Origin and significance of the SEM cathodoluminescence from zircon

In geochronological investigations, zircons are used frequently as a geological clock because of the small amounts of radioactive U isotopes and their decay products, e.g. Pb, incorporated into the crystal structure. However, the U or Pb concentration of zircon is not only dependent on the radioactive decay. One factor is the presence of old, inherited zircon cores in younger igneous zircons. This polyphase structure often cannot be recognized by conventional imaging methods, such as light microscopy. Dating such zircons, can therefore lead to ambiguous age results. However, zircons can be selected for dating purposes using cathodoluminescence (CL) in the scanning electron microscope. It is shown that zircon phases of different ages can be identified by their specific CL properties. Moreover, conclusions on the origin and on the development of the inherited phases can be drawn by comparison with detrital zircons. The zircon CL is formed by the superpostion of several broad bands. The shape of the spectra is dependent on the zircon genesis. By fitting the spectra with Gaussian curves, the individual CL bands can be separated. Using this methodology, it is possible to trace back the essential parts of the zircon CL to a superpostion of the quartz and zirconia (ZrO₂) CL. In addition, CL phenomena due to dysprosium impurities can be separated from intrinsic zircon CL properties.     

High-Resolution scanning electron micrographs of freeze-cracked cells in testes from normal and irradiated rats at different stages of gonadal development

Newly developed techniques in high-resolution scanning electron microscopy (SEM) and for tissue-processing procedures have been applied to an investigation of structures of various cells in rat testes at different stages of gonadal maturation. A series of high-resolution SEM micrographs are presented which survey the surfaces of different types of testis cells during normal development, and which also illustrate ultrastructural features of some of their intracellular organelles. In addition, a series of high-resolution SEM micrographs are presented which compare the structural features of Sertoli cells in normal testes with those in germ-cell-depleted testes obtained from rats killed at varying times after having been irradiated in utero. We describe our observations on the structural properties of surfaces and intracellular organelles in Sertoli cells, Leydig cells, peritubular myoid cells, and some classes of germinal cells. We also consider the possible role of Sertoli cell apical cytoplasmic processes in lumen formation. Similarities are pointed out between the structure of germ-cell-depleted testes, resulting from irradiation in utero, and the structure of germ-cell-depleted testes in seasonal breeders during periods of involution. Finally, we discuss advantages and disadvantages of methods employed to reveal the fine structure of intracellular organelles in cells of the testis.     

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.     

Initial experimental results on a magnetic beam separator spectrometer for the SEM

This article describes preliminary experiments to test the concept of scanning electron microscope (SEM) using a round magnetic beam separator to perform energy spectroscopy. Two experiments with an add-on attachment inside a conventional SEM were performed, one to estimate the sector image aberrations and the other to capture an energy spectrum of scattered electrons. The experiments show that the sector image aberrations lie well below 2 nm and that it is possible to capture the energy spectrum of secondary electrons.     

Simple collectors for cathodoluminescence in the SEM made from aluminium foil

Inexpensive cathodoluminescence collectors for scanning electron microscopes can easily be made from aluminium foil fashioned as tubes which abut against the window of the photo-multiplier at one end and shroud the specimen at the other. Their use in the study of fluorescent labelled mineralized tissues is illustrated.     

Low-temperature SEM for detection of fungicide activity

Low-temperature scanning electron microscopy (LTSEM) combined with cryopreparation methods provided images of well-preserved biological surfaces and structures on a routine basis. Fractures of wheat leaves revealed epidermal and parenchymatous cells and masses of fungal hyphae growing in intercellular spaces. Freeze-fractured plant cells contained haustoria of the brown rust fungus Puccinia triticina. Extrahaustorial matrices were clearly distinguishable and at infection sites granular material was found. Activity of the triazole fungicide cyproconazole was mainly directed towards fungal hyphae and sporogenic tissue, resulting in a stronger branching and swelling of hyphal tips and collapse of fungal cells. Cryofixation methods combined with the use of a cryopreparation unit (Balzers SCU 020) were more reliable in interpreting the observed biological events through easier discrimination between evidence and artefacts. More advantages in the use of the cryopreparation unit are described in detail.     

Application of the Laplacian filter to high-resolution enhancement of SEM images

Certain digital image-processing methods, which are useful for nonperiodic structural images, have been applied to high-resolution SEM images for the improvement of resolution. Samples utilized in the present study consisted of magnetic tape coated with gold, T4 phage coated with gold-palladium, and uncoated specimens of Prolamellar body (PLB) in Cucurbita moschata. These images were blurred and otherwise disturbed by electronic noise, though the images were taken at the limit of efficiency of intrinsic instrument. The major image-processing tool was the Laplacian filter, which subtracts the Laplacian from the original image. Noise, which is a serious problem in digital processing of high-resolution SEM images, was suppressed by the nonlinear type smoothing method. Also, the noise was evaluated by an autocorrelation function and a power spectrum of the image. By using these methods of “deblurring” and noise removal, we achieved better resolution, and structural details of our biological specimens were revealed.     

