effect of changing fluid particle size, tip (lateral) position, tip shape, and the lyocompatability of the tip and surface materials. The atomic-scale distribution of fluid-surface forces is examined for various positions of the tip, and the extent to which the fluid can act as a “cushion” by increasing the effective area of the tip-surface interaction is studied. The effect of a fluid on AFM imaging is investigated by generating “fluid images”, which are shown to be comparable in magnitude to the direct tip-surface interaction in the noncontact mode. We compare images generated by defective and defect-free surfaces, and analyse the fluid-tip forces acting in a lateral direction. An image formed from fluid forces acting in the direction of the surface normal does not show the presence of a vacancy, but an image formed from lateral fluid forces does.     

I-V curve oscillation observed by atomic force microscopy

Oscillation on the current-voltage curve measured by atomic force microscopy is observed when the distance between the tip and sample is large enough and beyond a critical value. The appearance of the oscillation is attributed to the excitation of electron standing waves between the tip and sample. From the first peak position and the voltage difference between the first two peaks on the current-voltage curve, the value of the work function at the detected point on silver film surface and the distance between the tip and the detected point can be calculated.     

Observation of multicellular spinning behavior of Proteus mirabilis by atomic force microscopy and multifunctional microscopy

This study aimed to observe the multicellular spinning behavior of Proteus mirabilis by atomic force microscopy (AFM) and multifunctional microscopy in order to understand the mechanism underlying this spinning movement and its biological significance. Multifunctional microscopy with charge-coupled device (CCD) and real-time AFM showed changes in cell structure and shape of P. mirabilis during multicellular spinning movement. Specifically, the morphological characteristics of P. mirabilis, multicellular spinning dynamics, and unique movement were observed. Our findings indicate that the multicellular spinning behavior of P. mirabilis may be used to collect nutrients, perform colonization, and squeeze out competitors. The movement characteristics of P. mirabilis are vital to the organism's biological adaptability to the surrounding environment.     

Possibility of measuring exchange force through force microscopy

This article describes the possibility of measuring exchange force through atomic force microscopy (AFM), based on the results of first-principles calculations for the exchange force between two magnetic Fe(001) films. We observed strong variation of the exchange force relative to the surface site. The magnitude of the force variation was larger than the force sensitivity of conventional AFM. These results suggest that a surface magnetic image with atomic resolution can be achieved by measuring the exchange force.     

Eutectic alloy microstructure: atomic force microscopy analysis

The exciting microstructures found in several eutectic alloys are a result of a cooperative growth, which is connected to the atomic transfer in the melt ahead the solid/liquid interface. In a eutectic system, the growth of solid phases depends on the simultaneous rejection of constituents to the liquid phase, which causes adjustments of the melt composition and hence, mass transport by diffusion normal to the growth direction. Generally, eutectic microstructures are examined by using optical (OM) and scanning electron microscopy (SEM). While OM may not provide the necessary resolution, the eutectic microstructure may present three-dimensional features, as a result of etching, which is not always possible to be observed by SEM. As an alternative, this paper describes the use of atomic force microscopy (AFM) in understanding micro-scale feature of a eutectic microstructure. For such a purpose, directionally solidified samples of a Ni–Al–V lamellar eutectic, a Ni–Al–Mo fibrous eutectic and a Ni–Al–Nb three-phase eutectic were examined. The results obtained provided a new picture of multi-phase microstructures and allows one to understand their new characteristics.     

Calculation of the optimal imaging parameters for frequency modulation atomic force microscopy

True atomic resolution of conductors and insulators is now routinely obtained in vacuum by frequency modulation atomic force microscopy. So far, the imaging parameters (i.e., eigenfrequency, stiffness and oscillation amplitude of the cantilever, frequency shift) which result in optimal spatial resolution for a given cantilever and sample have been found empirically. Here, we calculate the optimal set of parameters from first principles as a function of the tip–sample system. The result shows that the either the acquisition rate or the signal-to-noise ratio could be increased by up to two orders of magnitude by using stiffer cantilevers and smaller amplitudes than are in use today.     

Lateral force microscopy in low normal force limit

We have studied frictional force between SiN tip and Si surface by using lateral force microscopy. The cantilever we have used has very low stiffness of 0.006 N/m, and the normal force acting on the surface was much lower than the attractive force such as van der Waals force. In this low normal force limit, it was found that the frictional force did not depend on the normal force. We suggest a calibration method to estimate the attractive force from the lateral force data in this limit. The estimated attractive force between Si sample and SiN tip with radius of 10 nm was 0.4 nN in flat region and 0.65 nN at the corner of a rectangular hole.     

