Atomic force microscopy study of DNA deposited on poly l-ornithine-coated mica

Analyses of individual biomolecules, like DNA, or DNA–protein complexes, via atomic force microscopy, require ‘gentle’ methods to immobilize DNA on surfaces, which allow the ensemble of molecules to adopt conformations dictated primarily by their physical characteristics, and which possibly permit the use of a wide selection of buffers. We show that poly‐l ‐ornithine‐coated mica is a good substrate for fast, reliable deposition of DNA for wet or dry imaging. The surface firmly secures DNA, which retains the B‐form helical rise (0.34 nm bp^?1). The conformations of DNA that result are reminiscent of three‐dimensional random coils projected on to a plane. The contrast is good, especially in solution, and buffers with physiological concentrations of salt with or without divalent cations may be used. This is important for comparison of scanning probe microscopy results with those obtained by different techniques.     

A method of PSF generation for 3D brightfield deconvolution

This paper addresses the problem of 3D deconvolution of through focus widefield microscope datasets (Z‐stacks). One of the most difficult stages in brightfield deconvolution is finding the point spread function. A theoretically calculated point spread function (called a ‘synthetic PSF’ in this paper) requires foreknowledge of many system parameters and still gives only approximate results. A point spread function measured from a sub‐resolution bead suffers from low signal‐to‐noise ratio, compounded in the brightfield setting (by contrast to fluorescence) by absorptive, refractive and dispersal effects. This paper describes a method of point spread function estimation based on measurements of a Z‐stack through a thin sample. This Z‐stack is deconvolved by an idealized point spread function derived from the same Z‐stack to yield a point spread function of high signal‐to‐noise ratio that is also inherently tailored to the imaging system. The theory is validated by a practical experiment comparing the non‐blind 3D deconvolution of the yeast Saccharomyces cerevisiae with the point spread function generated using the method presented in this paper (called the ‘extracted PSF’) to a synthetic point spread function. Restoration of both high‐ and low‐contrast brightfield structures is achieved with fewer artefacts using the extracted point spread function obtained with this method. Furthermore the deconvolution progresses further (more iterations are allowed before the error function reaches its nadir) with the extracted point spread function compared to the synthetic point spread function indicating that the extracted point spread function is a better fit to the brightfield deconvolution model than the synthetic point spread function.     

Static and dynamic experiments in cryo-electron microscopy: comparative observations using high-vacuum, low-voltage and low-vacuum SEM

We carried out a unique comparative study between three modes of cryo‐scanning electron imaging: high‐vacuum, low‐voltage and low‐vacuum, using ice cream as a model system. Specimens were investigated both with and without a conductive coating (Au/Pd) and at temperatures for which ice either remains fully frozen (< ?110 °C) or undergoes sublimation (?110 to ?90 °C). At high magnification, high‐vacuum imaging of coated specimens gave the best results for ‘static’ specimens (i.e. containing fully frozen ice). Low voltages, such as 1 kV, could be used for imaging uncoated specimens at high vacuum, although slight ‘classical’ charging artefacts remained an issue, and the reduced electron beam penetration tended to decrease the definition between different microstructural features. However, this mode was useful for observing in situ sublimation from uncoated specimens. Low‐vacuum mode, involving small partial pressures of nitrogen gas, was particularly suited to in situ sublimation work: when sublimation was carried out in low vacuum in the absence of an anti‐contaminator plate, sublimation rates were significantly reduced. This is attributed to a small partial pressure of sublimated water vapour remaining near the specimen surface, enhancing thermodynamic stability.     

Exploring dynamic surface processes during silicate mineral (wollastonite) dissolution with liquid cell TEM

