A computational procedure for the analysis of electron images of nucleic acid molecules

A new procedure is described for analyzing electron images of labeled nucleic acid molecules. The method makes use of one-dimensional digital image information and determines the best relative orientation of the linear molecules. The analysis is performed with the computer program CLUSTER, which combines the information from each molecule stepwise in an iterative procedure, so that finally a label distribution is obtained, which is the combination of all information available. The rationale behind the analysis is the calculation of similarity coefficients, which are a measure of the probability for the relative orientation of each molecule pair. The method has been thoroughly tested and compared with other procedures described in the literature in order to indicate its performance and power. A biological application concerning the distribution of the protein RNA-polymerase on DNA of phage Mu is presented.     

Scanning (atomic) force microscopy imaging of earthworm haemoglobin calibrated with spherical colloidal gold particles

Scanning (atomic) force microscopy (SFM) permits high-resolution imaging of a biological specimen in physiological solutions. Untreated extracellular haemoglobin molecules of the common North American earthworm, Lumbricus terrestris, were imaged in NH₄Ac solution using calibrated SFM. Individual molecules and their top and side views were clearly identified and were comparable with the images of the same molecule obtained by scanning transmission electron microscopy (STEM). A central depression, the presumed mouth of the hole, was detected. We analysed 75 individual molecules for their lateral dimensions. Compression varied for different molecules, presumably because of the variation of the interaction between the SFM tip and the protein molecule. Two effective heights which correspond to the heights of the points of the haemoglobin molecules first and last touched by the tip, h₁ and h₂, respectively, were measured for each protein and ranged between 1.58 and 16.2 nm for h₁ and 1.23 and 13.6 nm for h₂. The apparent diameter was measured and ranged from 44.9 to 86.6 nm (63.2±10.5 nm, n =75), which is about twice the diameter of the molecule reported by STEM for the top view orientation. The higher the measured effective heights, the worse was the tip convolution effect. In order to determine the tip parameters (semivertical angle, curvature of radius and the cut-off height) and to calibrate images of earthworm haemoglobin molecules, spherical gold particles were scanned as standards. The tip sectional radii at distances of h₁ and h₂ above the tip apex were subtracted from the apparent diameter of the protein. The calibrated lateral dimension was 29.1 ±3.85 nm, which is close to the reported scanning transmission electron microscopy data 30.0 ±0.8 nm. The results presented here demonstrate that the calibration approach of imaging gold particles is practical and relatively accurate. Calibrated SFM imaging can be applied to the study of other biomacromolecules.     

Electron microscope images of mercury atoms bound to DNA filament.

Ultra-fine grids with holes of a few hundred angstroms in diameter were made from tropomyosin for specimen supporting media. DNA was mounted on the grid. Microscope images were obtained demonstrating one double-stranded DNA filament bridging a hole. Using mercurated DNA a few filamentous images were observed in a bridge, and series of faint fine sports were recognized along an edge of a hole. These spots were interpreted as the images of single mercury atoms bound to the filament. The control experiment was made on unstained DNA, some images of which showed only uniform intensity over the bridge, whereas others were disturbed to some extent by noise presumably due to extraneous molecules coating the filament.     

Effects of tip-sample forces and humidity on the imaging of DNA with a scanning force microscope

We have investigated the effects of tip-sample forces and relative humidity when using a scanning force microscope (SFM) to image DNA molecules adsorbed on fresh mica. As the force between the tip and the sample increases, the apparent height of the DNA molecules decreases. After being imaged with high forces, the DNA molecules recover partially in their apparent height, indicating that a plastic deformation of the DNA has been induced by the scanning tip. At low humidities, DNA molecules can be imaged with a force up to 150 nN during the scanning without obvious damages. At higher humidities, however, the DNA molecules can be dissected or swept away by the tip even at a tip-sample force of 30 nN. The net force between the tip and the molecules is the vector sum of several forces, the dominant components of which are the elastic force due to the cantilever bending and the capillary force resulting from the water meniscus formed between the tip and the sample surface. When the relative humidity of the imaging environment is increased, the capillary force becomes stronger.     

