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.     

Magnification variations in reflection electron microscopy using diffracted beams

In reflection electron microscope images obtained using electron beams diffracted at a small angle from the surfaces of bulk specimens, it is observed that the magnification in directions almost parallel to the incident beam appears to increase rapidly with distance from the in-focus position in both directions. An explanation for this effect is offered in terms of the curvature of the lines of energy flow around the cross-over of the electron beam formed by the condenser action of the fore-field of the objective lens.     

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.     

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.     

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.     

应用于纳米级聚焦离子束系统的静电透镜设计

在不同束流和能量下对离子光柱体进行了优化设计。给出了用阻尼最小二乘法设计的单个透镜和透镜系统,并研究了像平面处的离子束性能。采用先分解再组合的方法确定单个透镜参数,并以大束流、无限大放大倍数下像差系数与焦距的比为优化目标。选取工作模式时综合考虑了系统的光学性能和可实现性,大束流下采用平行模式,小束流下采用交叉模式,设计的透镜系统在大、小束流下分别选轴上像差和放大倍数为优化目标。计算表明,2 nA束流下像差为16.33 nm,放大倍数为-0.539 095 5,束斑直径为31.52 nm;2.5 pA束流下像差为2.15 nm,放大倍数为0.084 359 9,束斑直径为4.73 nm。此离子光柱体能够获得纳米量级的离子束,并且只需调整第二透镜第二、第三电极之间的距离以及第二电极电位(对源)就能改变样品处的束能,增加了光柱体的应用范围,实现了一套系统内同时具有刻蚀、沉积、注入和离子成像等功能。     

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.     

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.     

Influence of the elliptical illumination on acquisition and correction of coherent aberrations in high-resolution electron holography

In high-resolution off-axis electron holography, the interpretable lateral resolution is extended up to the information limit of the electron microscope by means of a correcting phase plate in Fourier space. A plane illuminating electron wave is generally assumed. However, in order to improve spatial coherence, which is essential for holography, the object under investigation is illuminated with an elliptically shaped electron source. This special illumination imposes a variation of beam directions over the field of view. Therefore, due to the interaction of beam tilt and coherent wave aberration, the effective aberrations vary over the field of view yielding a loss of isoplanicity. Consequently, in the past the aberrations were only corrected successfully for a small part of the field of view. However, a thorough analysis of the holographic imaging process shows that the imaging artifacts introduced by the elliptical illumination can be corrected under reconstruction by means of a phase curvature, which models the illuminating wave front. Applied in real space, this phase curvature is seamlessly incorporated into the correction process for coherent wave aberration resulting in an improvement of interpretable lateral resolution up to the information limit for the whole field of view.     

Köhler illumination in the TEM: Fundamentals and advantages

Köhler illumination is the most favourable design for the illumination path of an electron microscope with a condenser objective lens. The new illumination system of the EM 910 and EM 912 OMEGA allows both wide area (Köhler) illumination for TEM operation and spot illumination for analytical investigations. Compared to conventional systems and objective lenses with a condenser mini lens, this system offers many advantages. In addition to the homogeneous, highly coherent and parallel illumination of every point in the specimen, it offers advantages for selected area diffraction and spot scan mode. Combined with the electron optical selection of a condenser aperture, this illumination system provides the flexibility necessary to achieve optimum illumination for the specimen.     

A straightforward specimen holder modification for remnant magnetic-field measurement in TEM

We report a simple means to modify an analytic sample holder to perform ambient Hall probe measurements of the sample area inside a transmission electron microscope (TEM). These measurements are important in the case of electron microscopy studies involving magnetic materials. We characterize the magnetic field of the JEOL 2100F-LM, a microscope dedicated in design to perform magnetic imaging, and also of the JEOL 3000F FEG-TEM operated in Lorentz mode. In the case of the 3000F, we measure vertical remnant field about 300 Oe due to the objective lens of the microscope. In the case of the 2100F, design of the objective lens reduces the remnant field to about 4 Oe. We characterize the field along two orthogonal directions, and spatial characteristics of the field profile are made for both microscopes during all stages of specimen entry into the column. In the case of the 2100F, we additionally measure the field conditions as a function of objective lens excitation, which is important for in situ magnetization experiments. Finally, we provide experimental results illustrating the importance of these measurements.     