Most liquid cell transmission electron microscopy (LC TEM) studies focus on nanoparticles or nanowires, in large part because the preparation and study of materials in this size range is straightforward. By contrast, this is not true for samples in the micrometre size range, in large part because of the difficulties associated with sample preparation starting from a ‘bulk’ material. There are also many advantages inherent to the study of micrometre‐sized samples compared to their nanometre‐sized counterparts. Here, we present a liquid cell transmission electron study that employed an innovative sample preparation technique using focused ion beam (FIB) milling to fabricate micrometre‐sized electron transparent lamellae that were then welded to the liquid cell substrate. This technique, for which we have described in detail all of the fabrication steps, allows for samples having dimensions of several square micrometres to be observed by TEM in situ in a liquid. We applied this technique to test whether we could observe and measure in situ dissolution of a crystalline material called wollastonite, a calcium silicate mineral. More specifically, this study was used to observe and record surface dynamics associated with step and terrace edge movement, which are ultimately linked to the overall rate of dissolution. The wollastonite lamella underwent chemical reactions in pure deionized water at ambient temperature in a liquid cell with a 5‐m‐spacer thickness. The movement of surface steps and terraces was measured periodically over a period of almost 5 h. Quite unexpectedly, the one‐dimensional rates of retreat of these surface features were not constant, but changed over time. In addition, there were noticeable quantitative differences in retreat rates as a function crystallographic orientation, indicating that surface retreat is anisotropic. Several bulk rates of dissolution were also determined (1.6–4.2 ? 10^?7 mol m^?2 s^?1) using the rates of retreat of representative terraces and steps, and were found to be within one order of magnitude of dissolution rates in the literature based on aqueous chemistry data.     

Stereological measures of trabecular bone structure: comparison of 3D micro computed tomography with 2D histological sections in human proximal tibial bone biopsies

Stereology applied on histological sections is the ‘gold standard’ for obtaining quantitative information on cancellous bone structure. Recent advances in micro computed tomography (µCT) have made it possible to acquire three-dimensional (3D) data non-destructively. However, before the 3D methods can be used as a substitute for the current ‘gold standard’ they have to be verified against the existing standard. The aim of this study was to compare bone structural measures obtained from 3D µCT data sets with those obtained by stereology performed on conventional histological sections using human tibial bone biopsies. Furthermore, this study forms the first step in introducing the proximal tibia as a potential bone examination location by peripheral quantitative CT and CT. Twenty-nine trabecular bone biopsies were obtained from autopsy material at the medial side of the proximal tibial metaphysis. The biopsies were embedded in methylmetacrylate before µCT scanning in a Scanco µCT 40 scanner at a resolution of 20 × 20 × 20 µm³, and the 3D data sets were analysed with a computer program. After µCT scanning, 16 sections were cut from the central 2 mm of each biopsy and analysed with a computerized method. Trabecular bone volume (BV/TV) and connectivity density (CD) were estimated in both modalities, whereas trabecular bone pattern factor (TBPf) was estimated on the histological sections only. Trabecular thickness (Tb.Th), number (Tb.N) and separation (Tb.Sp), and structure model index (SMI) were estimated with the µCT method only. Excellent correlations were found between the two techniques for BV/TV (r = 0.95) and CD (r = 0.95). Additionally, an excellent relationship (r = 0.95) was ascertained between TBPf and SMI. The study revealed high correlations between measures of bone structure obtained from conventional 2D sections and 3D µCT data. This indicates that 3D µCT data sets can be used as a substitute for conventional histological sections for bone structural evaluations.     

Lamellar subcomponents of the cuticular cell membrane complex of mammalian keratin fibres show friction and hardness contrast by AFM

There is a substantial body of information indicating that 18‐methyleicosanoic acid (18‐MEA) is covalently linked to the outer surface of all mammalian keratin fibres and also forms the outer β‐layer of the cuticular cell membrane complex (CCMC) which separates the cuticle cells from each other. Low cohesive forces are expected between the lipid‐containing outer β‐layer and the δ‐layer of the CCMC, thus providing a weak point for cuticular delamination and presenting a fresh layer of 18‐MEA to the newly exposed surface. We have used lateral force microscopy and force modulation atomic force microscopy (AFM) to examine human hair fibres in which the non‐covalently linked fatty acids have been removed. Examination of the lateral force images of new cuticle surfaces revealed by the attrition of overlying cuticle layers showed three separate zones of clearly defined frictional contrast. These are thought to correspond with the δ‐layer, the proteinaceous epicuticle and outer β‐layers of the CCMC. The δ‐layer was found to have a thickness of 16 nm (SD = 1 nm, n = 25), comparable to the 18.0 nm thickness measured from transverse cross‐sections of fibres with transmission electron microscopy. Force modulation AFM showed that the outer β‐layer was softer than the epicuticle and the δ‐layer. The frictional contrast was removed following treatment with methanolic KOH (0.1 mol dm^?3) at 25 °C for 30 min, suggesting the hydrolysis of the thioester linkage and removal of 18‐MEA from the surface.     

