DESIGN AND PERFORMANCE OF TWO ORTHOGONAL EXTRACTION TIME-OF-FLIGHT SECONDARY ION MASS SPECTROMETERS FOR FOCUSED ION BEAM INSTRUMENTS

The design and performance of two orthogonal extraction time-of-flight mass spectrometers are reported that were adapted to existing focused ion beam microscopes for secondary ion mass spectrometry. The performances of these designs were compared to that of a prototype previously described by our group. The differences include newly designed transfer ion optics and in the use of a larger microscope chamber. The two new prototypes allow a mass resolving power of either 600 Th/Th (compact design) or 3000 Th/Th (high resolution design) while simultaneously achieving a lateral spatial resolution of less than 50 nm. The spectrometers and their performance (effective ion yield, mass resolving power, lateral, and depth resolution) are described and compared. Additionally, example applications are presented with multivariate statistical methods to visualize the data sets. Both time-of-flight mass analyzers use orthogonal extraction which avoids the need to pulse the primary ion beam, and the of use monoisotopic gallium to preserve the mass resolution. The goal of the design was a cost-effective accessory to augment typical focused ion beam-scanning electron microscopy applications as an alternative to the cost of a dedicated secondary ion mass spectrometer. The modified instrument allows excellent non destructive imaging and easy sample access, and benefits from the presence of complementary non destructive analytical and imaging techniques that exploit the presence of an electron microscope.     

Retro‐fitting an older (S)TEM with two Cs aberration correctors for 80 kV and 60 kV operation

For the characterization of light materials using transmission electron microscopy, a low electron acceleration voltage of 80 kV or even 60 kV is attractive due to reduced beam damage to the specimen. The concomitant reduction in resolving power of the microscope can be restored when using spherical aberration (C_s) correctors, which for the most part are only available in the latest and most expensive microscopes. Here, we show that upgrading of existing TEMs is an attractive and cost‐effective alternative. We report on the low‐voltage performance on graphitic material of a JEOL JEM‐2010F built in the early 1990s and retro‐fitted with a conventional imaging C_s corrector and a probe C_s corrector. The performance data show C_s retro‐fitted instruments can compete very favourably against more modern state‐of‐the‐art instruments in both conventional imaging (TEM) and scanning (STEM) modes.     

Historical perspective and current trends in emission microscopy, mirror electron microscopy and low-energy electron microscopy. An introduction to the proceedings of the Second International Symposium and Workshop on Emission microscopy and Related Techniques

Emission microscopes and related instruments comprise a specialized class of electron microscopes that have in common an acceleration field in combination with the first stage of imaging (i.e., an immersion objective lens, also called a cathode lens or emission lens). These imaging techniques include photoelectron emission microscopy (PEEM or PEM), electron emission induced by heat, ions, or neutral particles, mirror electron microscopy (MEM), and low-energy electron microscopy (LEEM), among others. In these instruments the specimen is placed on a flat cathode or is the cathode itself. The low-energy electrons that are emitted, reflected, or backscattered from the specimen are first accelerated and then imaged by means of an electron lens system resembling that of a transmission electron microscope. The image is formed in a parallel mode in all of the above instruments, in contrast to the image in scanning electron microscopes, where the information is collected sequentially by scanning the specimen. A brief history and introduction to emission microscopy, MEM, and LEEM is presented as a background for the Proceedings of the Second International Symposium and Workshop on this subject, held in Seattle, Washington, August 16-17, 1990. Current trends in this field gleaned from the presentations at that meeting are discussed.     

Current developments in high voltage electron microscopy

In respect of instrument design two main developments are taking place in high voltage electron microscopy: towards even higher operating voltages (3–5 MV) and towards higher resolving power at moderate voltages (250–600 kV). Applications of existing instruments (650 kV-1.2 MV) are still primarily in metallurgy, especially for radiation damage studies, but their usefulness for biological research is now being actively explored. Microchambers have been developed for observing specimens, both metallurgical and biological, in a controlled gaseous or liquid environment. The prospects for observing living material, except at low magnifications, remain very doubtful on account of ionization effects, but informative work with wet specimens should be possible.     

