Automatic focus correction for spot-scan imaging of tilted specimens.

The variation in defocus within an image of a highly tilted specimen can be a serious source of artifact. Spot-scan imaging can be combined with dynamic focusing to greatly reduce this range of defocus. A protocol is described for determining the parameters required for the automatic focus compensation during the recording of a spot-scan image. Images of a gold test specimen demonstrate the efficacy of this procedure in extending the area of the image that contains high-quality data. In case the tilt angle or resolution is high enough that the height difference of the specimen within each small illuminated area is larger than the depth of field, the image must be treated to compensate for the focus variation. The same principle is used as was developed for compensation of conventional images of tilted specimens.     

Absolute calibration of image plates for electrons at energy between 100 keV and 4 MeV

We measured the absolute response of image plate (Fuji BAS SR2040) for electrons at energies between 100 keV and 4 MeV using an electron spectrometer. The electron source was produced from a short pulse laser irradiated on solid density targets. This paper presents the calibration results of image plate photon stimulated luminescence per electron at this energy range. The Monte Carlo radiation transport code MCNPX results are also presented for three representative incident angles onto the image plates and corresponding electron energy depositions at these angles. These provide a complete set of tools that allows extraction of our absolute calibration to other spectrometer setting at this electron energy range.     

Near-field Raman imaging of morphological and chemical defects in organic crystals with subdiffraction resolution

In this study we report on the application of an aperture near‐field optical microscope for Raman imaging of organic materials. Spectral analysis and detailed Raman imaging are performed with integration times of 100 ms per point, without the aid of field enhancement effects. The studied samples consist of two high Raman‐efficiency molecular samples: a 7,7′,8,8′‐tetracyanoquinodimethane crystal showing surface defects and a 7,7′,8,8′‐tetracyanoquinodimethane thin film characterized by the presence of submicrometre‐sized organometallic copper–salt complexes. In the first case, the effect of the surface deformation was studied, whereas in the second sample we were able to chemically image the formation of salt complexes. Subdiffraction resolution was achieved in both studies.     

Interpretation of the fading of diffraction patterns from organic substances irradiated with 100 keV electrons at 10-300 K

The fading curves of Debye-Scherrer ring intensities have been recorded for evaporated films of some aliphatic and aromatic compounds at temperatures between T_s=14-66 K and room temperature. The lowest real specimen temperature T_s was calibrated by the condensation of different gas atoms and depends on the current density used, which has to be raised with increasing resistivity to radiation damage. The fading curves for the lowest specimen temperature were compared with a theory of Clark et al. modified by a multiple-hit model. By fitting the theory to experimental results, values of the cross-section σ for a single hit, the mean number m of hits necessary to destroy a sensitive unit and the ratio b of the damaged to the undamaged structure amplitude of a unit cell can be established. There are considerable differences in these quantities for different compounds. The temperature dependence of σ for tetracene and other compounds can be explained by an activation energy of the order of a few tens of one meV.     

Negative staining permits 4.0 A resolution with low-dose electron diffraction of catalase crystals.

Low-dose electron diffraction of thin single crystals of catalase that are negatively stained with the light-atom compound, dipotassium glucose-1,6-diphosphate, reveals Bragg reflections extending to 4.0A (= 0.40 nm). Under the same conditions, negative staining with the traditional heavy-metal salt, ammonium molybdate, also gives diffraction spots extending to 4.0 A. These results establish that negative staining of protein crystals preserves periodic structural information into the high-resolution range, unlike the widely accepted current belief that this methodology can give a resolution limited to only 20-25 A.     

Imaging streptavidin 2D crystals on biotinylated lipid monolayers at high resolution with the atomic force microscope

Streptavidin crystals were grown on biotinylated lipid monolayers at an air/water interface and transferred onto highly oriented pyrolytic graphite (HOPG). These arrays could be imaged to a resolution below 1 nm using the atomic force microscope. The surface topographs obtained were compared with negative-stain electron microscopy images and the atomic model as determined by X-ray crystallography. The streptavidin tetramer (60 kDa) exposes two free biotin-binding sites to the buffer solution, while two are occupied by linkage to the lipid monolayer. Therefore, the streptavidin 2D crystals can be used as nanoscale matrices for binding biotinylated compounds. Furthermore, this HOPG-based preparation method provides a general novel approach to study the structure of protein arrays assembled on lipid monolayers with the AFM.     

The use of backscattered electrons for imaging purposes in a scanning electron microscope.

It is shown that the use of a very large detector for backscattered electrons can provide a signal comparable to that obtained in the secondary emission mode and that the resolution available is also comparable. A technique is described whereby the difference is taken between these two signals, and the use of this difference signal can significantly enhance surface details. It is believed that the use of such a signal should ultimately prove advantageous in increasing resolution.     