Chemical and morphological changes in human dentin after Er:YAGlaser irradiation: EDS and SEM analysis

Sixty samples of human dentin were divided into six groups (n = 10) and were irradiated with Er:YAG laser at 100 mJ–19.9 J/cm², 150 mJ–29.8 J/cm², 100 mJ–35.3 J/cm², 150 mJ–53.0 J/cm², 200 mJ–70.7 J/cm², and 250 mJ–88.5 J/cm², respectively, at 7 Hz under a water spray. The atomic percentages of carbon, oxygen, magnesium, calcium, and phosphorus and the Ca‐to‐P molar ratio on the dentin were determined by energy dispersive X‐ray spectroscopy. The morphological changes were observed using scanning electron microscopy. A paired t‐test was used in statistical analysis before and after irradiation, and a one‐way ANOVA was performed (P ≤ 0.05). The atomic percent of C tended to decrease in all of the groups after irradiation with statistically significant differences, O and Mg increased with significant differences in all of the groups, and the Ca‐to‐P molar ratio increased in groups IV, V, and VI, with statistically significant differences between groups II and VI. All the irradiated samples showed morphological changes. Major changes in the chemical composition of dentin were observed in trace elements. A significant increase in the Ca‐to‐P ratio was observed in the higher energy density groups. Morphological changes included loss of smear layer with exposed dentinal tubules. The changes produced by the different energy densities employed could have clinical implications, additional studies are required to clarify them. Microsc. Res. Tech. 78:1019–1025, 2015. © 2015 Wiley Periodicals, Inc.     

Long polymeric tips of atomic force microscopy for large biological samples

We show a new atomic force microscopy technique for obtaining high‐resolution topographic images of large bio‐samples. To obtain high‐resolution topographic images for the samples, we fabricated a long polymeric tip with a small protrusion using two‐photon adsorbed photo‐polymerization techniques. The obtained tip length was over 50 µm, and the tip was used directly to visualize COS‐1 and 293 cells. Compared with commercial tips, the long tip made it easier to obtain topographic images of the large cells. In the magnified topographic images, the sub‐100‐nm resolution was confirmed with the long tips. This long probe tip is expected to broaden large sample‐related studies and applications in the future.     

Chromosome morphology after long-term storage investigated by scanning near-field optical microscopy

Fluorescence in situ hybridization coupled with far‐field fluorescence microscopy is a commonly used technique to visualize chromosomal aberrations in diseased cells. To obtain the best possible results, chromatin integrity must be preserved to ensure optimal hybridization of fluorescence in situ hybridization probes. However, biological samples are known to degrade and storage conditions can be critical. This study concentrates its investigation on chromatin stability as a function of time following fluorescence in situ hybridization type denaturing protocols. This issue is extremely important because chromatin integrity affects the fluorescence response of the chromosome. To investigate this, metaphase chromosome spreads of human lymphocytes were stored at both ?20 and ?80 °C, and were then imaged using scanning near‐field optical microscopy over a nine month period. Using the scanning near‐field optical microscope's topography mode, chromosome morphology was analysed before and after the application of fluorescence in situ hybridization type protocols, and then as a function of storage time. The findings revealed that human chromosome samples can be stored at ?20 °C for short periods of time (～ several weeks), but storage over 3 months compromises chromatin stability. Topography measurements clearly show the collapse of the stored chromatin, with variations as large as 60 nm across a chromosome. However, storage at ?80 °C considerably preserved the integrity with variations in topography significantly reduced. We report studies of the fluorescent response of stored chromosomes using scanning near‐field optical microscopy and their importance for gaining further understanding of chromosomal aberrations.     