On the suitability of the available cooling holders for low-dose work with the Philips EM400

The resolution that is routinely obtained with three liquid-nitrogen-cooled cooling holders (Philips types PW6591 and PW6599 and Gatan) presently available for or in common use with the Philips 400 series of electron microscopes has been assessed from the extent of Thon rings present in optical transforms of micrographs of a carbon foil imaged at 36,000 X. Although high resolution results (better than 10 Å) can be obtained for all types of holders, it is shown that for those holders with a directly attached liquid nitrogen reservoir the resolution obtained in the direction of the right-hand specimen translate is limiting (sometimes already at 20 Å) and strongly microscope-dependent.     

A flexible instrument control and image acquisition system for a scanning electron microscope

We have developed an instrument control and image acquisition system for use with scanning electron microscopes. By making the system flexible over a wide range of operating voltages, scan generation and image acquisition modes can be easily accommodated to a wide range of instruments. We show the implementation of this system for use with a custom‐built low‐voltage scanning electron microscope. We then explore the simple modifications that are required for control of two instruments intended for use as free electron lasers.     

Arrangement and stability of atoms at the apex of a scanning tip

A field ion microscope was used to examine the stability of the atomic arrangement at tip apexes. Although a single W atom at the top layer of a [111]-orientated tip apex protrudes from the underneath layer by only 0·91 Å, the present study suggests that the (111)-orientated W tip is the most desirable tip for scanning tunnelling microscopes because a large activation energy for surface diffusion on the (111) plane immobilizes the apex atom while the tip scans over a specimen surface and the tip apex can be resharpened by simply heating the tip.     

Prerequisites for a Cc/Cs-corrected ultrahigh-resolution TEM

After the introduction of a corrector to compensate for the spherical aberration of a TEM and the acceptance of this new instrumentation for high-resolution CTEM (conventional transmission electron microscope) and STEM (scanning transmission electron microscope) by the electron microscopy community, a demand for even higher resolution far below 1A has emerged. As a consequence several projects around the world have been launched to make these new instruments available and to further push the resolution limits down toward fractions of 1A. For this purpose the so-called TEAM (transmission electron aberration-corrected microscope) has been initiated and is currently under development. With the present paper we give a detailed assessment of the stability required for the base instrument and the electric stability, the manufacturing precision, and feasible semi-automatic alignment procedures for a novel C(c)/C(s)-corrector in order to achieve aberration-free imaging with an information limit of 0.5A at an acceleration voltage of 200 kV according to the goals for the first TEAM instrument. This new aberration corrector, a so-called Achroplanat, in combination with a very stable high-resolution TEM leads to an imaging device with unprecedented resolving power and imaging properties.     

A hot stage SEM for gas-solid reaction studies

An ultra-high vacuum, field emission source, scanning electron microscope (SEM) equipped with a gas cell to permit the in situ study of high temperature gas-solid reactions is described. High resolution SEM (50 Å) and scanning Auger microscopy (300 Å resolution) can be carried out at specimen temperatures up to 1000°C. Reaction products and processes can be imaged continuously in the SEM at pressures up to ～0.2 Torr. Gas pressures up to 50 Torr are possible but with some limitations. Methods of specimen mounting, problems of temperature measurement, and the microscope performance at high specimen temperatures and gas pressures are discussed. Examples of metal surface preparation and impurity segregation under high temperature annealing and gas exposure are reported.     

Quality assessment of atomic force microscopy probes by scanning electron microscopy: correlation of tip structure with rendered images

While image quality from instruments such as electron microscopes, light microscopes, and confocal laser scanning microscopes is mostly influenced by the alignment of optical train components, the atomic force microscope differs in that image quality is highly dependent upon a consumable component, the scanning probe. Although many types of scanning probes are commercially available, specific configurations and styles are generally recommended for specific applications. For instance, in our area of interest, tapping mode imaging of biological constituents in fluid, double ended, oxide-sharpened pyramidal silicon nitride probes are most often employed. These cantilevers contain four differently sized probes; thick- and thin-legged 100 microm long and thick- and thin-legged 200 microm long, with only one probe used per cantilever. In a recent investigation [Taatjes et al. (1997) Cell Biol. Int. 21:715-726], we used the scanning electron microscope to modify the oxide-sharpened pyramidal probe by creating an electron beam deposited tip with a higher aspect ratio than unmodified tips. Placing the probes in the scanning electron microscope for modification prompted us to begin to examine the probes for defects both before and after use with the atomic force microscope. The most frequently encountered defect was a mis-centered probe, or a probe hanging off the end of the cantilever. If we had difficulty imaging with a probe, we would examine the probe in the scanning electron microscope to determine if any defects were present, or if the tip had become contaminated during scanning. Moreover, we observed that electron beam deposited tips were blunted by the act of scanning a hard specimen, such as colloidal gold with the atomic force microscope. We also present a mathematical geometric model for deducing the interaction between an electron beam deposited tip and either a spherical or elliptical specimen. Examination of probes in the scanning electron microscope may assist in interpreting images generated by the atomic force microscope.     

