Real time computer simulation of electron microscope images with tilted illumination: Grain boundary applications

Computer programs have been developed to simulate electron microscope images from digitized graphically represented model structures. Via a television rate image processing system, these programs allow real time, interactive modification of the microscope objective lens parameters, incident beam inclination, and incident beam energy. In addition to explaining the computational methods, the need for using tilted beam illumination is explored to extend microscope resolution. For this study, the subject of grain boundary imaging is analyzed for a copper σ = 5,36.9°,(310) tilt boundary with a [001] common rotation axis. The Cu {200} lattice spacings of ～1.8Å on both sides of the interface cannot be reliably resolved under axial illumination conditions in a 200 kV microscope. Therefore, either tilted beam modes or higher incident beam energies were explored and the types of image features correlated with atomic position data through the digital frame store system.     

Illumination conditions in microsphere-assisted microscopy

White-light microsphere-assisted microscopy is a full-field and label-free imaging promising technique making it possible to achieve a subdiffraction lateral resolution. However, performance of this technique depends not only on the geometrical parameters but also on the illumination conditions of the optical system. In the present work, experimental measurements and computer simulations have been performed in air in order to determine the influence of the two diaphragm apertures of the Köhler arrangement and the spectral width of the light source on both the depth-of-focus of the microsphere and the optimization of the imaging contrast. Furthermore, the super-resolution phenomenon is demonstrated and the cumulated optical aberrations are shown through the measurement of the optical transfer function for the different arrangements of the illumination part.     

Design considerations and performance of an analytical stem

A VG Microscope HB5 has been modified in collaboration with the manufacturers. The major modifications are the addition of three post specimen lenses, a second condenser lens and a z-lift on the specimen stage. The requirements of the various parts of the microscope are reviewed and the compromises made necessary their interaction are discussed. The performance of the double condenser system, the z-lift stage and the high excitation objective lens are discussed briefly. A more detailed discussion of the expected performance of the post-specimen optics is given and preliminary experimental results are presented. The discussion is in two parts. The first part considers the recording of diffraction patterns and the use of detectors in the diffraction plane. The major effect is the distortion introduced by the lens system. By a suitable choice of operating conditions, this can be minimised. The second part considers the use of an electron spectrometer. Large chromatic effects can occur if a wide range of electron energies is involved. Provided that the correct lens configuration is used, these may prove to be an advantage since it is possible to produce an increase in collection efficiency with increasing energy loss. Finally, the effect of using an electron spectrometer with lower aberrations in conjunction with the post-specimen lenses is considered briefly.     

基于微透镜阵列生成特定矩形光斑的TIR组合透镜设计与仿真

LED功率的不断增大使其逐渐替代传统光源,并广泛用于各种照明光源。在某些室内照明以及道路照明设计中,通常需要借助LED透镜产生矩形光斑从而充分利用光能避免浪费。利用Light Tools软件设计了一种全内反射(total internal reflection,TIR)透镜与特定微型透镜阵列的组合透镜,设置TIR准直透镜透射曲面和全反射曲面为二次曲面,并对其进行准直优化。然后建立单个微型透镜,设置单个矩形微透镜长宽比分别为1∶1、3∶2和4∶3,并将其阵列化。通过改变微型透镜的尺寸以及球面曲率半径进行进一步优化,最后生成与微型透镜长宽比对应的均匀矩形光斑,从而满足不同矩形照明区域的照明需求。     

A rapid method to correct objective lens astigmatism in a TEM

This work describes a rapid method to correct the two-fold astigmatism of transmission electron microscope (TEM) objective lens employing caustic curve when no objective aperture is inserted. The method makes use of rounding the caustic curve via the objective lens stigmators after the condenser lens astigmatism has been corrected. It has many advantages over other methods, it is fast, straightforward, and does not need holes or an amorphous material.     

Very low voltage electron microscopy.

We conclude that a 150 V scanning microscope with a resolution of 10 A is quite feasible and could have considerable value. It might consist of a field emission source, an electron gun to decelerate the electrons, a condenser lens to produce a parallel beam, a multipole corrector and a short focal length objective lens. Electrons reflected from the specimen surface would pass through a spectrometer whose principal features would be a large collecting power and low (1/200) energy resolution. Finally, we should add that such a microscope presents a considerable challenge and new opportunities for the electron optician in both lens and spectrometer design. We cannot refrain from pointing out that the Scherzer theorem does not necessarily hold for such a lens since the constraints of the theorem do not apply to this case.     

