Electron microscopy and atomic force microscopy studies of chromatin and metaphase chromosome structure

The folding of the chromatin filament and, in particular, the organization of genomic DNA within metaphase chromosomes has attracted the interest of many laboratories during the last five decades. This review discusses our current understanding of chromatin higher-order structure based on results obtained with transmission electron microscopy (TEM), cryo-electron microscopy (cryo-EM), and different atomic force microscopy (AFM) techniques.Chromatin isolated from different cell types in buffers without cations form extended filaments with nucleosomes visible as separated units. In presence of low concentrations of Mg²⁺, chromatin filaments are folded into fibers having a diameter of ∼30 nm. Highly compact fibers were obtained with isolated chromatin fragments in solutions containing 1–2 mM Mg²⁺. The high density of these fibers suggested that the successive turns of the chromatin filament are interdigitated. Similar results were obtained with reconstituted nucleosome arrays under the same ionic conditions. This led to the proposal of compact interdigitated solenoid models having a helical pitch of 4–5 nm. These findings, together with the observation of columns of stacked nucleosomes in different liquid crystal phases formed by aggregation of nucleosome core particles at high concentration, and different experimental evidences obtained using other approaches, indicate that face-to-face interactions between nucleosomes are very important for the formation of dense chromatin structures.Chromatin fibers were observed in metaphase chromosome preparations in deionized water and in buffers containing EDTA, but chromosomes in presence of the Mg²⁺ concentrations found in metaphase (5–22 mM) are very compact, without visible fibers. Moreover, a recent cryo-electron microscopy analysis of vitreous sections of mitotic cells indicated that chromatin has a disordered organization, which does not support the existence of 30-nm fibers in condensed chromosomes. TEM images of partially denatured chromosomes obtained using different procedures that maintain the ionic conditions of metaphase showed that bulk chromatin in chromosomes is organized forming multilayered plate-like structures. The structure and mechanical properties of these plates were studied using cryo-EM, electron tomography, AFM imaging in aqueous media, and AFM-based nanotribology and force spectroscopy. The results obtained indicated that the chromatin filament forms a flexible two-dimensional network, in which DNA is the main component responsible for the mechanical strength observed in friction force measurements. The discovery of this unexpected structure based on a planar geometry has opened completely new possibilities for the understanding of chromatin folding in metaphase chromosomes. It was proposed that chromatids are formed by many stacked thin chromatin plates oriented perpendicular to the chromatid axis. Different experimental evidences indicated that nucleosomes in the plates are irregularly oriented, and that the successive layers are interdigitated (the apparent layer thickness is 5–6 nm), allowing face-to-face interactions between nucleosomes of adjacent layers. The high density of this structure is in agreement with the high concentration of DNA observed in metaphase chromosomes of different species, and the irregular orientation of nucleosomes within the plates make these results compatible with those obtained with mitotic cell cryo-sections. The multilaminar chromatin structure proposed for chromosomes allows an easy explanation of chromosome banding and of the band splitting observed in stretched chromosomes.     

Statistical estimation of atomic positions from exit wave reconstruction with a precision in the picometer range

The local structure of Bi4W2/3Mn1/3O8Cl is determined using quantitative transmission electron microscopy. The electron exit wave, which is closely related to the projected crystal potential, is reconstructed and used as a starting point for statistical parameter estimation. This method allows us to refine all atomic positions on a local scale, including those of the light atoms, with a precision in the picometer range. Using this method one is no longer restricted to the information limit of the electron microscope. Our results are in good agreement with x-ray powder diffraction data demonstrating the reliability of the method. Moreover, it will be shown that local effects can be interpreted using this approach.     

Adsorption and manipulation of carbon onions on highly oriented pyrolytic graphite studied with atomic force microscopy

Carbon onions produced by DC arc discharge method were deposited on highly oriented pyrolytic graphite (HOPG) surface and their adsorption and manipulation was studied using an atomic force microscopy (AFM). Well-dispersed adsorption of carbon onions on HOPG surface was obtained and aggregations of onions were not observed. The van der Waals interaction between the onion and HOPG surface and that between two onions, were calculated and discussed using Hamaker's theory. The manipulation of adsorbed onions on HOPG surface was realized using the AFM in both the raster mode and the vector mode. The controllability and precision of two manipulation modes were compared and the vector mode manipulation was found superior, and is a useful technique for the construction of nano-scale devices based on carbon onions.     

Surface structure investigations using noncontact atomic force microscopy

Surfaces of several A_IIIB_V compound semiconductors (InSb, GaAs, InP, InAs) of the (0 0 1) orientation have been studied with noncontact atomic force microscopy (NC-AFM). Obtained atomically resolved patterns have been compared with structural models available in the literature. It is shown that NC-AFM is an efficient tool for imaging complex surface structures in real space. It is also demonstrated that the recent structural models of III-V compound surfaces provide a sound base for interpretation of majority of features present in recorded patterns. However, there are also many new findings revealed by the NC-AFM method that is still new experimental technique in the context of surface structure determination.     

