Prospects for aberration-free electron microscopy

Future aberration-corrected electron microscopes that will enable sub-Angstroem spatial and sub-eV energy resolution are outlined . The sub-Angstroem transmission electron microscope (SATEM) only compensates for the spherical aberration and reduces the chromatic aberration disc by means of a monochromator. In order to correct for both aberrations, two novel correctors, the ultracorrector and the superaplanator are proposed which will yield a resolution limit of about 0.5A and a large field of view of more than 4 x 10(6) image points. The superaplanator is best suited for obtaining an achromatic aplanat required for the realization of the high-performance in situ electron microscope of the TEAM project.     

The future of the electron microscope*

The future of electron microscopy lies as much with the conventional as with the newer instruments. The point resolving power of the latter is likely to be pushed to 1 Å, but only after a considerable effort in solving problems of mechanical and electrical stability. Progress in correcting the lens aberrations is even slower. Techniques of specimen preparation and microscope operation are continuously being improved, but will need even greater refinement if proper use is to be made of a 1 Å resolving power, e.g. for identifying bases in a nucleic acid molecule. Extension of the working voltage to 1 MeV and above is increasing the usefulness of the conventional electron microscope, particularly in metallurgy, and plans are now being made for even higher voltages. Of the newer, unconventional instruments, the scanning electron microscope is already establishing a place for itself, especially in industrial applications where surface conditions on the microscale are important. It is likely to find increasing use in micro-circuitry, but also in some branches of biological research, even if its resolving power cannot be brought below 100 Å. The combination of X-ray spectrometry with electron microscopy holds promise of wide application, in the form of a hybrid electron microscope-microanalyser. Secondary-emission microscopes are slowly finding a place for themselves, mainly in applied science and technology. Mirror microscopes, for surface investigations, and ion microscopes, for microanalysis, are still in an early stage but have interesting possibilities. The field ion microscope, simplest in principle but sophisticated in technique, is unique in showing the position of individual atoms in a metal tip. It is already finding applications, especially in studies of radiation damage.     

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.     

Minimization of dose as a criterion for the selection of imaging modes in electron microscopy of amorphous specimens.

A fundamental limitation in electron microscopy of organic specimens is radiation damage by the electron beam. To minimize damage it is necessary to have maximum information collection for a given dose. Various modes of operation of conventional and scanning transmission microscopes are compared with respect to their ability to detect small changes in specimen thickness or density with a given signal to noise ratio. Incoherent imaging is assumed, and this is expected to apply to amorphous specimens under a variety of microscope conditions. For either very thin or very thick specimens, the scanning transmission microscope is found to require nearly 10 times less dose than a conventional microscope for the same signal to noise ratio in the image. For specimens of intermediate thickness, scanning and conventional transmission electron microscopes are roughly equivalent.     

PHOEBE,a prototype scanning laser-feedback microscope for imaging biological cells in aqueous media

Based on the principle of laser-feedback interferometry (LFI), a laser-feedback microscope (LFM) has been constructed capable of providing an axial (z) resolution of a target surface topography of ～ 1 nm and a lateral (x, y) resolution of ～ 200 nm when used with a high-numerical-aperture oil-immersion microscope objective. LFI is a form of interferometry in which a laser's intensity is modulated by light re-entering the illuminating laser. Interfering with the light circulating in the laser resonant cavity, this back-reflected light gives information about an object's position and reflectivity. Using a 1-mW He–Ne (λ = 632·8 nm) laser, this microscope (PHOEBE) is capable of obtaining 256 × 256-pixel images over fields from (10 μm × 10 μm) to (120 μm × 120 μm) in ～ 30 s. An electromechanical feedback circuit holds the optical pathlength between the laser output mirror and a point on the scanned object constant; this allows two types of images (surface topography and surface reflectivity) to be obtained simultaneously. For biological cells, imaging can be accomplished using back-reflected light originating from small refractive-index changes (> 0·02) at cell membrane/water interfaces; alternatively, the optical pathlength through the cell interior can be measured point-by-point by growing or placing a cell suspension on a higher-reflecting substrate (glass or a silicon wafer). Advantages of the laser-feedback microscope in comparison to other confocal optical microscopes include: the simplicity of the single-axis interferometric design; the confocal property of the laser-feedback microscope (a virtual pinhole), which is achieved by the requirement that only light that re-enters the laser meeting the stringent frequency, spatial (TEM₀₀), and coherence requirements of the laser cavity resonator mode modulate the laser intensity; and the improved axial resolution, which is based on interferometric measurement of optical amplitude and phase rather than by use of a pinhole as in other types of confocal microscopes.     