超高亮度LED微型投影仪照明系统设计

为了提高微型投影仪系统的性能,设计了一种超高亮度LED光源口袋式微型投影仪照明系统.其光源采用具有大面积发光面的RGB三色LED芯片.照明系统中最为关键的集光系统包含一个CPC和两个聚光透镜.在LightTools软件中追迹2×10(6)条光线进行仿真,DMD面上光照均匀性为92%,光通量为450lm.该照明系统比传统...     

应用于工业扫描枪的照明系统设计

针对工业扫描枪扫描时对被扫描面的照明要求,对扫描枪照明模块进行设计。与传统的商用小型扫描枪比,设计的工业扫描枪可实现更大面积均匀矩形的照明光斑,灵敏度与照明面的锐利度都有所提升。采用两种设计方案:一种方案通过拟合一个自由曲面,使光源经过自由曲面后得到所需光斑;另一种基于传统非成像原理设计的照明光路,使用复眼匀光得到所需光斑;根据市场需求,两种设计方案均可在被扫描面距离扫描枪350 mm处呈350 mm×230 mm的均匀矩形照明面,且照明轴上点的强度大于0.2 W/cm2,全部照明面均匀度大于75%。     

面板检测用显微镜光学系统设计

为了实现对面板上缺陷精细分析和分类,设计了可见光波段、数值孔径为0.42的复消色差平场显微物镜和照明光学系统.通过合理结构优化、光焦度分配和材料的选择,优化出平场复消色差物镜,使其MTF曲线接近衍射极限.采用科勒照明匀光方案,设计照明光学系统,并用Lighttools软件对照明光学系统进行仿真.实验表明,光学系统分辨率...     

Coherent nano-area electron diffraction

We describe the new coherent nano-area electron diffraction (NED) and its application for structure determination of individual nanostructures. The study is motivated by the challenge and the general lack of analytical techniques for characterizing nanometer-sized, heterogeneous phases. We show that by focusing electrons on the focal plane of the pre-objective lens using a 3rd condenser lens and a small condense aperture, it is possible to achieve a nanometer-sized highly parallel illumination or probe. The high angular resolution of diffraction pattern from the parallel illumination allows over-sampling and consequently the solution of phase problem based on the recently developed ab initio phase retrieval technique. From this, a high-contrast and high-resolution image can be reconstructed at resolution beyond the performance limit of the image-forming objective lens. The significance of NED for nanostructure characterization will be exemplified by single-wall carbon nanotubes and small metallic clusters. Imaging from diffraction patterns, or diffractive imaging, will be demonstrated using double-wall carbon nanotubes.     

A method for producing hollow cone illumination electronically in the conventional transmission microscope.

An electronic device manipulates the primary beam in the conventional transmission microscope to produce a hollow cone of illumination with its apex located at the specimen. The device uses the existing tilt coils of the microscope, and modulates the D.C. signals to both x and y tilt directions simultaneously with various waveforms to produce Lissajous figures in the back-focal plane of the objective lens. Electron diffraction patterns can be recorded which reflect the manner in which the direct beam is tilted during exposure of a micrograph. In the bright-field imaging mode the device provides a microscope transfer function without zeros in all spatial directions and has been used to obtain high resolution images which are also free from the effect of chromatic aberration. A standard second condenser aperture is employed and the width of the cone annulus is readily controlled by defocusing the second condenser lens. The cone azimuthal angle is also controlled electronically; hence the device can also be used in the dark-field imaging mode. This device has been applied to imaging both amorphous and crystalline materials including biomolecular specimens.     

视网膜细胞显微镜的照明系统

为满足视网膜细胞成像照明光束的高亮度、窄谱宽要求,提出了一种用于视网膜细胞成像的照明系统,将半导体激光器发出的632.8nm激光耦合到芯径为105μm的多模光纤中,用聚光镜将多模光纤的出光端汇聚到一个旋转毛玻璃上,再用一个投射物镜将毛玻璃上的光源像投射到眼睛里照亮视网膜。试验发现该照明方法很好地消除了激光散斑,满足视网膜显微成像对高亮度光源的要求,光源有较窄的谱宽,成像系统色差很小,成像质量优于采用传统氙灯做照明光源的图像。该照明系统能较好地满足视网膜显微成像。     

Contrast enhancement of negatively stained macromolecules and biomembranes by single sideband phase contrast interference.

