The use of specimen tilt in transmission electron microscopy of the central nervous system

Thin sections of nervous tissue were viewed at different tilt angles using a transmission electron microscope equipped with a eucentric goniometer stage. In a comparison study of various degrees of tilt, one can observe additional morphological features within synaptic profiles, define subsynaptic structures such as Taxi-bodies, and clearly see the crystalline formation of cytochemical tracers. This study demonstrates the value of tilting thin-sections in the analysis of synapses and other biological material at the ultrastructural level.     

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.     

Direct measurement of beam size in a spectroscopic ellipsometry setup

Spectroscopic ellipsometry signals used in thin film analysis are dependent on the beam probe size. In this work, we report a technique to determine the beam size that uses the existing detection facilities in a spectroscopic ellipsometry setup without the need to rearrange the optical components. The intensity signal recorded with the technique comprises a coupled boundary diffraction and knife edge wave that can be isolated using nonlinear fitting. This then permitted an accurate measurement of the beam size with the stronger knife edge component. The technique has the added advantage of picking up chromatic aberration in the probing lens which may be a factor in ellipsometry measurement.     

Chromatic two-photon excitation fluorescence imaging

We report on a chromatic axial scanning method for two-photon excitation fluorescence imaging. Effective axial scanning is achieved by incorporating a Fresnel lens in the system, which has large chromatic aberration and can therefore focus the excitation beam to different axial positions depending on its wavelength. We experimentally demonstrated this technique and used it to image the cross-section of fluorescent microspheres.     

Parallax measurements on stereomicrographs of hydrated single molecules: Their accuracy and precision at high magnification

Stereoimaging of hydrated single complex macromolecules requires thin freeze-etch platinum-carbon replicas (⩽200 Å) and that the transmission electron microscope (TEM) be equipped with a tilt-rotation eucentric goniometer stage. The original parallax equation is an accurate approximation for high-magnification work, micrographs (10⁵ ×) being less than 0.3% in error. In addition, we have derived formulas for high-magnification work to measure heights, lateral distances, and the object tilt angle for an object not lying flat on the film surface. The accuracy of the height measurements is evaluated on spermidine-condensed DNA toruses. By using the maximum error equation derived from the original parallax equation, we discuss methods to improve the height measurement precision (95% fractile) to the 5–10 Å range.     

Towards automated diffraction tomography: part I--data acquisition

The ultimate aim of electron diffraction data collection for structure analysis is to sample the reciprocal space as accurately as possible to obtain a high-quality data set for crystal structure determination. Besides a more precise lattice parameter determination, fine sampling is expected to deliver superior data on reflection intensities, which is crucial for subsequent structure analysis. Traditionally, three-dimensional (3D) diffraction data are collected by manually tilting a crystal around a selected crystallographic axis and recording a set of diffraction patterns (a tilt series) at various crystallographic zones. In a second step, diffraction data from these zones are combined into a 3D data set and analyzed to yield the desired structure information. Data collection can also be performed automatically, with the recent advances in tomography acquisition providing a suitable basis. An experimental software module has been developed for the Tecnai microscope for such an automated diffraction pattern collection while tilting around the goniometer axis. The module combines STEM imaging with diffraction pattern acquisition in nanodiffraction mode. It allows automated recording of diffraction tilt series from nanoparticles with a size down to 5nm.     

Studies of a magnetically focused electrostatic mirror. II. Aberration corrections

A magnetically focused electrostatic mirror is shown to be able to correct the spherical and chromatic aberrations of a probe forming system simultaneously. The probe forming system comprises a uniform magnetic lens and a uniform electrostatic mirror. Previous theoretical investigations showed that the spherical and chromatic aberration coefficients of these two components are the same values but with opposite sign, whose combination will therefore be free from aberrations. The experimental arrangement used a solenoid to produce a uniform magnetic field, and a series of plate electrodes to produce a uniform electrostatic field. These fields are shown to satisfy the experimental requirements. By deliberately changing the extraction voltage to defocus the electron beam, the author is able to observe correction of chromatic aberration by one order of magnitude. By deliberately changing the lens field and the mirror field, the author is able to observe the reduction of the asymmetry caused by the spherical aberration, which the author believes also indicates correction by one order of magnitude.     

High resolution at low beam energy in the SEM: resolution measurement of a monochromated SEM

The resolution of secondary electron low beam energy imaging of a scanning electron microscope equipped with a monochromator is quantitatively measured using the contrast transfer function (CTF) method. High-resolution images, with sub-nm resolutions, were produced using low beam energies. The use of a monochromator is shown to quantitatively improve the resolution of the SEM at low beam energies by limiting the chromatic aberration contribution to the electron probe size as demonstrated with calculations and images of suitable samples. Secondary electron image resolution at low beam energies is ultimately limited by noise in the images as shown by the CTFs.     