Improving the quality of electron backscatter diffraction (EBSD) patterns from nanoparticles

In this study, we investigated the relative contributions of atomic number (Z) and density (ρ) to the degradation of the electron backscatter diffraction (EBSD) pattern quality for nanoparticles < 500 nm in diameter. This was accomplished by minimizing the diffuse scattering from the conventional thick mounting substrate through the design of a sample holder that can accommodate particles mounted on thin‐film TEM substrates. With this design, the contributions of incoherently scattered electrons that result in the diffuse background are minimized. Qualitative and quantitative comparisons were made of the EBSD pattern quality obtained from Al₂O₃ particles approximately 200 nm in diameter mounted on both thick‐ and thin‐film C substrates. For the quantitative comparison we developed a ‘quality’ factor for EBSD patterns that is based on the ratio of two Hough transforms derived from a given EBSD pattern image. The calculated quality factor is directly proportional to the signal‐to‐noise ratio for the EBSD pattern. In addition to the comparison of the thick and thin mounting substrates, we also estimated the effects of Z and ρ by comparing the EBSD pattern quality from the Al₂O₃ particles mounted on thin‐film substrates with the quality of patterns obtained from Fe–Co nanoparticles approximately 120 nm in diameter. The results indicate that the increased background generated in EBSD patterns by the electrons escaping through the bottom of the small particles is the dominant reason for the poor EBSD pattern quality from nanoparticles < 500 nm in size. This was supported by the fact that we were able to obtain usable EBSD patterns from Al₂O₃ particles as small as 130 nm using the thin‐film mounting method.     

Fluorescence relaxation in 3D from diffraction-limited sources of PAGFP or sinks of EGFP created by multiphoton photoconversion

The relaxation of fluorescence from diffraction‐limited sources of photoactivatable green fluorescent protein (PAGFP) or sinks of photobleached enhanced GFP (EGFP) created by multiphoton photo‐conversion was measured in solutions of varied viscosity (η), and in live, spherical Chinese hamster ovary (CHO) cells. Fluorescence relaxation was monitored with the probing laser fixed, or rapidly scanning along a line bisected by the photoconversion site. Novel solutions to several problems that hamper the study of PAGFP diffusion after multiphoton photoconversion are presented. A theoretical model of 3D diffusion in a sphere from a source in the shape of the measured multiphoton point‐spread function was applied to the fluorescence data to estimate the apparent diffusion coefficient, D_ap. The model incorporates two novel features that make it of broad utility. First, the model includes the no‐flux boundary condition imposed by cell plasma membranes, allowing assessment of potential impact of this boundary on estimates of D_ap. Second, the model uses an inhomogeneous source term that, for the first time, allows analysis of diffusion from sources produced by multiphoton photoconversion pulses of varying duration. For diffusion in aqueous solution, indistinguishable linear relationships between D_ap and η⁻¹ were obtained for the two proteins: for PAGFP, D_aq= 89 ± 2.4 μm² s⁻¹ (mean ± 95% confidence interval), and for EGFP D_aq= 91 ± 1.8 μm² s⁻¹. In CHO cells, the application of the model yielded D_ap= 20 ± 3 μm² s⁻¹ (PAGFP) and 19 ± 2 μm² s⁻¹ (EGFP). Furthermore, the model quantitatively predicted the decline in baseline fluorescence that accompanied repeated photobleaching cycles in CHO cells expressing EGFP, supporting the hypothesis of fluorophore depletion as an alternative to the oft invoked ‘bound fraction’ explanation of the deviation of the terminal fluorescence recovery from its pre‐bleach baseline level. Nonetheless for their identical diffusive properties, advantages of PAGFP over EGFP were found, including an intrinsically higher signal/noise ratio with 488‐nm excitation, and the requirement for ∼1/200th the cumulative light energy to produce data of comparable signal/noise.     

Advances and problems with TEM characterization of Cr/CrN multilayer coatings

Multilayer Cr/CrN/Cr/Cr(N,C) and Cr/CrN with 8 and 32 layer coatings were deposited on austenite substrates using pulsed laser deposition (PLD) technique. The microstructure observations were performed using Philips CM20?, TECNAI G² F20 – TWIN? and JEOL EX4000? transmission microscopes. The performed experiments indicated that lowering the argon flow from 60 to 30 cm³/s during chromium ablation changes buffer layers microstructure from nearly amorphous to nano‐crystalline. The nitride or carbo‐nitride layers turned out to be less sensitive to changes in nitrogen flow during deposition. The columnar microstructure of Cr layers is coarser than those in CrN ones under the same deposition condition. This observation proved also that relying on PLD technique as thin as 30 nm layers might be formed within multilayer Cr/CrN coatings.     