Broadband dielectric microwave microscopy on micron length scales

We demonstrate that a near-field microwave microscope based on a transmission line resonator allows imaging in a substantially wide range of frequencies, so that the microscope properties approach those of a spatially resolved impedance analyzer. In the case of an electric probe, the broadband imaging can be used in a direct fashion to separate contributions from capacitive and resistive properties of a sample at length scales on the order of one micron. Using a microwave near-field microscope based on a transmission line resonator we imaged the local dielectric properties of a focused ion beam milled structure on a high-dielectric-constant Ba(0.6)Sr(0.4)TiO(3) thin film in the frequency range from 1.3 to 17.4 GHz. The electrostatic approximation breaks down already at frequencies above approximately 10 GHz for the probe geometry used, and a full-wave analysis is necessary to obtain qualitative information from the images.     

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.     

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.     

Electron cyclotron resonance ion source plasma chamber studies using a network analyzer as a loaded cavity probe

A method and first results utilizing a network analyzer as a loaded cavity probe to study the resonance properties of a plasma filled electron cyclotron resonance ion source (ECRIS) plasma chamber are presented. The loaded cavity measurements have been performed using a dual port technique, in which two separate waveguides were used simultaneously. One port was used to ignite and sustain the plasma with a microwave source operating around 11 GHz and the other was used to probe the cavity properties with the network analyzer using a frequency range around 14 GHz. The first results obtained with the JYFL 14 GHz ECRIS demonstrate that the presence of plasma has significant effects on the resonance properties of the cavity. With plasma the frequency dependent behavior is strongly damped and this trend strengthens with increasing microwave power.     

Reflection electron energy-loss spectroscopy and imaging for surface studies in transmission electron microscopes.

A review is given on the techniques and applications of high-energy reflection electron energy-loss spectroscopy (REELS) and reflection electron microscopy (REM) for surface studies in scanning transmission electron microscopes (STEM) and conventional transmission electron microscopes (TEM). A diffraction method is introduced to identify a surface orientation in the geometry of REM. The surface dielectric response theory is presented and applied for studying alpha-alumina surfaces. Domains of the alpha-alumina (012) surface initially terminated with oxygen can be reduced by an intense electron beam to produce Al metal; the resistance to beam damage of surface domains initially terminated with Al+3 ions is attributed to the screening effect of adsorbed oxygen. Surface energy-loss near-edge structure (ELNES), extended energy-loss fine structure (EXELFS), and microanalysis using REELS are illustrated based on the studies of TiO2 and MgO. Effects of surface resonances (or channeling) on the REELS signal-to-background ratio are described. The REELS detection of a monolayer of oxygen adsorption on diamond (111) surfaces is reported. It is shown that phase contrast REM image content can be significantly increased with the use of a field emission gun (FEG). Phase contrast effects close to the core of a screw dislocation are discussed and the associated Fresnel fringes around a surface step are observed. Finally, an in situ REM experiment is described for studying atomic desorption and diffusion processes on alpha-alumina surfaces at temperatures of 1,300-1,400 degrees C.     

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。     

Determination of dielectric constant and loss of high-K thin films in the microwave frequencies

A method to determine the dielectric constant and loss of high-K thin film dielectrics in the microwave frequency region using the extended cavity perturbation technique is presented. The feasibility of the technique is demonstrated by the determination of the dielectric constant and loss for reactively sputtered TiO₂ thin films on borosilicate glass substrates. The dielectric constant and loss is measured at 8.98, 9.96 and 10.97 GHz using a TE_10n rectangular cavity. Using this technique, the dielectric properties of TiO₂ films deposited under varying oxygen percentage in the sputtering atmosphere from 20% to 100% were measured. The dielectric constant and loss are found to be dependent on both the oxygen partial pressure as well as frequency of measurement. The film deposited at 50% of oxygen had a higher dielectric constant, ε_r = 44.35 at 8.98 GHz, where as the film deposited at 100% oxygen showed the lowest value of dielectric constant, ε_r = 21.36 at 10.97 GHz. The dielectric loss tangent varied from 0.004 to 0.019 depending on frequency and oxygen partial pressure. However this technique is applicable only for thin films coated on low K dielectric substrates.     

Integration of a high‐NA light microscope in a scanning electron microscope

We present an integrated light‐electron microscope in which an inverted high‐NA objective lens is positioned inside a scanning electron microscope (SEM). The SEM objective lens and the light objective lens have a common axis and focal plane, allowing high‐resolution optical microscopy and scanning electron microscopy on the same area of a sample simultaneously. Components for light illumination and detection can be mounted outside the vacuum, enabling flexibility in the construction of the light microscope. The light objective lens can be positioned underneath the SEM objective lens during operation for sub‐10 μm alignment of the fields of view of the light and electron microscopes. We demonstrate in situ epifluorescence microscopy in the SEM with a numerical aperture of 1.4 using vacuum‐compatible immersion oil. For a 40‐nm‐diameter fluorescent polymer nanoparticle, an intensity profile with a FWHM of 380 nm is measured whereas the SEM performance is uncompromised. The integrated instrument may offer new possibilities for correlative light and electron microscopy in the life sciences as well as in physics and chemistry.     

