扫描电镜的不同含水量植物叶片样品的处理及观察方法研究

为探讨不同含水量植物叶片进行扫描电镜观察的最合适的样品处理方法,及不同电子束加速电压对扫描电镜图像清晰度的影响,对不同含水量叶片进行了比较性研究。并通过理论分析得出：含水量小于75％的植物叶片可不经处理,直接进行观察,大于79％的叶片必须经过样品制备后观察;5—15KV加速电压时获得的图像最清晰。     

Changes in particle area measurements due to SEM accelerating voltage and magnification

Scanning electron microscopy (SEM) images of polymer blends followed by digital image analysis is a rapid and easy method for the measurement of particle size and dispersion. The particle size determination is done with appropriate off-line image analysis software. However, it is necessary to understand how machine parameters involved in the formation of the SEM image influence area measurements of morphological features. In this work, the influence of the accelerating voltage used during image acquisition was examined with standard samples and with polymer blend samples. A systematic study centered on two mutually exclusive assumptions of area variation or no area variation with accelerating voltage was carried out. The off-line image analysis software was then calibrated according to the assumptions. The main conclusion of this study was that kV has an important influence on area measurement in SEM images. This effect was observed for different standard materials (metallic and polymeric) and for the range of magnifications used. The higher the accelerating voltages, the greater the error at high magnification for polymer samples. As the beam energy increases, the primary electrons penetrate more deeply into the solid specimen, producing low-resolution signals. These signals degrade the image and surface details, which became less well defined. Therefore, images of polymer samples must be taken at lower accelerating voltages so the desired surface details can be imaged clearly. To avoid area measurement errors, particle measurement must be done with the calibration of the off-line image analysis software corresponding to the accelerating voltage and magnification used for the acquired images.     

Thermal effects of the electron beam and implications of surface damage in the analysis of bone tissue

Electron beam interactions with specimens in the scanning electron microscope (SEM) can lead to increased surface temperatures and damage. These changes may have significant consequences in the analysis of bone tissue. An investigation was performed to measure the surface temperature changes associated with the electron beam on a thermocouple with systematic variations in operating conditions. Probe currents, magnifications, and accelerating voltages were incrementally adjusted to measure the temperature changes and to make assessments for determining optimal operating conditions for the SEM in future analyses of bone tissue. Results from this study suggest that thermal effects were minimal at lower accelerating voltages (< 20 kV), lower probe currents (< 10 nA), and lower magnifications, but surface damage may still occur during the analysis of bone tissue.     

Modification of a commercial SEM with a computer controlled cathode stabilized power supply

Modification of the high-voltage circuitry of the thermionic electron gun of a commercial scanning electron microscope (SEM) to a fully computer-controlled, cathode-stabilized system is described. The modification permits automatic filament saturation and monitoring. Using this system, variations and drifts in the emission current over time can be adjusted and compensated automatically with a feedback loop in the computer-controlled system. This change also enables the accurate determination and setting of the primary electron beam energy. Therefore, the accelerating voltage is known for comparison to Monte Carlo computer modeling of the electron beam specimen interaction.     

A novel method for the discharge of electrostatic mirror formations in the scanning electron microscope

A method for the removal of electrostatic mirror formations in the scanning electron microscope (SEM) was conceptualised and implemented. This method utilises the controlled rampdown of the primary beam voltage to achieve mirror discharge. It is found that the new technique is effective in enabling imaging from high-beam voltages (20 kV) down to low voltages (1 kV) without the loss of imaging capability due to electrostatic mirrors. The entire discharge cycle could be performed in ≤ 4 min depending on sample and operating conditions. Hence, this technique compares favourably with the time needed to air the specimen chamber for mirror removal, but without the disadvantage of disrupting SEM operation. This method holds great promise in applications where the imaging of insulating samples at both high and low kV ranges are required.     