Signal to noise enhancement in dark field electron micrographs of vasopressin: filtering of arrays of images in reciprocal space

A technique for filtering arrays of images of dispersed molecules is presented which takes advantage of the crystallographic properties of regular arrays and the rapidity of optical treatment. A filter in reciprocal space consisting of perforations in a square or rectangular lattice, as determined by the image arrangement, reduces noise and averages the images simultaneously by transmitting only regularly recurring image structure. The filter is universal for all image contents and introduces no additional biases. The signal to noise improvement approaches √N for N independent images of the same molecule in the same configuration. The procedure, which is simple, rapid and inexpensive, is demonstrated with the aid of dark field electron micrographs of the protein vasopressin and combined with iodination of that molecule to elucidate its structure.     

Circular harmonic averaging of noisy single-molecule images

A new method for averaging of noisy single-molecule images has been developed. Averaging is done over the circular harmonic components of the images instead of over the images themselves. Those images which show different molecules or molecules in different projections can be separated into classes by comparing the power of their circular harmonic components, which is rotationally invariant. Orientational alignment, based on the rotational correlation of each image with all others, and averaging is performed separately for each class. Applying a quality measure to the circular harmonic averages allows separation of significant and nonsignificant components. Computer simulations indicate that the method is quite stable against noise. One of the main advantages of "circular harmonic averaging" over existing methods is that no reference images are needed in the averaging procedure.     

一维矩形光栅AFM图像盲探针表面重建模拟研究

探针结构参数的合理选取将直接决定扫描图像及其盲探针修正图像的失真程度。基于此,以一维矩形模拟光栅为典型案例,对该模拟光栅的原子力显微镜(AFM)扫描成像过程与盲探针修正过程进行了仿真,阐明了探针结构参数对扫描成像过程与盲探针修正过程的影响规律。通过建立线宽变化度与半高宽相结合的图像重建误差评价指标,确定了针对该模拟光栅的AFM探针建议结构参数,并取得了良好的光栅图像重建效果。研究表明,应用线宽变化度结合半高宽来综合评价光栅的AFM测量和图像重建过程,有利于提升实际光栅AFM图像盲探针重建的准确度。     

Theoretical analysis of effect of tip atomic species on apparent barrier height on Al(100) surface

We have analyzed the effect of the tip atomic species on the tip-sample separation and the bias-voltage dependence of apparent barrier height (ABH) on an Al(100) surface using the boundary-matching scattering-state density functional method, which can be used to calculate electron states under applied bias voltages self-consistently within the density functional theory. We found that, from the dependence of the tip-sample separation, the difference between measurements with the two tip atomic species is larger in the ABH than in the maximum barrier height evaluated from the calculated potential profile. Furthermore, we found that the bias-polarity dependence of the ABH measured with the Na tip shows behavior opposite to that shown by the ABH measured with the Al tip. These results can be understood from the difference in the degree of lateral confinement of tunneling electrons in the tunneling barrier region between the two atomic species.     

A new approach to the interpretation of atom probe field-ion microscopy images.

The field distribution and the ion trajectories close to the tip surface are known to mainly control the contrast of field-ion microscopy and the resolution of the three-dimensional atom probe. The proper interpretation of images provided by these techniques requires the electric field and the ion trajectories to be determined accurately. A model has been developed in order to compute the ion trajectories close to a curved emitting surface modelled at the atomic scale. In this model, both the gradual change of the tip surface and the chemical nature of atoms were taken into account. Predictions and results given by this approach are shown to be in excellent agreement with experiments. The calculated electric field at the tip surface is consistent with field-ion microscopy contrasts. The preferential retention of surface atoms and the order of evaporation were correctly simulated. The ion trajectories were successfully described. In this way, the crucial problem of trajectory overlap and local magnification could be investigated. These simulations not only lead to a new understanding of the physical basis of image formation, but also have a predictive value.     