A comparison study of scratch and wear properties using atomic force microscopy

The patterning technique that uses an AFM (atomic force microscopy) tip as a scratch tool, also known as AFM scratching, has been a vital technique for nanofabrication because of its low cost and potential to reach a resolution into the sub-nanometer domain. The AFM scratching technique was first used to study the scratch characteristics of silicon, with an emphasis on establishing its scratchability or the nanoscale machinability. The effects of the scratch parameters, including the applied tip force and number of scratches, on the size of the scratched geometry were specifically evaluated. The primary property that measures the scratchability was identified and assessed. To illustrate its suitability and reliability, the value of the scratchability, based on the present Si scratching experiments, was compared with the values based on the data available in the literatures for different scratching conditions or for materials other than Si. Since AFM scratching is in some aspects similar to the nanoscale wear test, the scratchability property identified is also compared with two major wear resistance indicators, wear coefficient and hardness. All comparison results indicate that the scratchability property identified, the scratch ratio, is an appropriate manufacturability indicator for measuring the degree of the ease or difficulty of a material scratched by an AFM tip and more suitable than the wear coefficient and hardness to gauge the nanoscale AFM scratchability.     

An atomic force microscopy investigation of cyanophage structure

Marine viruses have only relatively recently come to the attention of molecular biologists, and the extraordinary diversity of potential host organisms suggests a new wealth of genetic and structural forms. A promising technology for characterizing and describing the viruses structurally is atomic force microscopy (AFM). We provide examples here of some of the different architectures and novel structural features that emerge from even a very limited investigation, one focused on cyanophages, viruses that infect cyanobacteria (blue-green algae). These were isolated by phage selection of viruses collected from California coastal waters. We present AFM images of tailed, spherical, filamentous, rod shaped viruses, and others of eccentric form. Among the tailed phages numerous myoviruses were observed, some having long tail fibers, some other none, and some having no visible baseplate. Syphoviruses and a podovirus were also seen. We also describe a unique structural features found on some tailed marine phages that appear to have no terrestrial homolog. These are long, 450 nm, complex helical tail fibers terminating in a unique pattern of 3+1 globular units made up of about 20 small proteins.     

Electron microscopy study of Hispanic Terra Sigillata

Two Hispanic Terra Sigillata pottery samples from different workshops – Tricio and Andújar – have been characterized by means of electron microscopy and associated techniques and X-ray diffraction data. The combined information from transmission electron microscopy images, electron diffraction patterns and microchemical analysis has revealed the nature and distribution of the precipitates of the ceramic piece slip, which is a very important part in the characterization of these kind of ceramic wares. Both samples present homogeneously dispersed α-Fe_2-xAl_xO₃ (corundum-type structure) particles embedded in a glassy matrix of SiO₂-Al₂O₃. The Si : Al ratio of the matrix is different in each case, with a higher Al content in the Andújar ceramic sample. Crystallites of spinel – Mg(Al,Fe)₂O₄ – and Al_2-xFe_xO₃ are also detected in both cases. In addition, ilmenite phase (FeTiO₃) and TiO₂ (rutile-type) were observed less frequently. PACS  68.37.Lp; 79.20.Uv; 61.10.Nz     

Imaging the elastic properties of coiled carbon nanotubes with atomic force microscopy

Coiled carbon nanotubes were produced catalytically by thermal decomposition of hydrocarbon gas. After deposition on a silicon substrate, the three-dimensional structure of the helix-shaped multiwalled nanotubes can be visualized with atomic force microscopy. Helical structures of both chiralities are present in the nanotube deposits. For larger coil diameters ( >170 nm), force modulation microscopy allows one to probe the local elasticity along the length of the coil. Our results agree with the classical theory of elasticity. Similar to the case of straight nanotubes, the Young modulus of coiled multiwalled nanotubes remains comparable to the very high Young modulus of hexagonal graphene sheets.     

Thermomechanical properties of polymer nanolithography using atomic force microscopy

The temperature-dependent mechanical properties of polyethylene terephthalate (PET) polymers are investigated using force-distance curves, adhesion force, and atomic force microscope (AFM) nanolithography combined the heating techniques. The results show that the width of grooves on the polymers at 20-60 °C were in the range of 14-363 nm. The wear depth of the polymers increased with increasing heating temperature. A volume of 251.85-2422.66 μm(3) at a load of 30-50 nN with heating to 30-60 °C was removed, as compared to that of 26.60-70.30 μm(3) obtained at room temperature. The contact forces of PET started increasing at 9 nN, whereas the size of the holes was average at a pressure. The results may be of importance in explaining the heating relationship among adhesion force, volume removal rate, and pressure.     

Analysis of the structure of block copolymer films by atomic force microscopy

The structure of thin films of the polysterene-polymethylacrylate-polysterene triblock copolymer was studied. Universal algorithms to analyze atomic-force-microscopy images of thin block-copolymer films were developed.     