显微观测技术的新进展及其应用

依据显微观测技术的发展过程 ,介绍了普通的光学显微镜和 2 0世纪流行的电子显微镜 ,详细阐述了以扫描隧道显微镜和激光扫描共焦显微镜为代表的新型显微镜系列的发展 ,以及各类显微镜的基本工作原理和应用情况     

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.     

Scanning probe microscopy of biological samples and other surfaces

Scanning probe microscopes derived from the scanning tunnelling microscope (STM) offer new ways to examine surfaces of biological samples and technologically important materials. The surfaces of conductive and semiconductive samples can readily be imaged with the STM. Unfortunately, most surfaces are not conductive. Three alternative approaches were used in our laboratory to image such surfaces. 1. Crystals of an amino acid were imaged with the atomic force microscope (AFM) to molecular resolution with a force of order 10^?8 N. However, it appears that for most biological systems to be imaged, the atomic force microscope should be able to operate at forces at least one and perhaps several orders of magnitude smaller. The substitution of optical detection of the cantilever bending for the measurement by electron tunnelling improved the reliability of the instrument considerably. 2. Conductive replicas of non-conductive surfaces enabled the imaging of biological surfaces with an STM with a lateral resolution comparable to that of the transmission electron microscope. Unlike the transmission electron microscope, the STM also measures the heights of the features. 3. The scanning ion conductance microscope scans a micropipette with an opening diameter of 0·04-0·1 μm at constant ionic conductance over a surface covered with a conducting solution (e.g., the surface of plant leaves in saline solution).     

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.     

Construction and characterization of the fringe field monochromator for a field emission gun

Although some microscopes have shown stabilities sufficient to attain below 0.1 eV spectral resolution in high-resolution electron energy loss spectroscopy, the intrinsic energy width of the high brightness source (0.3-0.6 eV) has been limiting the resolution. To lower the energy width of the source to 50 meV without unnecessary loss of brightness, a monochromator has been designed consisting of a short (4 mm) fringe field Wien filter and a 150 nm energy selection slit (nanoslit) both to be incorporated in the gun area of the microscope. A prototype has been built and tested in an ultra-high-vacuum setup (10(-9) mbar). The monochromator, operating on a Schottky field emission gun, showed stable and reproducible operation. The nanoslits did not contaminate and the structure remained stable. By measuring the current through the slit structure a direct image of the beam in the monochromator could be attained and the monochromator could be aligned without the use of a microscope. Good dispersed imaging conditions were found indicating an ultimate resolution of 55 meV. A Mark II fringe field monochromator (FFM) was designed and constructed compatible with the cold tungsten field emitter of the VG scanning transmission microscope. The monochromator was incorporated in the gun area of the microscope at IBM T.J. Watson research center, New York. The monochromator was aligned on 100 kV and the energy distribution measured using the monochromator displayed a below 50 meV filtering capability. The retarding Wien filter spectrometer was used to show a 61 meV EELS system resolution. The FFM is shown to be a monochromator which can be aligned without the use of the electron microscope. This makes it directly applicable for scanning transmission microscopy and low-voltage scanning electron microscopy, where it can lower the resolution loss which is caused by chromatic blur of the spot.     

Broadband electron spin resonance at 4-40 GHz and magnetic fields up to 10 T

A broadband electron spin resonance spectrometer is described which operates at frequencies between 4 and 40 GHz and can be used in superconducting magnets. A tunable cylindrical cavity is connected to a vector network analyzer via coaxial cables, and the radiation is fed into the cavity by a coupling loop. No field modulation is employed. Resonance frequencies below 14 GHz are obtained by inserting dielectrics with different permittivities into the cavity. The setup allows for measurements with the microwave magnetic field either parallel or perpendicular to the external field.     

Effect of a confocal pinhole in two-photon microscopy.

We investigated the effect of a finite-sized confocal pinhole on the performance of nonlinear optical microscopes based on two-photon excited fluorescence and second-harmonic generation. These techniques were implemented using a modified inverted commercial confocal microscope coupled to a femtosecond Ti:sapphire laser. Both the transverse and axial resolutions are improved when the confocal pinhole is used, albeit at the expense of the signal level. Therefore, the routine use of a confocal pinhole of optimized size is recommended for two-photon microscopy wherever the fluorescence or harmonic signals are large.