纳米级针孔矢量衍射波前误差分析

应用哈特曼-夏克(H-S)波前检测仪检测大数值孔径(NA)透镜时,需要采用纳米级针孔产生参考球面波前对H-S传感器进行标定.为了制作出满足要求的高质量针孔,本文对影响参考波前质量的各种要素进行了仿真计算和分析,以获得最优针孔加工参数.基于矢量衍射理论,在会聚高斯光束照射下,计算了针孔厚度和直径大小对衍射波前误差的影响,衍射波前中的像差成份、能量透过率、强度均匀性、针孔加工误差及光束相对针孔中心发生平移、离焦、倾斜时衍射波前误差的变化.分析计算显示,在NA为0.6时,为了使相对于理想球面波的波峰波谷值(P-V)偏差不大于0.005λ(λ=193 nm),在实际针孔的加工制作中,应选取材料铬,并取厚度200 nm,直径180 nm为适宜.     

Design of an aberration corrected low-voltage SEM

The low-voltage foil corrector is a novel type of foil aberration corrector that can correct for both the spherical and chromatic aberration simultaneously. In order to give a realistic example of the capabilities of this corrector, a design for a low-voltage scanning electron microscope with the low-voltage foil corrector is presented. A fully electrostatic column has been designed and characterised by using aberration integrals and ray tracing calculations. The amount of aberration correction can be adjusted relatively easy. The third order spherical and the first order chromatic aberration can be completely cancelled. In the zero current limit, a FW50 probe size of 1.0 nm at 1 kV can be obtained. This probe size is mainly limited by diffraction and by the fifth order spherical aberration.     

Aberration-compensated large-angle rocking-beam electron diffraction

The application of convergent beam electron diffraction (CBED) to determine symmetry, refine structure factors, and measure specimen thickness requires rather thick specimen and is very difficult or even impossible in the case of large unit cell materials. The large-angle rocking-beam electron diffraction (LARBED) technique introduced in this paper gives access to the kind of experimental data contained in CBED patterns but over a much larger angular range. In addition to symmetry determination and thickness measurement even for thin samples this technique also allows, in principle, very accurate measurements of structure factors. Similar to precession electron diffraction (PED), LARBED uses the illumination tilt coils to sequentially change the angle of incidence of the electron beam over a very large range. I will present results obtained by a recently developed self-calibrating acquisition software which compensates for aberration-induced probe shifts during the acquisition of LARBED patterns and keeps the probe within a few nm, while covering a tilt range from 0 to 100 mrad. This paper is dedicated to Prof. John C. H. Spence on the occasion of his 65th birthday.     

Automated microscopy for electron tomography.

Instrumentation and methodology for the automatic collection of tomographic tilt series data for the three-dimensional reconstruction of single particles is described. The system consists of a Philips EM 430 TEM, with a Gatan 673 cooled slow-scan CCD camera and a Philips C400 microscope computer control unit attached. The procedure for data collection includes direct digital recording of the images on the CCD camera and the automatic measurement and correction of (a) image shifts resulting from tilting the specimen, (b) variation of defocus and (c) the eucentric height position of the specimen. Experiments are described illustrating the possibilities and limitations of automatic data collection. Data collection at a magnification of 30k shows that the exposure time of the specimen to the beam is reduced by a factor of 10-100 compared to manual operation of the TEM.     

Breaking the spherical and chromatic aberration barrier in transmission electron microscopy

Since the invention of transmission electron microscopy (TEM) in 1932 (Z. Physik 78 (1932) 318) engineering improvements have advanced system resolutions to levels that are now limited only by the two fundamental aberrations of electron lenses; spherical and chromatic aberration (Z. Phys. 101 (1936) 593). Since both aberrations scale with the dimensions of the lens, research resolution requirements are pushing the designs to lenses with only a few mm space in the pole-piece gap for the specimen. This is in conflict with the demand for more and more space at the specimen, necessary in order to enable novel techniques in TEM, such as He-cooled cryo electron microscopy, 3D-reconstruction through tomography (Science 302 (2003) 1396) TEM in gaseous environments, or in situ experiments (Nature 427 (2004) 426). All these techniques will only be able to achieve Angstrom resolution when the aberration barriers have been overcome. The spherical aberration barrier has recently been broken by introducing spherical aberration correctors (Nature 392 (1998) 392, 418 (2002) 617), but the correction of the remaining chromatic aberrations have proved to be too difficult for the present state of technology (Optik 57 (1980) 73). Here we present an alternative and successful method to eliminate the chromatic blur, which consists of monochromating the TEM beam (Inst. Phys. Conf. Ser. 161 (1999) 191). We show directly interpretable resolutions well below 1A for the first time, which is significantly better than any TEM operating at 200 KV has reached before.     