Magnetic refinement of tips for magnetic force microscopy

Silicon cantilever probes with monolithically integrated tips are commercially available and are routinely used for atomic force microscopy (AFM). For such probes, amagnetic refinement of the silicon tip has been developed and results in a deposition of ferromagnetic material such as nickel or CrCoTa in the top area of the tip. The method consists of essentially three steps: (1) A broad-area sputter deposition of a ferromagnetic material; (2) a selective electron beam-induced carbon deposition at the top of the tip; (3) a broad-area ion-beam sputter etching, which removes the magnetic layer everywhere except underneath the carbon cap. The method allows to control the total amount and extension of the magnetic material left at the tip. It is applicable to all kinds of ferromagnetic materials which can be deposited as a thin layer by sputter deposition or evaporation. Experiments indicate that the method is reliable and improves the resolution of magnetic force microscopy (MFM). With such magnetically refined tips on silicon cantilevers, MFM measurements have been performed in contact mode as well as in dynamic and static noncontact modes. In this paper, the method for magnetic tip refinement is described and MFM measurements with these tips are presented.     

Shear force imaging of DNA in a near-field scanning optical microscope (NSOM)

A near-field scanning optical module has been constructed as an accessory for a Nanoscope IIIa commercial scanning probe microscope. Distance feedback and topographic registration are accomplished with an uncoated optical fibre scanning tip by implementation of the shear force technique. The tip is driven by a piezoelectric actuator at a resonance frequency of 8–80 kHz. A laser diode beam is scattered by the tip and detected by a split photodiode, with lock-in detection of the difference signal. The amplitude ( r ) and phase (τ) responses were characterized as a function of the calibrated tip–sample separation. Using an r cos τ feedback signal, imaging of pUC18 relaxed circular plasmid DNA spread on mica precoated with cetylpyridinium chloride was achieved. The apparent width (28 ± 5 nm) was approximately four times that achieved by scanning force measurements with the same instrument; the apparent height of the DNA (0.6 ± 0.3 nm) was similar with the two techniques. These results demonstrate the applicability of the shear force signal for imaging biological macromolecules according to topography and in conjunction with the optical signals of a near-field scanning optical microscope (NSOM).     

Characterization of AC mode scanning ion-conductance microscopy.

A scanning ion-conductance microscope (SICM) with a vibrating probe has been recently developed (vSICM). In this system, the amplitude of the AC ionic current is detected by using a lock-in amplifier locked to the vibration frequency of the probe. Such a scheme allows for a better control of the tip position because the AC ionic current is more sensitive to the probe-surface distance than the DC ionic current used previously. In this paper, we demonstrate the utility of this technique to the imaging of topographically rough specimens and high-resolution imaging over selected small areas. We also show that it is possible to record the DC ionic current simultaneously during the scan, which can reveal additional information not apparent in the images obtained with the AC ionic current.     

Use of multivariate statistics in analysing the images of biological macromolecules

We have developed a new technique of analysis that allows automatic classification of molecule images according to subtle differences. Computer alignment and multivariate statistical methods were used to analyze electron micrographic images of horseshoe crab hemocyanin half-molecules. The molecule projections fell into four distinct classes related to four different positions of the molecule on the grid. Averages obtained for each images subset are interpreted in terms of a three-dimensional model arrangement for the four subunits forming the half-molecule.     

Automatic selection of molecular images from dark field electron micrographs

A method is described which can be used objectively to select putative molecular images from dark field electron micrographs of unstained molecules. The only characteristic of the molecule required for automatic selection is an estimate of molecular weight. Structures are selected from micrographs by a series of steps including: low pass filtering, edge detection and mass determination. The procedure is shown to be reliable for images with signal-to-noise ratios of at least 4.0. Moreover, the method is insensitive to both the shape and the number of molecules in the image. Five different molecules with molecular weights between MW 330,000 and MW 4000 are successfully selected from low dose STEM and high dose tilt beam dark field electron micrographs.     

Feasibility of multi-walled carbon nanotube probes in AFM anodization lithography

Multi-walled carbon nanotube (CNT) tips were used in atomic force microscope (AFM) anodization lithography to investigate their advantages over conventional tips. The CNT tip required a larger threshold voltage than the mother silicon tip due to the Schottky barrier at the CNT-Si interface. Current-to-voltage curves distinguished the junction property between CNTs and mother tips. The CNT-platinum tip, which is more conductive than the CNT-silicon tip, showed promising results for AFM anodization lithography. Finally, the nanostructures with high aspect ratio were fabricated using a pulsed bias voltage technique as well as the CNT tip.     