Primary considerations for image enhancement in high-pressure scanning electron microscopy

The mechanisms of electron beam scattering are examined to evaluate its effect on contrast and resolution in high-pressure scanning electron microscopy (SEM) techniques reported in the literature, such as moist-environment ambient-temperature SEM (MEATSEM) or environmental SEM (ESEM). The elastic and inelastic scattering cross-sections for nitrogen are calculated in the energy range 5–25 keV. The results for nitrogen are verified by measuring the ionization efficiency, and measurements are also made for water vapour. The effect of the scattered beam on the image contrast was assessed and checked experimentally for a step contrast function at 20 kV beam voltage. A considerable degree of beam scattering can be tolerated in high-pressure SEM operation without a significant degradation in resolution. The image formation and detection techniques in high-pressure SEM are considered in detail in the accompanying paper.     

On the improved phase-grating method

A new method for computing scattering amplitudes in high resolution transmission electron microscopy has been examined. The method, which is called the improved phase-grating (IPG) method, is shown to produce reasonable results only for very small specimen thicknesses and diverges for thicknesses larger than 20–40 Å in copper [001] for accelerating voltages between 200 kV and 1 MV. The validity of the method is discussed and is shown to depend on electron wavelength, slice thickness, the number of reflections that are included in the calculation and the choice of specimen. It is also shown that the method does not readily allow for slice thicknesses smaller than the specimen periodicity along the incident electron beam direction.     

The observation of amorphous materials at high voltage and high resolution

Thin films of amorphous carbon, silicon and germanium have been examined at high resolution at accelerating voltages up to 575 kV with the Cambridge University high resolution electron microscope. The directly interpretable resolution has been demonstrated to extend to 0?22 nm, so that the microscope is capable of providing unambiguous structural information at the atomic level. The observations of both carbon and silicon were, however, somewhat disappointing in that no significant specimen detail was revealed despite the improved performance compared with that of conventional 100 kV instruments. Some of the factors involved in observation and interpretation of these images are discussed.     

SEM electron channelling patterns as a technique for the characterization of ion-implantation damage

Electron channelling patterns (ECPs) formed in back-scattered images in the scanning electron microscope (SEM) have been used occasionally to confirm surface amorphization during ion implantation. In order to place such observations on a more quantitative basis, the study reported here has explored the variation of ECP appearance with both specimen damage levels (and thus subsurface structures) and SEM accelerating voltage (i.e. sampled depth). Polished and annealed (0001) single crystal sapphire discs were implanted to various damage levels up to both subsurface and full surface amorphization. Damage levels were measured independently by Rutherford back-scattering (RBS). Selected-area ECPs were obtained in a Jeol-840 electron microscope operating over the range 5–40 kV in 5-kV steps. Progressive ECP degradation—in terms of high-order line disappearance—was observed with increasing dose, culminating in total pattern loss when full surface amorphization occurred. However, ECP information could still be obtained from the damaged near-surface material even when a subsurface amorphous layer was present, thus demonstrating the shallow retrieval depth of information from the ECP technique. Indeed, because the spatial distribution of damage from ion implantation is both calculable and measurable, these experiments have also allowed us, for the first time, to explore and demonstrate the shallow sample depths from which the majority of ECP contrast originates (< 150 nm in sapphire at an accelerating voltage of 35 kV), even when the beam penetration is considerable by comparison (～ 5 μm). Furthermore, the way in which this sampled depth varies with SEM accelerating voltage is both demonstrated and shown to be a powerful diagnostic technique for studying the distribution of near-surface structural damage.     

Imaging of specimens at optimized low and very low energies in scanning electron microscopes.

The modern trend towards low electron energies in scanning electron microscopy (SEM), characterised by lowering the acceleration voltages in low-voltage SEM (LVSEM) or by utilising a retarding-field optical element in low-energy SEM (LESEM), makes the energy range where new contrasts appear accessible. This range is further extended by a scanning low-energy electron microscope (SLEEM) fitted with a cathode lens that achieves nearly constant spatial resolution throughout the energy scale. This enables one to optimise freely the electron beam energy according to the given task. At low energies, there exist classes of image contrast that make particular specimen data visible most effectively or even exclusively within certain energy intervals or at certain energy values. Some contrasts are well understood and can presently be utilised for practical surface examinations, but others have not yet been reliably explained and therefore supplementary experiments are needed.     

