Scanning tunneling microscope combined with scanning electron microscope

A scanning tunneling microscope (STM) has been installed in a usual scanning electron microscope (SEM) with a vacuum of 10⁻⁶ Torr. The STM image is displayed on the cathode ray tube of the SEM, 512 × 512 pixels, with a scanning rate of 80 s/picture. The spatial resolution of the STM is about 1 Å, while that of the SEM is several tens of ångströms. The combined scanning microscope covers a wide magnification range from 10 to 10⁷, where STM covers the high magnification region from 10⁵ to 10⁷.     

A wet stage modification to a scanning electron microscope.

A modification to the vacuum system of a JSM2 scanning electron microscope has enabled hydrated specimens to be placed inside the specimen chamber of the instrument and to be surronded by water vapour at a pressure up to approximately I 3-kPa (10 Torr). The surface topography was observed by detecting the backscattered electrons using a wide angle backscattered electron detector placed close to the specimen. The microscope was operated in the normal scanning mode which allowed the examination of the surface topography of the specimens, whilst still retaining the depth of focus which is a feature of the SEM. This modification has enabled a resolution of approximately 0.2 mum to be obtained from biological specimens partially immersed in water at temperatures just above 0 degrees C.     

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.     

Early results using high-resolution,low-voltage,low-temperature SEM

Recent advances in the design of the scanning electron microscope (SEM) column, such as the coupling of a field-emission gun to a low-aberration immersion lens and the availability of a high-stability cryo-transfer stage, make low-temperature, low-voltage SEM (LTLVSEM) possible at very high resolution. We have used this combination to obtain results with uncoated biological specimens. The trichocyst from a Paramecium was used as a test specimen to observe the shrinkage of this structure as the temperature is raised from 170 K to room temperature following freeze-drying. High-magnification stereo images were obtained of trichocysts that had been prepared by freezing, freeze-substitution and critical-point drying and which were subsequently viewed by LTLVSEM to reduce beam damage and contamination.     

Soft X-ray microscopy with a cryo scanning transmission X-ray microscope: I. Instrumentation, imaging and spectroscopy

We have developed a cryo scanning transmission X-ray microscope which uses soft X-rays from the National Synchrotron Light Source. The system is capable of imaging frozen hydrated specimens with a thickness of up to 10 μm at temperatures of around 100 K. We show images and spectra from frozen hydrated eukaryotic cells, and a demonstration that biological specimens do not suffer mass loss or morphological changes at radiation doses up to about 10¹⁰ Gray. This makes possible studies where multiple images of the same specimen area are needed, such as tomography ( Wang et al. (2000 ) Soft X-ray microscopy with a cryo scanning transmission X-ray microscope: II. Tomography. J. Microsc . 197, 80–93) or spectroscopic analysis.     

Analytical scanning electron microscopy for solid surface

A scanning electron microscope of ultra-high-vacuum (UHV-SEM) with a field emission gun (FEG) is operated at the primary electron energies of from 100 eV to 3 keV. The instrument can form the images that contain information on surface chemical composition, chemical bonding state (electronic structure), and surface crystal structure in a microscopic resolution of several hundred angstroms (Å) using the techniques of scanning Auger electron microscope, scanning electron energy loss microscope, and scanning low-energy electron diffraction (LEED) microscope. A scanning tunneling microscope (STM) also has been combined with the SEM in order to obtain the atomic resolution for the solid surface. The instrumentation and examples of their applications are presented both for scanning LEED microscopy and STM.     

A study of living plant specimens by low-temperature scanning electron microscopy

Young fresh Tradescantia reflexa stamen hair cells were used to clarify the optimal conditions for direct viewing and taking photographs with a scanning electron microscope (SEM) equipped with a cryo-system. The rate of protoplasmic streaming in the cells was measured under an optical microscope after examining and photographing them in the SEM over a period of a few minutes. Almost the same rate of streaming (5.5 μm/second, 20°C) was observed in nonirradiated control cells and irradiated cells photographed in the SEM using an accelerating voltage of 10 kV with the cryo-stage at a temperature of – 15°C. (The specimen holder and specimen were not at this temperature, but, rather, probably somewhat higher.) Fresh plant organs, tissues, and cells were also tested under the same conditions. The fine structure was well preserved in detail. The procedures were as follows: (1) prompt attachment of fresh plant materials on an aluminum specimen holder with double-faced adhesive Scotch tape or a small amount of plastic adhesive for woodcraft; (2) setting the holder on the cryo-stage cooled to –15°C in advance and rapid evacuation; and (3) quick SEM examination and photography (within several minutes). The advantages of this method are summarized as follows: (1) high possibility of viewing living materials; (2) minimal artifacts: freedom from chemical fixation and additional procedures utilized in ordinary SEM specimen preparation; and (3) simplicity, speediness, and economy in preparation for viewing. Since the specimens were not likely to be frozen during quick examination and photography, this method might well be called “low-temperature SEM” (LT-SEM) as distinguished from “cryo-SEM”.     