Computer-controlled testing system for investigating the dynamic characteristics of contactors with A.C. electromagnet drives

An original computer-controlled measurement system with electrodynamic sensor is presented in this paper. The system was used to examine simultaneously dependencies of time related electromagnet coil current, electromagnet coil voltage, electric power supplied to electromagnet coil, voltage across contacts, acceleration, speed, path and kinetic energy of contactor moving element. An additional software allows diagrams of the following parameters to be obtained: maximum and minimum acceleration, maximum speed and kinetic energy of contactor moving element, electric energy supplied to electromagnet coil and times typical of alternating current electromagnetic drive for selected values of the coil supply voltages and various switching on angles of this voltage (within the range 0–170°). Results of the investigation enable also to take a view on such parameters of the designed equipment as, for example, making time or rebounds of the contactor contacts. The system is very useful to verify the results of investigation of computer simulated contactor closing.     

Contrast analysis of cryo-images of n-paraffin recorded at 400***kV out to 2·1 Å resolution

N-Paraffin was used as a test specimen for evaluating the relative merits of 400-kV versus 100-kV electron microscopy in recording data for electron crystallographic analysis of beam-sensitive materials. The parameter used for comparison, the relative contrast R, is the ratio of amplitudes from the computed Fourier transform of images and amplitudes from an electron diffraction pattern from the same crystal. R will thus be a measure of the contrast from an experimental image relative to that of a perfect image. Electron diffraction patterns and bright-field images were recorded at 400 kV at a specimen temperature of ?167°C. Using the flood-beam imaging technique the best R-value is 0 08 for all reflections in the resolution zone from 4 to 3 Å. This value is equivalent to that found at 100 kV. In the resolution zone from 3 to 2 Å we have found R — 0 02. Using the spot-scan imaging technique, on the other hand, R was measured to be 0·42 for the reflections between 4- and 3-Å resolution. This amount of relative contrast is 1·7 times that observed at 100 kV. Reflections at 3–2 Å displayed an R-value of 0 05. Besides obtaining higher R-values when applying the spot-scan imaging technique at 400 kV, we observe a higher yield of images with isotropic diffraction and/or higher resolution reflections. Various contrast-attenuating factors, including the modulation transfer function of the photographic film and the cryo-holder, envelope functions for spatial and temporal coherence and lens and high-tension instabilities, the contrast transfer function and lastly the radiation damage effects, have been considered in interpreting the observed image contrast. Overall, use of 400 kV in combination with spot-scan does offer important improvements in contrast levels, which can be very useful in determining the three-dimensional structure from protein crystals.     

Picosecond pulsed two-photon imaging with repetition rates of 200 and 400 MHz

We show the viability of high-resolution two-photon fluorescence imaging of fixed and live cells by exciting the fluorophores with a train of near-infrared pulses with duration in the picosecond range. This is exemplified with a compact, diode-pumped Nd:YVO₄ laser, emitting trains of 7-ps pulses at a wavelength of 1064 nm, with a repetition rate of 200 MHz at two separate outputs. Incoherent combination of the outputs enabled two-photon excitation with a repetition rate of 400 MHz. For a numerical aperture of 1.4 (oil), we used an average illumination power of up to 20–40 mW at the sample. The pulses were coupled into a beam scanning microscope, either directly or through a single mode glass fibre. Compared with standard femtosecond titanium–sapphire excitation conditions, our experiments were performed with a 2.5 or 5 times higher repetition rate, 30–70 times longer pulses and 10–35 times lower pulse peak intensity. The experiments indicate the possibility of significantly relaxing the temporal pulse width constraints for a series of applications.     

17.1 nm波段光电成像系统分辨率的实验研究

设计了17.1 nm波段光电成像系统,用于对波长为17.1 nm的极紫外光进行成像。该成像系统分为三大部分:光源、单色仪和探测系统。一个调Q的Nd∶YAG激光器(Continuum 9000)用来产生激光等离子体,掠入射单色仪由2块球面聚焦镜和1块600 L/mm的球面掠入射光栅组成,经单色仪分光后得到波长为17.1 nm的单色光,探测器是微通道板(MCP)和荧光屏组件共同组成,Kodak400型胶卷被用于记录狭缝的像。结果获得了一宽度为3 mm的狭缝的像,实验测得130 μm的成像系统的空间分辨率,好于相同条件下文献[6]的结果。     

Electron microscopy of tRNA crystals. I. Thin crystals negatively stained with uranyl acetate

The first attempt to study crystal structures of tRNA by electron microscopy is described. Sufficiently thin crystals were prepared from yeast tRNAphe. The thickness of the thinnest was estimated at 130 A corresponding to a bilayer of the molecules. The L-shaped structure seemed to be maintained even after the negative staining with uranyl acetate. Optically filtered images from electron micrographs were compared with those simulated from the drawing of the molecular model by optical transform. The results suggest that the observed images reflect the real molecular arrangements within the crystal lattice although the shape of tRNA molecules seems to be somewhat modified by the uneven staining.     