In situ atomic force microscopy studies of reversible light-induced switching of surface roughness and adhesion in azobenzene-containing PMMA films

Thin films in the range 40-80 nm of a blend of PMMA with an azobenzene derivative have been studied directly during UV and blue light irradiation by atomic force microscopy (AFM), revealing highly reversible changes in the surface roughness and the film adhesion. UV light induces an ≈80% increase in surface roughness, whereas illumination by blue light completely reverses these changes. Based on the observed surface topography and transition kinetics a reversible mass flow mechanisms is suggested, where the polarity changes upon switching trigger a wetting-dewetting transition in a surface segregation layer of the chromophore. Similar AFM measurements of the pull-off force indicate a decrease upon UV and an increase after blue light illumination with a complex kinetic behavior: a rapid initial change, attributed to the change in the cis isomer fraction of the azobenzene derivative, and a more gradual change, indicative of slow structural reorganization.     

New control methodology of microcantilevers in atomic force microscopy

We apply an external feedback control technique to vibrating microcantilevers in atomic force microscopy. Here we have no difficulty in getting information on periodic orbits required for application of the external feedback control unlike controlling chaos since stable orbits are used as reference ones. This approach enables us not only to control vibrations of the cantilevers but also to measure the sample surfaces (surface topographies) simultaneously. The efficiency and validity of our approach is demonstrated by numerical simulations and a theoretical analysis with the assistance of numerical computations.     

Quantitative imaging of electrospun fibers by PeakForce Quantitative NanoMechanics atomic force microscopy using etched scanning probes

Electrospun polymeric submicron and nanofibers can be used as tissue engineering scaffolds in regenerative medicine. In physiological conditions fibers are subjected to stresses and strains from the surrounding biological environment. Such stresses can cause permanent deformation or even failure to their structure. Therefore, there is a growing necessity to characterize their mechanical properties, especially at the nanoscale.Atomic force microscopy is a powerful tool for the visualization and probing of selected mechanical properties of materials in biomedical sciences. Image resolution of atomic force microscopy techniques depends on the equipment quality and shape of the scanning probe. The probe radius and aspect ratio has huge impact on the quality of measurement.In the presented work the nanomechanical properties of four different polymer based electrospun fibers were tested using PeakForce Quantitative NanoMechanics atomic force microscopy, with standard and modified scanning probes. Standard, commercially available probes have been modified by etching using focused ion beam (FIB). Results have shown that modified probes can be used for mechanical properties mapping of biomaterial in the nanoscale, and generate nanomechanical information where conventional tips fail.     

Morphology of synthetic DOPA-eumelanin deposited on glass and mica substrates: An atomic force microscopy investigation

Bright field microscopy and atomic force microscopy techniques are used to investigate morphological properties of synthetic eumelanin, obtained by oxidation of l-DOPA solution, deposited on glass and mica substrates. Deposits of eumelanin are characterized by aggregates with different shape and size. On a micrometric scale, filamentous as well as granular structures are present on glass and mica substrates, with a larger density on the former than on the latter. On a nanometric scale, filamentous aggregates, several microns long and about 100 nm wide and high, and granular aggregates, ∼50 nm high and 100 nm wide, are found on both substrates, whereas point-like deposits less than 10 nm high and less than 50 nm wide are found on mica substrate. Dynamic light scattering measurements and atomic force microscopy images support the evidence that eumelanin presents only nanometric point-like aggregates in aqueous solution, whereas such nanoaggregates organize themselves according to granular and filamentous structures when deposition occurs, as a consequence of interactions with the substrate surface.     

Interpretation of scanning tunneling microscopy and atomic force microscopy images of 1T-TiS2

The surface of 1T-TiS₂ was examined by scanning tunneling microscopy (STM) and atomic force microscopy (AFM). The STM and AFM images of this compound were interpreted on the basis of the partial electron density ρ(r,E_F) and total electron density ρ(r) of a slab which consists of six (001) 1T-TiS₂ layers. Electronic structure calculations were performed using the ab-initio Hartree–Fock program crystal. It was found that the bright spots in experimental STM images correspond to sulfur atoms at both positive and negative bias voltages. The AFM image showed a periodicity which can be explained by the atomic corrugation at the surface. Structural defects on the surface were also investigated, and their interpretation constitutes experimental proof that only sulfur atoms were detected by scanning probe microscopies.     