Peculiarities of the valence band of pyrolytic carbon

The results of experimental and theoretical studies of the valence band of pyrolytic carbon, heat-treated beforehand at different temperatures ranging from 2300 to 3000°C, are presented. Analysis of the results indicates that the defectiveness of the material affects the electronic states at the top of the valence band.Translated from Izvestiya Vysshikh Uchebnykh Zaveenii, Fizika, No. 6, pp. 81–85, June, 1986.     

Micro-track structure analysis for 100 MeV Si ions in CR-39 by using atomic force microscopy

To analyze the micro-track structure of heavy ions in a polymer material,parameters including bulk etch rate,track etch rate,etch rate ratio,and track core size were measured.The pieces of CR-39 were exposed to 100 MeV Si ions with normal incidence and were etched in 6.25N NaOH solution at 70 C.Bulk etch rate was read out by a profilemeter after several hours of etching.The other parameters were obtained by using an atomic force microscope(AFM)after a short time of etching.We have measured the second etch pits and minute etch pits to obtain the track growth curve and three dimension track structures to track the core size and etch rate measurements.The local dose of the track core was calculated by theδ-ray theory.In our study,we figure out that the bulk etch rate Vb=(1.58±0.022)μm/h,the track etch rate Vt=(2.90±0.529)μm/h,the etch rate ratio V=1.84±0.031,and the track core radii r≈4.65 nm.In the meantime,we find that the micro-track development violates the traditional track-growth model.For this reason,a scenario is carried out to provide an explanation.     

Reflectivity function reconstruction using the frequency deconvolution technique

Deconvolution of ultrasonic echo signals improves resolution and quality of ultrasonic images. A frequency deconvolution algorithm depends on the Fast Fourier transform is proposed for ultrasonic data. The stability of the algorithm and the influence of the truncation effect on the deconvoluted results were investigated with respect to the duration time of reflectivity function reconstruction and the signal to noise ratio. Reliability of the separation of reconstructing the reflectivity of a biological tissue is estimated by frequency deconvolution of the echo ultrasound signals.     

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.     

Measurement of nanomechanical properties of biomolecules using atomic force microscopy

The capabilities of atomic force microscopy (AFM) have been rapidly expanding beyond topographical imaging to now allow for the analysis of a wide range of properties of diverse materials. The technique of nanoindentation, traditionally performed via dedicated indenters can now be reliably achieved using AFM instrumentation, enabling mechanical property determination at the nanoscale using the high spatial and force resolutions of the AFM. In the study of biological systems, from biomolecules to complexes, this technique provides insight into how mesoscale properties and functions may arise from a myriad of single biomolecules. In vivo and in situ analyses of native structures under physiological conditions as well as the rapid analysis of molecular species under a variety of experimental treatments are made possible with this technique. As a result, AFM nanoindentation has emerged as a critical tool for the study of biological systems in their natural state, further contributing to both biomaterial design and pharmacological research. In this review, we detail the theory and progression of AFM-based nanoindentation, and present several applications of this technique as it has been used to probe biomolecules and biological nanostructures from single proteins to complex assemblies. We further detail the many challenges associated with mechanical models and required assumptions for model validity. AFM nanoindentation capabilities have provided an excellent improvement over conventional nanomechanical tools and by integration of topographical data from imaging, enabled the rapid extraction and presentation of mechanical data for biological samples.     

Determination of interface atomic structure and its impact on spin transport using Z-contrast microscopy and density-functional theory

We combine Z-contrast scanning transmission electron microscopy with density-functional-theory calculations to determine the atomic structure of the interface in spin-polarized light-emitting diodes. A 44% increase in spin-injection efficiency occurs after a low-temperature anneal, which produces an ordered, coherent interface consisting of a single atomic plane of alternating Fe and As atoms. First-principles transport calculations indicate that the increase in spin-injection efficiency is due to the abruptness and coherency of the annealed interface.     

Surface structure of fluorine-graphite intercalation compounds observed by atomic force microscopy

Atomic force microscopy (AFM) has been used to image fluorine-graphite intercalation compounds (C_3.5F; stage 1 + 2, C_3.9F; stage 2, (CF)_n; stage 1). For all samples the atomic resolution is achieved. The AFM image of the C_3.5F compound exhibits a new orthorhombic superlattice structure (a = 0.49 nm, b = 0.42 n, ∠a−b = 90°). In the AFM images of C_3.5F and (CF)_n the protrusions are attributed to fluorine atoms. The AFM image of C_3.9F exhibits a centered hexagonal structure similar to highly oriented pyrolytic graphite (HOPG).     

Determination of silicone coating Young's modulus using atomic force microscopy

The polymerisation degree of thin polymer coatings was checked by following the variation of their local mechanical properties. Atomic force microscope (AFM) was used in an indentation mode to investigate the mechanical characteristics of silicone coatings on polycarbonate substrates. The evolution of Young's modulus of the silicone coatings was determined as a function of the polymer annealing time. We have used a relative method to measure Young's moduli, which involves a calibration step with a set of reference polymers. No variation was observed for the modulus of silicone coatings annealed during more than 40 min at 130 °C. This result indicates that over-heating does not modify the mechanical properties of the coating.