A method of using the incident beam tilt as a eucentric goniometer in transmission electron microscopy

The principal difficulties in constructing and operating a eucentric specimen tilting goniometer in a transmission electron microscope are discussed, together with the goniometric function of the incident beam tilt. The latter function is found easy to operate in a eucentric manner. The imaging beam then will have a non-axial path, which will increase particularly the field chromatic aberration. Earlier, however, a technique for the compensation of the chromatic aberration during displaced aperture dark field image formation has been developed. In combination with this technique, it proved possible to use the ordinary incident beam tilt as a eucentric goniometer. Image sequences were obtained, with accurately varied diffraction conditions. The tilt angles and the direction of the tilt axis can be very accurately determined from the displacements of the diffraction pattern.     

Enabling the detection of UV signal in multimodal nonlinear microscopy with catalogue lens components

Using an optical system made from fused silica catalogue optical components, third‐order nonlinear microscopy has been enabled on conventional Ti:sapphire laser‐based multiphoton microscopy setups. The optical system is designed using two lens groups with straightforward adaptation to other microscope stands when one of the lens groups is exchanged. Within the theoretical design, the optical system collects and transmits light with wavelengths between the near ultraviolet and the near infrared from an object field of at least 1 mm in diameter within a resulting numerical aperture of up to 0.56. The numerical aperture can be controlled with a variable aperture stop between the two lens groups of the condenser. We demonstrate this new detection capability in third harmonic generation imaging experiments at the harmonic wavelength of ～300 nm and in multimodal nonlinear optical imaging experiments using third‐order sum frequency generation and coherent anti‐Stokes Raman scattering microscopy so that the wavelengths of the detected signals range from ～300 nm to ～660 nm.     

In situ topography measurement in the SEM

An automatic technique for measuring heights in situ in the SEM based on a combination of stereo-metric and focusing methods has been developed. A pair of images of a surface element on the specimen is obtained by tilting the beam electrically in a manner such that the plane containing the tilt axis is coincident with the focal plane of the final lens. Cross-correlation is used to determine the parallax between the image pair which is then used to iteratively correct the height of the tilt axis by changing the lens focus. As a result, the lens focus tracks the specimen topography. With an appropriate specimen surface containing high resolution features for image correlation, the technique is capable of maintaining both its lateral and vertical resolutions over several decades of height displacement up to 100 μm. In an experimental system based on a commercial electron-optical column, spot, line profile, and three-dimensional measurements have been demonstrated.     

Inspection of refractive X-ray lenses using high-resolution differential phase contrast imaging with a microfocus X-ray source

A refractive x-ray lens was characterized using a magnifying cone beam setup for differential phase contrast imaging in combination with a microfocus x-ray tube. Thereby, the differential and the total phase shift of x rays transmitted through the lens were determined. Lens aberrations have been characterized based on these refractive properties.     

Electron optical property of a charged foil,observed with a transmitted electron detector device in an SEM

Experimental evidence is presented for the electron optical behaviour of a charged foil area, using the transmitted electron detection device of the scanning electron microscope JSM 50 A (JEOL). The primary electron beam scanning a thin pioloform foil on the one hand produces a charged foil region which on the other hand acts as an electron lens to the primary and scattered electrons. Scanning electron microscopical investigations of air particulates in the submicron size range can be eased by using a transmitted electron detection device both of the bright and dark field operation mode. The image contrast thus may be improved by orders of magnitude, also allowing on line operation of an image analysis system. Using a special preparation technique, depositing the particles on a thin supporting foil which is also used for LAMMA analysis – Wieser et al. 1980, the x-ray spectra of single particles provided by an energy dispersive x-ray spectrometer may be quantitatively interpreted on the basis of the peak-to-background method (Statham and Pawley 1978, Small et al. 1979). Figure 1 shows a schematic of the transmission detector device of the JSM 50 A when operated in the dark field mode. Geometrical dimensions and apertures also are given in Fig. 1. The dark field diaphragm (DFD) on the optical axis of the microscope blocks all electrons (primary electrons and scattered electrons) within an angle of about 10^?2 rad from contributing to the video signal. As long as magnifications above about 350 × are used the primary electron beam hits the DFD thus yielding a transmission scanning electron micrograph in dark field mode. Below this limit or above the corresponding maximal scanning angle (about 7 × 10^?3 rad) of the primary electron beam the rim of the DFD becomes visible in the displayed image as shown in Fig. 2a. At the same magnification Figure 2b shows the sharpened contours of the DFD as obtained by focussing the primary electron beam to the plane of the DFD by lowering the objective lens excitation. By means of the thin bar attached to the DFD (left hand upper corner of Fig. 2b) the DFD may be centered to the optical axis or exchanged to the bright field aperture. Looking to the circular center of Fig. 2a, we recognize the black grid bars and a few black particles whereas the supporting foil looks bright. No video signal can be obtained, because both the grid bars, and to a lesser extent the particles, are not transparent to the primary electrons of 15 keV energy. On the other hand all electrons scattered by the thin foil to an angle of more than 10^?2 rad are seen by the scintillator and hence accumulate a measurable video signal: This is also favoured by the large solid angle outside the DFD, which is about 30 times the solid angle of the DFD itself.     