Freeze-fracture of 3T3 cells for high-resolution scanning electron microscopy

Triton-extracted, freeze-fractured 3T3 cells have been examined in the Hitachi S-900 field-emission SEM, after light platinum coating, at low beam voltage to evaluate the performance of the microscope under these conditions. For unstained material fixed in glutaraldehyde alone, high-resolution images can be obtained, at accelerating voltages of 1.5-5kV, after rotary deposition of platinum to an average thickness of 1.5-3nm. Comparisons are made between these results and those of studies by TEM of deep-etch replicas of similar material previously published.     

Collection deficiencies of scanning electron microscopy signal contrasts measured and corrected by differential hysteresis image processing

Limitations of scanning electron microscopy (SEM) image resolution and quality were measured in digital image data and their effect on image contrasts was analyzed and corrected by differential hysteresis (DH) processing. DH processing is a mathematical procedure that utilizes hysteresis properties of intensity variations in the image for a segmentation of differential contrast patterns. These patterns display contrast properties of the data as coherent full-frame images. The contrast segmentation is revertible so that the original image can be restored from the sum of the sequentially extracted DH contrast patterns. DH imaging enhances weak contrast components so that they are more easily recognizable and displays SEM image data free of signal collection efficiency contrasts. Example image data include environmental SEM (ESEM) and SEM images of low and mediumhigh magnifications where collection deficiencies included charging of the specimen surface, obstructions from specimen topography, and uneven signal collection properties of the detector. ESEM low-vacuum image data, which appear to be of high quality, contained local areas of reduced contrasts due to residual surface charging. In such areas, signal contrasts were reduced up to 80%, which suppressed most of the weak short-range contrasts. In low-magnification SEM images, up to 93% of the local high precision contrast was lost from the various adverse effects which diminished the pixel-related contrast resolution of the microscope and resulted in images with low detail. Also, at medium magnification, surface charging effects dramatically reduced the image quality because contrasts resulting from local electron beam/specimen interactions were reduced by as much as 71%. DH imaging restored the local contrast losses by elimination of the collected distorted fraction of signal contrasts and reconstitution of the collected maintained fraction. Restored DH images are of superior quality and enhance the imaging capability of the conventional SEM. DH contrast segmentation provides an improved basis for the measurement of various signal contrast components and detector performances. The DH analysis will ultimately facilitate a precise deduction of specimen properties from extracted contrast patterns.     

Generation of narrow focused beams in a plasma-cathode electron gun

The possibility of generating a narrow focused electron beam in a plasma-cathode electron gun has been studied. An operating mode in which the emitting plasma surface is deep in the emitting channel is optimal for obtaining the highest emission-current density. The beam diameter is reduced by a factor of 1.5–2.0 by choosing the operating mode of the plasma emitter. A two-lens focusing system is used to focus the electron beam. Studies have shown the possibility of generating beams with a power density five to seven times higher than that attained earlier in plasma-cathode guns. As a result of these studies, a plasma-cathode electron gun that allows generation of an electron beam with a diameter of ～260 μm and a power of 3 kW at an accelerating voltage of 30 kV has been developed.     

A linear accelerator-injector for the SIBERIA and TNK complexes of special synchrotron radiation sources

An 80-MeV electron linear accelerator-injector operates in a pulsed standing-wave mode. A 2.8-GHz disk-and-washer accelerating structure is composed of six 1-m-long regular sections with power input in the middle. The electron beam with parameters of 4 A/40 kV/18 ns/1 Hz from the diode gun enters the accelerating structure without prebunching. During acceleration, the beam is divided into bunches with a repetition frequency of 2.8 GHz and transported over the electron-optic channel to the booster ring. Today, the electron beam has an energy of 80 MeV, a current of 80 mA, and an energy spread of 1% and maintains continuous operation of the SIBERIA facility.     

Enhancement of the Breakdown Strength in Accelerators with Radio-Frequency Quadrupole Focusing

On the basis of experimental data on the effect of the electron loading in accelerators with radio-frequency quadrupole focusing and their analytical processing, a conclusion is drawn that the electrodes of the accelerating system are contaminated during the operation of an ion gun. Special measures, including the purification of the proton beam in the matching channel and cleaning of the electrodes of the accelerating system by RF conditioning, reduce the effect of the electron loading and enhance the breakdown strength of the accelerating structure up to 450 kV/cm.     