A charge coupled device camera with electron decelerator for intermediate voltage electron microscopy

Electron microscopists are increasingly turning to intermediate voltage electron microscopes (IVEMs) operating at 300-400 kV for a wide range of studies. They are also increasingly taking advantage of slow-scan charge coupled device (CCD) cameras, which have become widely used on electron microscopes. Under some conditions, CCDs provide an improvement in data quality over photographic film, as well as the many advantages of direct digital readout. However, CCD performance is seriously degraded on IVEMs compared to the more conventional 100 kV microscopes. In order to increase the efficiency and quality of data recording on IVEMs, we have developed a CCD camera system in which the electrons are decelerated to below 100 kV before impacting the camera, resulting in greatly improved performance in both signal quality and resolution compared to other CCDs used in electron microscopy. These improvements will allow high-quality image and diffraction data to be collected directly with the CCD, enabling improvements in data collection for applications including high-resolution electron crystallography, single particle reconstruction of protein structures, tomographic studies of cell ultrastructure, and remote microscope operation. This approach will enable us to use even larger format CCD chips that are being developed with smaller pixels.     

A precise stage arrangement for correlative microscopy for specimens mounted on glass slides,stubs or EM grids

The instrumentation necessary for precise and fast correlation of images derived from a light optical microscope (LM) and a scanning electron microscope (SEM) operated in the reflective mode, is described. The specimens can be mounted on standard microscope slides (25 × 75 mm), SEM-stubs (12 mm ø), or on transmission EM grids (3 mm ø). The instrumentation consists of two parts: an attachable precision stage for an LM, and an attachable slide carrier for the stage of an SEM. By taking into account the vernier readings of the stages of both microscopes (LM and SEM), identical particles in a specimen can be found instantaneously under either microscope. Therefore it is concluded that the use of this instrumentation in correlative microscopy (LM → SEM → LM) is time saving, and especially recommended on fragile biological specimens, which may deteriorate rapidly under the electron beam of an SEM.     

Visualization of substructure in ferritin molecules: an artifact

It has been suggested in the literature that the core of the ferritin molecule contains a tetrad or other substructure resolvable in electron micrographs. The work described in this paper indicates: 1. At a potential resolution of 5 Å, near-focus electron micrographs of ferritin molecules show no substructure in the core. 2. By defocusing the microscope a computed amount, electron micrographs of the same ferritin molecule can be produced which show apparent core substructure similar to that shown in several patterns reported in the literature. 3. Apparent ferritin molecules with or without substructure, depending upon the phase granularity in the negative, can be added in printing by over-exposing an area of the background equal in size to a ferritin molecule. 4. Underfocus contrast enhancement, frequently used by electron microscopists at lower magnifications without loss of resolution, does result in a loss of resolution at maximum useful magnifications. 5. The established physical principles of electron-optical image formation which should be applied to all high-resolution electron microscopy to distinguish substructure from artifact, are reviewed. This work is compatible with recent X-ray diffraction studies of ferritin molecules which indicate that iron-containing micelles in the core of ferritin are smaller than the practical resolution of the best electron microscopes and therefore neither they nor their substructure could be imaged.     

Contrast and resolution from biological objects examined in the electron microscope with particular reference to negatively stained specimens

For the practical biologist applying electron microscopy to the study of biological macromolecules, there are serious problems in obtaining high resolution images showing detail below 2.5–3.0 nm. The limitation in resolution from biological specimens can be attributed to support film thickness and granularity, specimen preparation, irradiation damage, focusing effects and possible contamination in the electron beam. Specimens possessing repeating features can be analysed and averaged by optical diffraction and image reconstruction methods which offer some improvement to the signal to noise ratio. The above problems, with particular reference to irradiation damage, still impose the basic limitation for high resolution applications. When considered together they offer formidable difficulties in practical terms in attempting to make full use of the potential resolving power of modern electron microscopes.     