A straightforward procedure is described for the production of contrast enhancement of negatively stained macromolecules and biological membranes by single sideband phase contrast interference (electron optical shadowing). The instrumental adjustment required to produce this type of phase contrast illumination is readily achieved by beam deflection from the strioscopic (dark field) mode. Part of the hollow cone of electrons from the annular condenser aperture that are unscattered by the specimen are permitted to pass through the objective aperture and interfere with the scattered beam. The electron optical shadowing effect is produced because only one side of the unscattered beam is used. Careful adjustment of the beam tilt control, with the ability to tilt in any azimuth, allows optimal illumination conditions to be achieved. The results presented show the increased image contrast obtained using as specimens the purified cylindrical macromolecule from human erythrocyte membranes, purified nuclear envelopes and collagen fibres.     

Condenser‐free contrast methods for transmitted‐light microscopy

Phase contrast microscopy allows the study of highly transparent yet detail‐rich specimens by producing intensity contrast from phase objects within the sample. Presented here is a generalized phase contrast illumination schema in which condenser optics are entirely abrogated, yielding a condenser‐free yet highly effective method of obtaining phase contrast in transmitted‐light microscopy. A ring of light emitting diodes (LEDs) is positioned within the light‐path such that observation of the objective back focal plane places the illuminating ring in appropriate conjunction with the phase ring. It is demonstrated that true Zernike phase contrast is obtained, whose geometry can be flexibly manipulated to provide an arbitrary working distance between illuminator and sample. Condenser‐free phase contrast is demonstrated across a range of magnifications (4–100×), numerical apertures (0.13–1.65NA) and conventional phase positions. Also demonstrated is condenser‐free darkfield microscopy as well as combinatorial contrast including Rheinberg illumination and simultaneous, colour‐contrasted, brightfield, darkfield and Zernike phase contrast. By providing enhanced and arbitrary working space above the preparation, a range of concurrent imaging and electrophysiological techniques will be technically facilitated. Condenser‐free phase contrast is demonstrated in conjunction with scanning ion conductance microscopy (SICM), using a notched ring to admit the scanned probe. The compact, versatile LED illumination schema will further lend itself to novel next‐generation transmitted‐light microscopy designs. The condenser‐free illumination method, using rings of independent or radially‐scanned emitters, may be exploited in future in other electromagnetic wavebands, including X‐rays or the infrared.     

STEM tomography for thick biological specimens

Scanning transmission electron microscopy (STEM) tomography was applied to biological specimens such as yeast cells, HEK293 cells and primary culture neurons. These cells, which were embedded in a resin, were cut into 1-microm-thick sections. STEM tomography offers several important advantages including: (1) it is effective even for thick specimens, (2) 'dynamic focusing', (3) ease of using an annular dark field (ADF) mode and (4) linear contrasts. It has become evident that STEM tomography offers significant advantages for the observation of thick specimens. By employing STEM tomography, even a 1-microm-thick specimen (which is difficult to observe by conventional transmission electron microscopy (TEM)) was successfully analyzed in three dimensions. The specimen was tilted up to 73 degrees during data acquisition. At a large tilt angle, the specimen thicknesses increase dramatically. In order to observe such thick specimens, we introduced a special small condenser aperture that reduces the collection angle of the STEM probe. The specimen damage caused by the convergent electron beam was expected to be the most serious problem; however, the damage in STEM was actually smaller than that in TEM. In this study, the irradiation damage caused by TEM- and STEM-tomography in biological specimens was quantitatively compared.     