Effects of hot electron emission on a low-conductivity tetracyanoethylene polymer layer including studies of the corrugation of the film surface

The effect of strong field electron emission (FEE) on a tetracyanoethylene (TCNE) polymer layer was studied by Field Ion Microscopy (FIM) using TCNE and Ne as the imaging gases. The TCNE polymer was formed on each tungsten tip by radical polymerisation before FEE. The FIM images show field emission spots all over the surface of the tip. The FEM images show a random distribution of several field emission areas at the onset of FEE. After sometime at a current of about microA, there is a transition to higher currents at the same voltage, in which the electron emission pattern changes to have only one emitting area. After this transition, two different types of FIM images were observed, depending on the imaging gas that was used. Neon FIM images at low tip voltages show spots in the areas where the electron emission current was greatest, and at much higher voltages these images show emission from other areas with lower surface corrugation. However, the FIM images with TCNE as the imaging gas do not show any differences between the areas with and without electron emission. The FIM images remain as before FEE, which can be explained by the formation of a new polymer by the reaction of the surface layer with the imaging gas. It is assumed that chemically reactive fragments at the polymer/vacuum interface, which are needed for the polymerisation reaction, are formed by pyrolysis and sputtering processes during FEE.     

Algorithm of fatigue crack detection and determination of its tip position in optical images

An algorithm of fatigue crack detection in optical images taken in fatigue tests of materials is proposed and tested. The algorithm is designed for automation of measurements of the crack propagation parameter and tracing the crack tip position in the course of cyclic loading for the purpose of shifting the optical system with respect to the examined sample surface to the “region of interest.” It is found that the coordinates of the image fragment containing the crack can be determined with a mean error of 1.93% of the total size of the raster. Testing of the algorithm on model images shows that the mean error of determining the crack tip position is smaller than 56 pixels.     

In-situ STM studies of electrochemical underpotential deposition of Pb on Au(111)

A modified scanning tunnelling microscope (STM) has been used to observe in-situ deposition and stripping of an electrochemical film. With STM tip and sample immersed in an acid electrolyte, single atomic steps on Au(111) have been imaged during the deposition and stripping of a monolayer-thick, underpotential deposit (UPD) of Pb. Integration of the electrochemical current passed during the film deposition and evidence from the STM images themselves confirm monolayer coverage. Our images show enhanced film growth at step edges and defect sites. Observations of single plating and stripping cycles indicate that the Au substrate returns unaltered. Except for atomic resolution images of Au(111), which we have not yet achieved in an electrolyte, all types of Au surface features seen in air are reproduced under the electrolytic solution. The modifications made to our STM in order to perform in-situ electrochemical experiments are described.     

Quantitative photogrammetric analysis and qualitative stereoscopic analysis of SEM images

This paper is concerned with methods for obtaining three-dimensional (3D) information from scanning electron microscope (SEM) images. It may come as a surprise that this topic merits separate discussion, because, it is generally accepted that every SEM image of a rough surface using the secondary electron emission mode gives apparent 3D information. This is true to some extent, since SEM images are comparable with photographs of rough surfaces illuminated with light coming from one direction, but our main concern here will be with 3D visualization from stereoscopic pairs of images. However, there are other ways in which 3D information may be obtained, even in the SEM, and these are considered first. The remainder of the paper is largely concerned with the use of stereoscopic parallaxes, whether or not they are visualized as such. Commonly used methods for viewing stereo-pairs are described, followed by simple methods for measuring stereoscopic parallaxes. Formulae for reducing linear measurements in the two photographs to real height differences are given.     

Theoretical comparison of illumination and collection mode images of magneto-optical dots

We compare theoretical images of the same sample obtained with two different scanning near-field optical microscopes. The sample is a two-dimensional periodic array of magnetic sub-micrometric dots. The magnetization is perpendicular to the sample plane (polar magnetization). The first configuration is a scanning tunnelling optical near-field microscope (STOM) where the tip is used in the detection mode and the sample is illuminated by total internal reflection. The second configuration is an inverted STOM: the tip is used in the emission mode and the diffracted field is far-field detected in one direction. We present the models used to describe the two configurations and then explain the main lines of the formalism used to calculate the diffracted fields by a magneto-optical sample.