Investigating biomolecular recognition at the cell surface using atomic force microscopy

Probing the interaction forces that drive biomolecular recognition on cell surfaces is essential for understanding diverse biological processes. Force spectroscopy has been a widely used dynamic analytical technique, allowing measurement of such interactions at the molecular and cellular level. The capabilities of working under near physiological environments, combined with excellent force and lateral resolution make atomic force microscopy (AFM)-based force spectroscopy a powerful approach to measure biomolecular interaction forces not only on non-biological substrates, but also on soft, dynamic cell surfaces. Over the last few years, AFM-based force spectroscopy has provided biophysical insight into how biomolecules on cell surfaces interact with each other and induce relevant biological processes. In this review, we focus on describing the technique of force spectroscopy using the AFM, specifically in the context of probing cell surfaces. We summarize recent progress in understanding the recognition and interactions between macromolecules that may be found at cell surfaces from a force spectroscopy perspective. We further discuss the challenges and future prospects of the application of this versatile technique.     

Atomic force microscopy evidence for K domains on freshly cleaved mica

The unit cell height in the c-direction of muscovite mica is well established at 10 Å. However, we have observed steps much lower than this whilst imaging freshly cleaved mica surfaces in an atomic force microscope. The steps, 1.0±0.05 Å high, are unstable and disappear in a period of minutes after cleavage. We propose that they are due to the presence of domains of residual K⁺ ions that form two matching patchworks on the cleaved faces. Upon cleavage, they relax inwards from the bulk equilibrium position 1.6 Å above the oxygen atoms of the tetrahedral silicate. Possible mechanisms for the disappearance of the steps are discussed.     

Metrology of electromagnetic static actuation of MEMS microbridge using atomic force microscopy

The objective of this paper is to describe application of atomic force microscopy (AFM) for characterization and calibration of static deflection of electromagnetically and/or thermally actuated micro-electromechanical (MEMS) bridge. The investigated MEMS structure is formed by a silicon nitride bridge and a thin film metal path enabling electromagnetic and/or thermal deflection actuation. We present how static microbridge deflection can be measured using contact mode AFM technology with resolution of 0.05 nm in the range of up to tens of nm. We also analyze, for very small structure deflections and under defined and controlled load force varied in the range up to ca. 32 nN, properties of thermal and electromagnetical microbridge deflection actuation schemes.     

DNA adsorption and desorption on mica surface studied by atomic force microscopy

The adsorption of DNA molecules on mica surface and the following desorption of DNA molecules at ethanol-mica interface were studied using atomic force microscopy. By changing DNA concentration, different morphologies on mica surface have been observed. A very uniform and orderly monolayer of DNA molecules was constructed on the mica surface with a DNA concentration of 30 ng/μL. When the samples were immersed into ethanol for about 15 min, various desorption degree of DNA from mica (0-99%) was achieved. It was found that with the increase of DNA concentration, the desorption degree of DNA from the mica at ethanol-mica interface decreased. And when the uniform and orderly DNA monolayers were formed on the mica surface, almost no DNA molecule desorbed from the mica surface in this process. The results indicated that the uniform and orderly DNA monolayer is one of the most stable DNA structures formed on the mica surface. In addition, we have studied the structure change of DNA molecules after desorbed from the mica surface with atomic force microscopy, and found that the desorption might be ascribed to the ethanol-induced DNA condensation.     

Study on the interactions between anti-cancer drug oxaliplatin and DNA by atomic force microscopy

Oxaliplatin is one of the most important anticancer drugs at present. However, the mechanism of action of oxaliplatin is still controversial. In this study, the interactions between oxaliplatin and a plasmid DNA have been studied so as to reveal the structural basis of its activity. The structural characteristic of pUC19 DNA (2 ng/μL) incubated with 100 μmol/L and 1000 μmol/L of oxaliplatin for the different time on a freshly cleaved highly ordered pyrolytic graphite (HOPG) surface was investigated by atomic force microscopy (AFM). High resolution AFM observation indicated that oxaliplatin can induce pUC19 DNA molecules change from the extended conformation to the entangled structures with many nodes, and finally to the compact particles. The present AFM results provide structural evidence about the interactions between oxaliplatin and circular duplex DNA containing multiple targets.