The influence of lens chromatic aberration on electron energy-loss spectroscopy quantitative measurements.

An investigation has been made into the effect of chromatic aberrations of a pre-spectrometer lens system on quantitative elemental analysis by electron energy loss spectroscopy (EELS). In transmission electron microscopy (TEM) diffraction mode, the measured effects are typically 150-330 times larger than if only objective-lens chromatic aberration were important. We discuss several methods of avoiding errors arising from chromatic aberration, including selection of a suitable optical mode (dependent on the desired spatial resolution), adjustment of the TEM imaging system so as to focus the system for a chosen energy loss, and analysis of a large area of a uniform specimen.     

色眼：新的人眼色差简化眼模型

应用双色游标对准技术提出了一种新的测量人眼色差的方法。测量结果用于确定新简化眼在可兑光谱范围内折射年的变化量。为了减小球差，进一步将新简化眼的折射面改为非球面。新简化服被称为色眼，它提供了人眼色差的两种形式：纵向与横向色差改进的考虑。     

On dark field techniques in transmission electron microscopy.

Different dark field techniques in transmission electron microscopy are investigated. In particular, a displaced aperture technique is developed, which produces high quality off-axis dark field images. The essential feature of the method is that the chromatic aberration of the objective and intermediate lenses is compensated for with the aid of a one-stage beam deflector in the object plane of the intermediate lens. In addition, the image distortion and the effect of the curvature of the image field are minimized by reducing the coefficient of sperical aberration of the intermediate lens. Diffracted beams, forming angles alpha up to 2.5 degrees with the optical axis, can be used for dark field imaging. Resolutions better than 30 A for alpha = 2 degrees, and 17 A for alpha = 1 degree, are obtained.     

Measurement of lens aberrations by means of image displacements in beam-tilt series

Electron microscope image aberrations are determined by means of the beam tilt/image displacement method with respect to the precision required to obtain a resolution of 1 A. The method simultaneously yields all image aberrations to the fourth order and it is independent of the material used for the procedure. The experimental procedure using amorphous carbon is described and errors in measuring beam-tilt angles, magnifications and image displacements can be kept sufficiently small to achieve the required accuracy. The method is applied to determine aberration constants of a CM300 FEG/UT microscope with correction of the three-fold astigmatism. The coefficient of spherical aberration and the modulus of the three-fold astigmatism were measured to 0.60 (+/- 0.02) mm and 150 (+/- 50) nm, respectively. The beam tilt/image displacement procedure is also computer simulated using an amorphous model structure yielding the same values for the lens aberrations which are used for imaging. However, a coefficient of spherical aberration of 0.67 mm is obtained by applying the focus variation/diffractogram analysis on the same model.     

低色差GRIN棒透镜的设计原则

对梯度折射率(GRIN)棒透镜色差的影响因素进行了讨论,根据Fantone模型,在光学玻璃Nd-V图中直观地表示了离子交换法制备低色差GRIN棒透镜时交换离子对的选择原则和基础玻璃光学性能确定原则.分析表明,以具有常用光学性质的玻璃作为基础玻璃,可以通过Na⁺/Li⁺交换或K⁺/Cs⁺交换获得低色差GRIN棒透镜,而K⁺/Tl⁺交换和Na⁺/Ag⁺交换所制作的GRIN棒透镜具有很大的色差.上述原则对低色差GRIN棒透镜的制作具有指导意义.     

CBED and LACBED characterization of crystal defects

Convergent‐beam electron diffraction (CBED) obtained with a focused incident beam is well known for the identification of the point and space groups but it can also be used for the analysis of stacking faults and antiphase boundaries. Large‐angle convergent‐beam electron diffraction (LACBED) is performed with a large defocused incident beam and is well adapted to the characterization of most types of crystal defects: point defects, perfect and partial dislocations, stacking faults, antiphase boundaries and grain boundaries. Among the advantages of these methods with respect to the conventional transmission electron microscopy methods, are that one or few patterns are required for a full analysis and the interpretations are easy and unambiguous. The LACBED technique is particularly useful for the analysis of dislocations present in anisotropic and beam‐sensitive materials.     

On the importance of fifth-order spherical aberration for a fully corrected electron microscope

Next generation aberration correctors will not only eliminate the third-order spherical aberration, but also improve the information limit by correction of chromatic aberration. As a result of these improvements, higher order aberrations, which have largely been neglected in image analysis, will become important. In this paper, we concern ourselves with situations where sub-A resolution can be achieved, and where the third-order spherical aberration is corrected and the fifth-order spherical aberration is measurable. We derive formulae to explore the maximum value of the fifth-order spherical aberration for directly interpretable imaging and discuss the optimum imaging conditions and their applicable range.