高分辨力光学微扫描显微热成像系统设计与实现

为提高已研制的基于非制冷焦平面探测器的显微热成像系统的空间分辨力,研究提出了一种基于光学平板旋转微扫描器的高分辨力显微热成像系统.分析了光学平板旋转微扫描的工作原理,给出了微扫描器相关的参数设计、加工容差,并与现有的非制冷显微热成像系统实现了一体化设计与加工,确定了光学微扫描显微热成像系统的技术指标.利用该系统实际采集的显微热图像与过采样处理结果表明系统整体设计的有效性,系统空间分辨力得到提高,可应用于高分辨力显微热分析.＃     

Combining accurate defocus with low-dose imaging in high resolution electron microscopy of biological material

High resolution (< 2 nm) electron microscopy of biological specimens requires three exacting conditions to be met simultaneously: (a) fine specimen detail must be protected from destruction by the electron beam (low dose), (b) the electron optics must be adjusted to be capable of imaging that detail interpretably (accurate defocus), and (c) a suitable field of interest must be identified. We describe a method encompassing all three with an 80% success rate using only minor modifications to a transmission electron microscope, and no expensive on-line computing.     

Electron microscopy of tRNA crystals: I. Thin crystals negatively stained with uranyl acetate

The first attempt to study crystal structures of tRNA by electron microscopy is described. Sufficiently thin crystals were prepared from yeast tRNA^phe. The thickness of the thinnest was estimated at 130 Å corresponding to a bilayer of the molecules. The L-shaped structure seemed to be maintained even after the negative staining with uranyl acetate. Optically filtered images from electron micrographs were compared with those simulated from the drawing of the molecular model by optical transform. The results suggest that the observed images reflect the real molecular arrangements within the crystal lattice although the shape of tRNA molecules seems to be somewhat modified by the uneven staining.     

Image acquisition with immersion objective lenses using electrons emitted with several tenths of an electron volt energies: towards high spatial resolution ESCA analysis

This study aims to evaluate the spatial resolution achievable with photoelectrons in order to perform localised UPS or XPS analyses on various heterogeneous samples. This investigation is intentionally restricted to direct image acquisition by immersion objective lenses, involving electrons ejected with initial energies of several tenths of an electron-volt. In order to characterise the contribution of all optical elements, analytical investigations were associated to numerical simulations based on SIMION 7 software. The acquisition of high-quality images implies a simultaneous reduction in spherical and chromatic aberrations by a narrow aperture stop placed at the output pupil of the objective. With such limitations in useful emission angles, it is shown that monochromatic electron beams build images with a resolution of about 1 nm, especially for the acceleration bias mode where the focussing electrode is biased at a positive high voltage. Even energy dispersed electron beams, limited by a 4 eV band pass spectrometer, can produce images convenient for highly localised ESCA analyses (resolution 3 nm), where the objective lens is associated with an aperture stop of 30 microm in diameter without using acceleration voltages above 5000 V.     

Advantages of stereo imaging of metallic surfaces with low voltage backscattered electrons in a field emission scanning electron microscope

High emission current backscattered electron (HC-BSE) stereo imaging at low accelerating voltages (≤ 5 keV) using a field emission scanning electron microscope was used to display surface structure detail. Samples of titanium with high degrees of surface roughness, for potential medical implant applications, were imaged using the HC-BSE technique at two stage tilts of + 3° and − 3° out of the initial position. A digital stereo image was produced and qualitative height, depth and orientation information on the surface structures was observed. HC-BSE and secondary electron (SE) images were collected over a range of accelerating voltages. The low voltage SE and HC-BSE stereo images exhibited enhanced surface detail and contrast in comparison to high voltage (> 10 keV) BSE or SE stereo images. The low voltage HC-BSE stereo images displayed similar surface detail to the low voltage SE images, although they showed more contrast and directional sensitivity on surface structures. At or below 5 keV, only structures a very short distance into the metallic surface were observed. At higher accelerating voltages a greater appearance of depth could be seen but there was less information on the fine surface detail and its angular orientation. The combined technique of HC-BSE imaging and stereo imaging should be useful for detailed studies on material surfaces and for biological samples with greater contrast and directional sensitivity than can be obtained with current SE or BSE detection modes.     

In-plane rigid-body vibration mode characterization with a nanometer resolution by stroboscopic imaging of a microstructured pattern

This article introduces an improved approach for the characterization of in-plane rigid-body vibration, based on digital processing of stroboscopic images of the moving part. The method involves a sample preparation step, in order to pattern a periodic microstructure on the vibrating device, for instance, by focused ion beam milling. An image processing method has then been developed to perform the optimum reconstruction of this a priori known object feature. In-plane displacement and rotation are deduced simultaneously with a high resolution (10-2 pixel and 0.5 x 10(-3) rad, respectively). The measurement principle combines phase measurements-that provide the high resolution-with correlation-that unwraps the phase with the proper phase constants. The vibration modes of a tuning fork are used for demonstrating the capabilities of the method. For applications allowing the sample preparation, the proposed methodology is more convenient than common interference methods or image processing techniques for the characterization of the vibration modes, even for amplitudes in the nanometer range.