Variable bright-darkfield-contrast, a new illumination technique for improved visualizations of complex structured transparent specimens

Variable bright-darkfield contrast (VBDC) is a new technique in light microscopy which promises significant improvements in imaging of transparent colorless specimens especially when characterized by a high regional thickness and a complex three-dimensional architecture. By a particular light pathway, two brightfield- and darkfield-like partial images are simultaneously superimposed so that the brightfield-like absorption image based on the principal zeroth order maximum interferes with the darkfield-like reflection image which is based on the secondary maxima. The background brightness and character of the resulting image can be continuously modulated from a brightfield-dominated to a darkfield-dominated appearance. When the weighting of the dark- and brightfield components is balanced, medium background brightness will result showing the specimen in a phase- or interference contrast-like manner. Specimens can either be illuminated axially/concentrically or obliquely/eccentrically. In oblique illumination, the angle of incidence and grade of eccentricity can be continuously changed. The condenser aperture diaphragm can be used for improvements of the image quality in the same manner as usual in standard brightfield illumination. By this means, the illumination can be optimally adjusted to the specific properties of the specimen. In VBDC, the image contrast is higher than in normal brightfield illumination, blooming and scattering are lower than in standard darkfield examinations, and any haloing is significantly reduced or absent. Although axial resolution and depth of field are higher than in concurrent standard techniques, the lateral resolution is not visibly reduced. Three dimensional structures, reliefs and fine textures can be perceived in superior clarity.     

An easily built diffuse illumination system effective at both very low and moderate magnifications, for observing in situ stained slides

Effective study of in situ stained sections often requires illumination that is difficult to achieve with commonly used research microscopes. One must be able to switch quickly and conveniently from the very lowest to moderate magnifications. At all magnifications contrast due to light scatter must be minimized, so that the weak staining that signifies low gene expression can be observed reliably. For the lowest power objectives (e.g., 1.25× or 2×) many microscopes require that the condenser be removed to illuminate the full field of view. This is not only very inconvenient when switching magnifications, but without a condenser the low numerical aperture of the illuminating light beam results in unwanted contrast due to light scatter. We have devised a simple system that diffusely illuminates the full field of view of the lowest power objective (1.25×) and has high enough numerical aperture for use with the 25× and 40× objectives. A key feature is the use of a large diameter ring light (internal diameter 5.8 cm), placed on the microscope base, to illuminate a large diameter diffuser placed just below the slide.     

First experimental test of a new monochromated and aberration-corrected 200 kV field-emission scanning transmission electron microscope

The first 200 kV scanning transmission electron microscope (STEM) with an imaging energy filter, a monochromator and a corrector for the spherical aberration (Cs-corrector) of the illumination system has been built and tested. The STEM/TEM concept with Koehler illumination allows to switch easily between STEM mode for analytical and TEM mode for high-resolution or in situ studies. The Cs-corrector allows the use of large illumination angles for retaining a sufficiently high beam current despite the intensity loss in the monochromator. With the monochromator on and a 3 microm slit in the dispersion plane that gives 0.26 eV full-width at half-maximum (FWHM) energy resolution we have obtained so far an electron beam smaller than 0.20 nm in diameter (FWHM as measured by scanning the spot quickly over the CCD) which contains 7 pA current and, according to simulations, should be around 0.12 nm in true size. A high-angle annular dark field (ADF) image with isotropic resolution better than 0.28 nm has been recorded with the monochromator in the above configuration and the Cs-corrector on. The beam current is still somewhat low for electron energy-loss spectroscopy (EELS) but is expected to increase substantially by optimising the condenser set-up and using a somewhat larger condenser aperture.     

Improvements that facilitate the operation of the Corinth 275 electron microscope

Details are given of modifications to the Corinth 275 electron microscope providing illumination of the magnification scale and locking of the astigmatism controls. The construction of a loading jig suitable for use with specimen rods of this microscope is described. Operational procedures that allow accurate setting of filament height and centering and also give some compensatory correction of condenser astigmatism are discussed.     

三维振动测量系统的照明模块设计

为了满足三维测量系统所需的大视场照明条件,设计了一种基于自由曲面的透镜阵列的匀光照明模块。根据能量对应关系计算得到自由曲面透镜的离散点数据,通过Rhino软件拟合离散点得到该自由曲面透镜实体模型,再将得到的自由曲面透镜面型导入Light Tools软件,进行阵列后得到匀光照明模块。仿真结果表明,该照明模块可使2.56m2的接收面上的亮度达到13 102lx,照明均匀度达到92.86%,且公差符合装配要求。