蠕墨铸铁气缸盖激光热冲击研究

试验研究:整形后的激光热冲击蠕墨铸铁气缸盖.通过红外测温仪监测特征点温度波动;采用CCD摄像头监控试样的表面状态.通过扫描电镜(SEM)、光学显微镜(OM)和硬度仪表征了热冲击试验后试样的组织和硬度变化.结果表明:蠕墨铸铁气缸盖通过2000次热冲击;热冲击试验后蠕墨铸铁气缸盖的组织和硬度没有明显变化,说明在热冲击试验温...     

Beam transfer characteristics of a commercial environmental SEM and a low vacuum SEM

Two commercial instruments that permit a gaseous environment in their specimen chamber have been investigated, namely, a 'FEI Quanta 600 FEG' environmental scanning electron microscope and a 'LEO SUPRA 35VP FESEM' low vacuum scanning electron microscope. The gas flow field is first computed by the direct simulation Monte Carlo method and the gas density gradient, speed, Mach number and temperature are found in the transition region from high pressure to vacuum. The electron beam transfer characteristics are then derived for different accelerating voltages and pressures and a comparison is made among different situations and with some published works. Certain physical parameters are analysed and discussed to establish a figure of merit that can be used as a standard performance specification for commercial environmental scanning electron microscope and low vacuum scanning electron microscope.     

A MEMS‐based heating holder for the direct imaging of simultaneous in‐situ heating and biasing experiments in scanning/transmission electron microscopes

The introduction of scanning/transmission electron microscopes (S/TEM) with sub‐Angstrom resolution as well as fast and sensitive detection solutions support direct observation of dynamic phenomena in‐situ at the atomic scale. Thereby, in‐situ specimen holders play a crucial role: accurate control of the applied in‐situ stimulus on the nanostructure combined with the overall system stability to assure atomic resolution are paramount for a successful in‐situ S/TEM experiment. For those reasons, MEMS‐based TEM sample holders are becoming one of the preferred choices, also enabling a high precision in measurements of the in‐situ parameter for more reproducible data. A newly developed MEMS‐based microheater is presented in combination with the new NanoEx?‐i/v TEM sample holder. The concept is built on a four‐point probe temperature measurement approach allowing active, accurate local temperature control as well as calorimetry. In this paper, it is shown that it provides high temperature stability up to 1,300°C with a peak temperature of 1,500°C (also working accurately in gaseous environments), high temperature measurement accuracy (<4%) and uniform temperature distribution over the heated specimen area (<1%), enabling not only in‐situ S/TEM imaging experiments, but also elemental mapping at elevated temperatures using energy‐dispersive X‐ray spectroscopy (EDS). Moreover, it has the unique capability to enable simultaneous heating and biasing experiments. Microsc. Res. Tech. 79:239–250, 2016. © 2016 Wiley Periodicals, Inc.     

Scanning reflection image from a solid specimen in the scanning electron microscope with a condenser-objective lens

To achieve the highest resolution in the scanning electron microscope (SEM) or in the scanning transmission electron microscope (STEM), the sample must be mounted in the high-field region of a condenser-objective lens. A secondary electron (SE) image can then be obtained using a collector before the lens. It is also possible to obtain a scanning reflection image by tilting the specimen so that the second half of the condenser-objective lens field deflects the forward-scattered electrons onto the transmission detector beyond the specimen. Experiments were made with an unmodified commercial SEM fitted with a condenser-objective in the upper stage and with a transmission detector, and it was found that the scanning reflection image from a solid sample can provide additional useful information when used in conjunction with the SE image.     