Compact, low power radio frequency cavity for femtosecond electron microscopy

Reported here is the design, construction, and characterization of a small, power efficient, tunable dielectric filled cavity for the creation of femtosecond electron bunches in an existing electron microscope without the mandatory use of femtosecond lasers. A 3 GHz pillbox cavity operating in the TM(110) mode was specially designed for chopping the beam of a 30 keV scanning electron microscope. The dielectric material used is ZrTiO(4), chosen for the high relative permittivity (ε(r) = 37 at 10 GHz) and low loss tangent (tan δ = 2 × 10(-4)). This allows the cavity radius to be reduced by a factor of six, while the power consumption is reduced by an order of magnitude compared to a vacuum pillbox cavity. These features make this cavity ideal as a module for existing electron microscopes, and an alternative to femtosecond laser systems integrated with electron microscopes.     

Automated monitoring to reduce electron microscope downtime

High-end transmission electron microscopes are complex and sensitive instruments. Failure of one of the external supplies, malfunction of the microscope hardware or maloperation are typical reasons for subsystems to fail. Especially if undiscovered for a longer period of time, this can cause unnecessary downtime, compromising user access and increasing operating costs. Utilizing the software introduced in this article (“MoniTEM”), we have succeeded to reduce downtime of an FEI Tecnai Polara by coupling constant monitoring of critical subsystems with automatic, remote feedback to the system supervisor, ensuring immediate problem solving. The software described here is freely available from http://www.imba.oeaw.ac.at/monitem/ and can be readily adapted for use with other FEI transmission electron microscopes.     

软X射线光学技术的某些进展

本文主要介绍软X射线等离子体光源、多层膜软X射线反射镜和软X射线显微镜。高强度的实验室用软X射线等离子体光源较之同步辐射具有体积小、造价低、光束大和单位脉冲光通量高等优点。最近发展的多层镀膜用于软X射线正入射光学元件,可以得到比掠入射光学元件好得多的分辨率。软X射线显微镜提供了生物样品分析的一种新工具,它填补了常用的光学显微镜和电子显微镜之间的空隙。     

On the extinction coefficient in confocal polarization microscopy

The extinction coefficient in conventional polarized light microscopes is non-zero, even with perfect polars, solely because of the image formation process in these instruments. The imaging in confocal microscopes is different from conventional instruments and it is shown that in this case an infinite extinction coefficient results in the ideal case whereas a finite value is always to be expected with conventional instruments. Images of the same microelectronic specimen taken in both conventional and confocal polarized light microscopes are compared.     

3-D Optical Interference Microscopy at the Lateral Resolution

For applications in micro- and nanotechnologies the lateral resolution of optical 3-D microscopes becomes an issue of increasing relevance. However, lateral resolution of 3-D microscopes is hard to define in a satisfying way. Therefore, we first study the measurement capabilities of a highly resolving white-light interference (WLI) microscope close to the limit of lateral resolution. Results of measurements and simulations demonstrate that better lateral resolution seems to be achievable based on the envelope evaluation of a WLI signal. Unfortunately, close to the lateral resolution limit errors in the measured amplitude of micro-structures appear. On the other hand, results of interferometric phase evaluation seem to be strongly low-pass filtered in this case.

Furthermore, the instrument transfer characteristics and the lateral resolution capabilities of WLI instruments are also affected by polarization. TM polarized light is less sensitive to edge diffraction and thus systematic errors can be avoided. However, apart from ghost steps due to fringe order errors, the results of phase evaluation seem to be closer to the real surface topography if TE polarized light is used. The lateral resolution can be further improved by combining WLI and structured illumination microscopy. Since the measured height of rectangular profiles close to the lateral resolution limit is generally too small compared to the real height, we introduce a method based on phase evaluation which characterizes the heights of barely laterally resolved rectangular gratings correctly.     

A small scanning tunnelling microscope with large scan range for biological studies

We have developed a scanning tunnelling microscope specially designed for biological applications presenting some new features: the scanner tube is mounted parallel to the surface of the sample which enables a high resolution optical microscope to be brought close to the sample when working in air or liquids. The maximum scan range is 5×20 μm with a vertical range of 20 μm and the total size of the system does not exceed 10×40 mm. The piezo-sensitivity of the scanner tube versus applied voltage was analysed by interferometry measurements and by using scanning tunnelling microscopes. We found a value for the piezoelectric constant d₁₃ of ?1·71 Å/V at low voltages (under a few volts) going up to ?2 Å/V for higher voltages. Large-scale images of a carbon grid showed a surprisingly good linearity of the scanner tube.