Measurement of spherical aberration,beam tilt and image rotation in tem with a focussed probe

When a crystalline specimen in a transmission microscope is illuminated by a small focussed probe parallel to a zone axis, electrons diffracted at large angles by planes in higher order Laue zones (HOLZ) are imaged as a circle surrounding the direct beam. The diameter of this HOLZ image circle is directly related to the spherical aberration coefficient C_s of the objective lens. For the Philips EM430 at voltages between 50 and 300 kV, the 〈111〉 zone axis in silicon was used to measure an averaged C_s of 2.12 ± 0.04 mm. A displacement of the HOLZ image circle relative to the central probe image is attributed to a small tilt of the beam axis induced by lens interactions when switching between image and diffraction modes. This tilt could affect the quality of lattice images, where we require that both the beam and zone axes should be parallel with the optic axis. Even in an aberration-free lens, multiple diffracted images are observed when the probe is focussed and imaged in a plane below the specimen. This effect is proposed as a convenient method for measuring the direction and sign of g in two-beam defect analysis.     

Effect of crystal and beam tilt on simulated high-resolution TEM images of interfaces.

The effects of crystal and beam tilt on high-resolution transmission electron microscope (HRTEM) images of planar coherent interfaces were investigated by multislice image simulations. It was found that a beam tilt of 0.5 Bragg angle (theta B) was sufficient to introduce detrimental artifacts into most images of interfaces in crystals only 1/8 xi 000 thick, while crystal tilt had a much smaller effect even for crystals 1 xi 000 thick. Effects produced in HRTEM images of interfaces by crystal and beam tilt included the introduction of additional periodicities and loss of compositional detail across a boundary, translation of a boundary from its actual position, and apparent mismatch of atomic planes across a perfectly coherent interface. These results indicate that alignment of the electron beam parallel to the optic axis is critical for reliable HRTEM imaging of interfaces in materials. Techniques for obtaining accurate alignment are also discussed.     

A standard video-rate confocal laser-scanning reflection and fluorescence microscope

A confocal laser-scanning microscope (CLSM) differs from a conventional microscope by affording an extreme depth discrimination, as well as a slightly improved resolution. These features afford improved imaging, and make possible new imaging techniques. The CLSM developed at TNO has standard video-rate imaging, and is capable of working in reflection and in fluorescence mode simultaneously. Nonconfocally the laser-scanning microscope can also be used in transmission mode. In addition to the evident advantages of a fast system when searching objects or studying living objects, the time needed to produce an image of extended depth of focus and high resolution is very short. Furthermore, the high-speed averaging of many images at low laser-power levels, and the short dwelling time of the focused laser beam (60 ns) obviate quenching effects in fluorescence microscopy and prevent damage to the object. In this article the TNO-CLSM system is outlined. The most important specifications are summarized, and some representative micrographs obtained with the system are shown. Furthermore, the performance of the system is illustrated by some experimental results.     

SEM imaging of air-sensitive specimens

A device has been built that allows four air-sensitive specimens to be examined in a Philips 501 scanning electron microscope (SEM) without interrupting the vacuum. The specimens are mounted onto a revolving four-faceted stage in an inert atmosphere. The sealed device is then mounted onto the goniometer substage of the SEM and interfaced with the stage rotation drive. After the microscope reaches the operating vacuum condition, the device is opened by turning the stage rotation knob. The device can be viewed with the SEM image during this operation. The same mechanism which opens and closes the device also rotates the four-faceted sample mount to the desired specimen. The x and y translations of the SEM sub-stage are used to move the specimen in the imaging plane. The operation of this device was tested with polyolefin catalysts which are extremely air-sensitive. The main advantage of this device is that it allows air-sensitive specimens to be routinely examined without compromising the other uses of the SEM. Specimens mounted on this device can be viewed from 0 to ± 90° tilt. With a minor adaption, this device can be used to view the total 360° surface of a specimen in a manner similar to that described by Herrmann (1979).