10-kV diffractive imaging using newly developed electron diffraction microscope

A new electron diffraction microscope based on a conventional scanning electron microscope (SEM), for obtaining atomic-level resolution images without causing serious damage to the specimen, has been developed. This microscope in the relatively low-voltage region makes it possible to observe specimens at suitable resolution and record diffraction patterns. Using the microscope we accomplished 10-kV diffractive imaging with the iterative phase retrieval and reconstructed the structure of a multi-wall carbon nanotube with its finest feature corresponding to 0.34-nm carbon wall spacing. These results demonstrate the possibility of seamless connection between observing specimens by SEM and obtaining their images at high resolution by diffractive imaging.     

A high-resolution add-on in-lens attachment for scanning electron microscopes

A compact add-on objective lens for the scanning electron microscope (SEM) has been designed and tested. The lens is < 35 mm high and can be fitted on to the specimen stage as an easy-to-use attachment. Initial results show that it typically improves the spatial resolution of the SEM by a factor of three. The add-on unit is based upon a permanent magnet immersion lens design. Apart from the extra attachment to the specimen stage, the SEM with the add-on lens functions in the normal way. The in-lens unit can comfortably accommodate specimen heights up to 10 mm. The new add-on lens unit opens up the possibility of operating existing SEMs in the high-resolution in-lens mode. By using a deflector at the top of the add-on lens unit, it can also operate as a quantitative multichannel voltage contrast spectrometer, capable of recording the energy spectrum of the emitted secondary electrons. Initial experiments confirm that a significant amount of voltage contrast can be obtained.     

High‐resolution cryo‐SEM allows direct identification of F‐actin at the inner nuclear membrane of Xenopus oocytes by virtue of its structural features

The nuclear envelope of Xenopus laevis stage VI oocytes was studied in a high‐resolution field emission cryo‐scanning electron microscope to compare the level of structural preservation obtainable by different procedures of specimen preparation. All approaches generally allowed frequent detection of long filaments of about 10 nm in diameter that were attached to the nuclear envelope's inner membrane facing the nuclear interior. Structural details of these 10‐nm filaments, however, could not be unveiled by standard procedures of specimen preparation and analysis, including critical point drying and imaging at room temperature. In contrast, after freeze‐drying and imaging at ?100°C, the 10‐nm filament type was found to be composed of distinct globular subunits of approximately 5 nm in diameter that were arranged in a helical manner with right‐handed periodicity. Stereoscopic images showed that some of these filaments were lying directly on the membrane whereas others appeared to hover at a certain distance above the nuclear envelope. The appearance of these filaments was highly similar to that of in vitro polymerized F‐actin analysed in parallel, and closely resembled the structural characteristics of F‐actin filaments described earlier. By virtue of their structural features we therefore conclude that these filaments at the nuclear periphery represent F‐actin. The high level of structural resolution obtainable by field emission cryo‐SEM illustrates the potential of this method for studying details of biological structures in a subcellular context.     

Freeze-fracturing for conventional and field emission low-temperature scanning electron microscopy: the scanning cryo unit SCU 020

A dedicated cryopreparation system, the SCU 020 (Balzers), is introduced and described in detail for use in low-temperature scanning electron microscopy (LTSEM). The basic unit consists of two parts: (i) a high-vacuum preparation chamber equipped with a cold-stage, motor-driven fracturing microtome, planar magnetron (PM) sputter source, quartz-crystal thin-film monitor, Meissner cold trap, and turbo molecular pump stand; and (ii) a second part (separated from the first by a sliding, high-vacuum valve) residing in the SEM chamber. This is equipped with an anti-contamination cold trap, a fully movable goniometer cold stage (having motor drives for x, y, and rotation) and replaces the SEM's original stage (Raith). The SCU 020 is entirely self contained allowing independence from, and synchroneity with, the SEM of choice. LTSEM micrographs of specimen (that are fully frozen hydrated or partially freeze-dried) surfaces or fracture faces, without or with various metal coatings, can be examined over a broad temperature range (-150 to +50°C). This is made possible by the combined application of the two, independently controlled, cold stages and the on-line, high-vacuum, specimen cryo transfer between them. In-situ etching is simple and straightforward. Intramembranous particles and membrane fracture steps, typically imaged in transmission electron microscopy (TEM), are resolved by PM sputtering with platinum at low specimen temperature and high-resolution LTSEM in a field emission microscope.     