Bitumen morphologies by phase-detection atomic force microscopy

Bitumen is a complex mixture of hydrocarbons for which microstructural knowledge is incomplete. In an effort to detail this microstructure, 13 bitumens were analysed by phase‐detection atomic force microscopy. Based on morphology, the bitumens could be classified into three distinct groups. One group showed fine domains down to 0.1 µm, another showed domains of about 1 µm, and a third group showed up to four different domains or phases of different sizes and shapes. No correlation was found between the atomic force microscopy morphology and the composition based on asphaltenes, polar aromatics, naphthene aromatics and saturates. A high correlation was found between the area of the ‘bee‐like’ structures and the vanadium and nickel content in bitumen, and between the atomic force microscopy groups and the average size of molecular planes made of fused aromatics. The morphology and the molecular arrangements in bitumen thus appear to be partly governed by the molecular planes and the polarity defined by metallic cations.     

Direct atomic force microscopy observations of monovalent ion induced binding of DNA to mica

Multivalent ions in solution are known to mediate attraction between two like‐charged molecules. Such attraction has proved useful in atomic force microscopy (AFM) where DNA may be immobilized to a mica surface facilitating direct imaging in liquid. Theories of DNA immobilization suggest that either ‘salt bridging’ or fluctuation in the positions of counter ions about both the mica surface and DNA backbone secure DNA to the mica substrate. Whilst both theoretical and experimental evidence suggest that immobilization is possible in the presence of divalent ions, very few studies identify that such immobilization is possible with monovalent ions. Here we present direct AFM evidence of DNA immobilized to mica in the presence of only monovalent ions. Our data depict E. coli plasmid pBR322 adsorbed onto the negatively charged mica both after short (10 min) and long (24 h) incubation periods. These data suggest the need to re‐explore current theories of like‐charge attraction to include the possibility of monovalent interactions. We suggest that this DNA immobilization strategy may offer the potential to image natural processes with limited immobilization forces and hence enable maximum conformational freedom of the immobilized biomolecule.     

Comparison of I5M and 4Pi-microscopy

The axial (z‐) resolution of ～100 nm provided by 4Pi and I⁵M fluorescence microscopy relies on the coherent addition of spherical wavefronts of two opposing high aperture angle lenses. Both microscopes feature a point‐spread function (PSF) with a sharp central spot that is accompanied by axially shifted sidelobes which leads to replication artefacts in the raw image data. In a 4Pi‐microscope the sidelobes are less pronounced than in I⁵M and without relevant lateral (x,y) substructure, making their posterior removal in the image reliable and fast. On the other hand, high speeds of raw data acquisition are more easily gained by I⁵M. Moreover, I⁵M features a stronger signal as compared to the commonly employed two‐photon excitation (2PE) 4Pi‐imaging mode. We investigate here the capability of both techniques to image (aqueous) specimens without artefacts. To this end, we consider the optical transfer function (OTF) of the two microscopes in conjunction with the signal‐to‐noise‐ratio (SNR) of the object to be imaged. The imaging of E. coli bacteria with an interconvertable setup enabled a direct comparison of the two imaging modes. As both systems rely on high aperture angles, water‐immersion lenses of the largest numerical aperture available (NA = 1.2) were employed. The experimental results are corroborated by simulations assuming the signal strength encountered in the experiment. The comparison of the theoretical with the experimental PSFs/OTFs showed that our setup operated close to theory in both imaging modes. Although I⁵M provided about 10 times brighter raw image data as compared to (2PE) 4Pi‐microscopy, the I⁵M data could not be entirely cleared of artefacts. In conclusion, with the current aperture angles and fluorescence signal strengths, it is not advisable to trade in the suppression of the sidelobes for a larger image signal.     