Low voltage scanning electron microscopy

The scanning electron microscope (SEM) is usually operated with a beam voltage, V₀, in the range of 10–30 kV, even though many early workers had suggested the use of lower voltages to increase topographic contrast and to reduce specimen charging and beam damage. The chief reason for this contradiction is poor instrumental performance when V₀=1–3 kV, The problems include low source brightness, greater defocusing due to chromatic aberration greater sensitivity to stray fields, and difficulty in collecting the secondary electron signal. Responding to the needs of the semiconductor industry, which uses low V₀ to reduce beam damage, considerable efforts have been made to overcome these problems. The resulting equipment has greatly improved performance at low kV and substantially removes the practical deterrents to operation in this mode. This paper reviews the advantages of low voltage operation, recent progress in instrumentation and describes a prototype instrument designed and built for optimum performance at 1 kV. Other limitations to high resolution topographic imaging such as surface contamination, the de-localized nature of the inelastic scattering event and radiation damage are also discussed.     

Aperture contrast in thick amorphous specimens using scanning transmission electron microscopy

The contrast observed in thick amorphous specimens using a scanning transmission electron microscope (STEM) can be considerably improved by the use of an optimum collector aperture angle. The size of this angle can be calculated by considering the variation of electron current transmitted through the specimen as a function both of the specimen thickness and of the angle of collection subtended at the specimen. Typically these calculations predict optimum angles to be several times the half-width of the elastic scattering distribution, often 10(-1) rad or more. Observations of biological sections of up to 2 micron in thickness using scanning attachments of commercial transmission microscopes have verifie these results at beam voltages of 50, 100 and 200 kV. Wide angle convergent beam diffraction patterns were used to give accurate values of the effective angles represented by the various collector apertures. Once the linearity of the detector-amplifier system had been established, operation in a line modulation mode enabled quantitative measurements to be made of the image contrast. Such measurements also offer a quick effective method of comparing electron beam penetrations.     

High-voltage electron microscopy of inorganic particles in intact blood cells

Preliminary results obtained by examining intact fixed human red and white blood cells containing inorganic particles under the high-voltage transmission electron microscope are described. Iron filings and ferritin ingested in vitro by granulocytes were observed as were inorganic particles in red cells from a case of lead poisoning. The nature of the particles in red cells is discussed. A faint intracellular network was seen in normal red cells and in cells from a case of lead poisoning. It was found possible to focus the electron microscope at different planes within the cell. The optimum accelerating voltage for red cells appeared to be around 750 kV, whereas a clear image of granulocytes was obtained at voltages between 750 and 1000 kV. The first results indicate that it is possible to examine intact blood cells under the high-voltage electron microscope. Further work is in progress to determine if more information can be acquired by this technique.     

Perspective and limitations of cryo-electron microscopy

We investigated the possibility of vitrifying temperature-sensitive lipid phases as well as (small) biological specimens. From a suspension of unilamellar vesicles, prepared from dipalmitoyl-phosphatidylcholine (DPPC), thin aqueous films were formed at various temperatures. With cryo-electron microscopy vesicles were found to be smooth, rippled and faceted or faceted only, depending on the temperature of thin-film formation (318, 312 and 296 K respectively). The morphology and the electron diffraction patterns indicate that membranes can by physically fixed by vitrification in their high-temperature configuration and studied at low temperature by cryo-electron microscopy. This finding suggests that it may also be possible to preserve, in their original state, the more complex membrane systems found in living organisms by initiating rapid-cooling at a physiological temperature. This was explored by vitrification of thin films formed on specimen grids with (human) blood platelets adhering to collagen fibres. Low-temperature observation with an acceleration voltage of 120 kV revealed subcellular details. More details were observed when using higher accelerating voltages (200 and 300 kV) of the electron beam. The results presented in this paper illustrate the great potential of cryo-electron microscopy in the study of membrane dynamics, both in relatively simple model membrane systems and in more complex biological membrane systems.