The use of light and electron microscopy in the study of experimentally altered spores and pollen grains

Plant spores and pollen grains were heated to different temperatures, from room temperature to 350°C at atmospheric pressure, in a nichrome-wire resistance furnace, and to different temperatures from 100 to 500°C at 1 kb pressure in a modified apparatus normally used for triaxial rock-deformation studies. In the temperature and pressure experiments, the grains were mixed with silica sand and sea water in the proportions of 1:4:5. For optical microscopy, the pollen grains were mounted in glycerine jelly: for SEM, they were mounted on aluminium stubs rinsed with water and sputter-coated with gold. STEM and HVEM were used on thick sections of spores of Lycopodium clavatum embedded in Epon-Araldite. The different layers of the spore or pollen-grain (i.e. exine+intine) degrade differently. The ornamented part of the exine (the sexine?mainly composed of sporopollenin, lipids and polysaccharides) begins to degrade at approximately 300–350°C and 1 kb pressure, exposing the non-ornamented nexine layer. The nexine seems able to withstand high temperatures and pressures. Between 300 and 500°C with 1 kb pressure, the grains begin to form an amorphous mass. Compared with other work in which the outer exine layer of L. clavatum was removed by oxidation with ozone, here the same layer was gradually removed by pressure and temperature effects. The smell of phenolic compounds is very prominent at higher temperatures (400°C with 1 kb pressure and upwards) and production of other gas, unidentified so far, is evident from about 300°C and 1 kb pressure. The degraded exposed nexine layer at 500°C resembles thin plates in the SEM. A similar structure was also found in L. clavatum heated at 300°C and 1 kb pressure. The exposed nexine of L. clavatum was sectioned and examined in the TEM, which showed the trilamellar units, of which the main bulk of the nexine is composed, to be very resistant to high temperatures and pressures. STEM and HVEM have been used to study globular structures observed in the L. clavatum spore cavity.     

Cryogenic EBSD on ice: preserving a stable surface in a low pressure SEM

Naturally deformed ice contains subgrains with characteristic geometries that have recently been identified in etched surfaces using high-resolution light microscopy (LM). The probable slip systems responsible for these subgrain boundary types can be determined using electron backscattered diffraction (EBSD), providing the etch features imaged with reflected LM can be retained during EBSD data acquisition in a scanning electron microscope (SEM). Retention of the etch features requires that the ice surface is stable. Depending on the pressure and temperature, sublimation of ice can occur. The equilibrium temperature for a low pressure SEM operating at 1 × 10(-6) hPa is about -112°C and operating at higher temperatures causes sublimation. Although charging of uncoated ice samples is reduced by sublimation, important information contained in the etch features are removed as the surface sublimes. We developed a method for collecting EBSD data on stable ice surfaces in a low pressure SEM. We found that operating at temperatures of <-112°C reduced sublimation so that the original etch surface features were retained. Charging, which occurred at low pressures (<1.5 × 10(-6) to 2.8 × 10(-5) hPa) was reduced by defocusing the beam. At very low pressures (<1.5 × 10(-6) hPa) the spatial resolution with a defocused beam at 10 kV was about 3 μm in the x-direction at -150°C and 0.5 μm at -120°C, because at higher temperature charging was less and only a small defocus was needed to compensate it. Angular resolution was better than 0.7° after orientation averaging. Excellent agreement was obtained between LM etch features and EBSD mapped microstructures. First results are shown, which indicate subgrain boundary types comprised of basal (tilt and twist) and nonbasal dislocations (tilt boundaries).     

Near-field observation of carrier diffusion in GaAs quantum structures under high magnetic fields

Photoluminescence from a two‐dimensional electron‐gas system in GaAs single hetero‐structures was investigated using a scanning near‐field optical microscope (SNOM) operated at cryogenic temperatures under high magnetic fields. The local intensity of the luminescence increased 600‐fold that at 0 T as the magnetic field was increased up to 6 T. The enhancement depended on the spatial resolution of the SNOM. These characteristics are explained by the suppression of the diffusion of photocarriers caused by the Lorentz force in magnetic fields.     

Simple modifications for accurate high-temperature gas exposures using scanning electron microscopy

Relatively low-cost modifications to standard commercial scanning electron microscopes (SEM) that allow accurate exposure of sample(s) to noncorrosive gases at ambient and high temperatures are outlined. Energy-dispersive spectroscopic analysis of sample(s) exposed to noncorrosive gases at high temperatures is demonstrated. Gas exposure is limited to pressures of less than 10^?4 torr (1.33 × 10^?2 Pa) as a result of limitations on SEM system operation and SEM safety interlocks. Gases are limited to noncorrosive types as a result of potential damage to system detection devices and internal mechanical parts.