An EELS-based study of the effects of pyrolysis on natural carbonaceous materials used for activated charcoal preparation

Electron energy‐loss spectroscopy (EELS) has been used to characterize the electronic structure of charcoal phases at the nanoscale, thus demonstrating that the technique can be applied to environmental science. Activated charcoal is extensively used to remove pollutants from liquid and gaseous sewage. It is mainly obtained by activation of coke or charcoal produced from ligneous precursors. The present study concerns the use of by‐products of local Caribbean agriculture, such as sugar cane bagasse, fruit stones and seeds, for use as activated charcoal precursors. Charcoal phases are prepared by high‐temperature pyrolysis of lignocellulosic raw materials under a nitrogen gas flow. With the aim of optimizing the pyrolysis temperature and duration and oxygen content, the concentration of carbon sp² hybridized chemical bonds and structural ordering have been followed by EELS for different treatment temperatures. To quantify the carbon sp² content, near edge structure (NES) at the carbon K edge has been measured to determine the strength of π → π* and 1s → π* transitions. Three precursors of plant origin, shells of Terminalia catappa and Acrocomia karukerana and seeds of Psidium guajava, with the pyrolysis temperatures between 600 and 900 °C, were investigated. The fraction of carbon sp² bonding is found to increase when the temperature rises from 600 °C to the range 700–750 °C and becomes stable at higher temperatures. For temperatures in excess of 700 °C, structural ordering probably occurs and well‐defined 1s → σ* NES is present, whose intensity increases with increasing preparation temperature. For the highest temperature of around 900 °C, the structure of the final product is less well organized than graphitized carbon but a few per cent of a highly ordered phase is found.     

New variance expressions for systematic sampling: the filtering approach

We present a collection of variance models for estimators obtained by geometric systematic sampling with test points, quadrats, and n‐boxes in general, on a bounded domain in n‐dimensional Euclidean space ?ⁿ, n = 1, 2, ... , and for systematic rays and sectors on the circle. The approach adopted ? termed the filtering approach ? is new and different from the current transitive approach. This report is only preliminary, however, because it includes only variance models in terms of the covariogram of the measurement function. The estimation step is in preparation.     

Nanoscale characterization of gold nanoparticles created by in situ reduction at a polymeric surface

Transmission Electron Microscopy is used as a quantitative method to measure the shapes, sizes and volumes of gold nanoparticles created at a polymeric surface by three different in situ synthesis methods. The atomic number contrast (Z‐contrast) imaging technique reveals nanoparticles which are formed on the surface of the polymer. However, with certain reducing agents, the gold nanoparticles are additionally found up to 20 nm below the polymer surface. In addition, plan‐view high‐angle annular dark‐field scanning transmission electron microscopy images were statistically analyzed on one sample to measure the volume, height and effective diameter of the gold nanoparticles and their size distributions. Depth analysis from high‐angle annular dark‐field scanning transmission electron microscopy micrographs also gives information on the dominant shape of the nanoparticles.     

Accurate measure of laser irradiance threshold for near-infrared photo-oxidation with a modified confocal microscope

Femtosecond mode‐locked lasers are now being used routinely in multiphoton fluorescence and autofluorescence spectroscopy, are just beginning to be used in refractive surgery, and may be used in the future diagnosis of skin cancer. Pulses from these lasers induce non‐linear effects in resultant tissue interactions. Using a modified confocal microscope with dispersion compensation and accurate measurements of beam diameter, a very low threshold was measured for photochemical oxidation in cultured cells. The measured threshold showed non‐linear photo‐oxidation at a peak irradiance and photon‐flux density of 8.4 × 10⁸ W cm^?2 and 3.4 × 10²⁷ photons cm^?2 s^?1, respectively (90‐fs pulse). The impact of these findings is significant to those using ultrashort lasers because they provide a tangible reference point (microscope‐independent) for the generation of photo‐oxidative stress in laser‐exposed tissues, and because they highlight the importance of dispersion compensation in minimizing collateral tissue damage.     

Is fast mapping good mapping? A review of the benefits of high‐speed orientation mapping using electron backscatter diffraction

Orientation mapping using automated electron backscatter diffraction (EBSD) is now a common technique for characterizing microstructures. Improvements in software and hardware have resulted in high‐speed mapping capabilities above 80 000 points h^?1. For ‘routine’ microstructural analyses of materials such as steel and aluminium (e.g. texture and grain size measurements and high angle boundary characterization), high‐speed orientation mapping is an ideal approach with minimal penalty on the final statistics. However, for the accurate analysis of very low angle boundaries and for routine analyses of more difficult materials (e.g. most rock samples), we advocate a more patient approach to orientation mapping with an emphasis on data accuracy and reliability. It is important that the objectives of any EBSD analysis are carefully considered before starting – in this way the maximum potential of an EBSD system can be achieved.