Practical method for noise removal in scanning electron microscopy

A new smoothing filter has been developed for noise removal of scanning electron microscopy (SEM) images. We call this the complex hysteresis smoothing (CHS) filter. It is much easier to use for SEM operators than any other conventional smoothing filter, and it rarely produces processing artifacts because it does not utilize a definite mask (which usually has processing parameters of size, shape, weight, and the number of iterations) like a common averaging filter or a complicated filter shape in the Fourier domain. Its criterion for distinguishing noise depends simply on the amplitude of the SEM signal. When applied to several images with different characteristics, it is shown that the present method has a high performance with some original advantages.     

Comparison of osmium/sonication and edta/sonication microdissection techniques in exposing the adepithelial basal lamina surface of developing rat colon

We have used two epithelial-stripping techniques in our studies of the basal lamina in the developing rat colon. The first involves prolonged osmication followed by sonication; the second uses chelation of calcium by EDTA followed by sonication. Both techniques remove the epithelium from the basal lamina; however, the EDTA/sonication technique appears to produce a cleaner adepithelial surface of the basal lamina. In addition, the fine structure of the basal lamina appears to be better preserved in specimens prepared by the EDTA/sonication technique. In contrast, the basal lamina of specimens prepared by the osmium/sonication technique has a shattered appearance that we believe is due to an increase in the fragility of the delicate fetal basal lamina.     

Sectionable microcarrier beads: An improved method for the preparation of cell monolayers for electron microscopy

Cross-linked dextran beads provide an excellent surface for tissue-cultured cell monolayers, and can be processed for transmission (TEM) and scanning (SEM) electron microscopy, as well as light microscopy (LM). Cells are grown to confluency on the surface of the microcarriers, where at any point aliquots can be removed and experimentally treated as desired (e.g. immunocytochemistry) providing a representative sample. Sample preparation for TEM follows standard procedures for any cell monolayer, but infiltration times must be at least doubled to allow penetration of the beads. The polymerized blocks can then be sectioned for TEM or LM with no additional steps required. SEM sample preparation involves attaching the fixed bead/cell suspension to a glass coverslip with poly-1-lysine, dehydration, critical point drying, and coating for conductivity. The fixed and dried sample can also be attached directly to the SEM stub as free beads and subsequently gold coated. These beads provide (1) an increased surface area of cells visible per area of thin section, (2) eliminates the careful orientation required for flat substrate methods of embedding, (3) decreases the amount of sample manipulation in the forms of re-embedding and gluing, and (4) decreases the amount of drying artifact seen as cracking in SEM monolayer preparations.     

A new method for cell culture on an electron-transparent melamine foil suitable for successive LM,TEM and SEM studies of whole cells

A new cell culture technique is described which is based on the observation that foils cast from the melamine resin hexamethylol-melamine-ether are suitable for the cultivation of beating heart muscle cells and fibroblasts of the rat. This foil can be flamed for sterilization, is about 80 nm in thickness, homogeneous and smooth, withstands dehydration and critical point-drying, can be removed from glass and permits the imaging of whole cells successively by light microscopy, transmission and scanning electron microscopy. The method is capable of narrowing the gap between light and electron microscopy, yielding excellent whole cell preparations in various kinds of microscopic studies to be performed on one and the same cell.     

Use of Peldri II (a fluorocarbon solid at room temperature) as an alternative to critical point drying for biological tissues

A new chemical, Peldri II, is evaluated as a compound for drying soft biological tissues for scanning electron microscopy. Peldri II, a fluorocarbon, is a solid at room temperature and is a liquid above 25°C. Cells or tissues are embedded in Peldri II by immersing them in the liquid form and allowing it to solidify. Once solidified, Peldri II will sublime with or without vacuum to dry tissues, probably without introducing surface tension. Several types of cells and tissues have been examined to compare preservation with Peldri II and critical point drying techniques. No differences were detected between the two techniques when normal surface structures were examined. Peldri II appears to be a significant improvement over hexamethyldisilazane as a drying agent for scanning electron microscopy. It is also very convenient for drying large numbers of samples.     

A method for exposing the internal anatomy of small and delicate tissues for correlated SEM/TEM studies using polyethylene glycol embedding

A method for preparing and handling large, clean, distortion-free cut surfaces through small and delicate tissues for correlated SEM/TEM examination is described. In this method, tissues are fixed according to conventional protocols; however, instead of critical-point-drying after fixation, tissues are first embedded in polyethylene glycol (PEG), a water-soluble waxy solid. Tissue blocks are easily oriented and sectioned to the desired regions, immersed in a solvent to remove PEG, critical-point-dried, and examined with an SEM. The same tissue blocks can be reworked for TEM by immersing in propylene oxide and embedding in an epoxy resin.     

A new method for investigating the undersurface of cell monolayers by scanning electron microscopy

Glow discharge is commonly used for cleaning the inside of coating units and for cleaning hard surfaces before carbon or metal evaporation procedures. In this study it has been used to remove the embedding medium to reveal, for scanning electron microscope (SEM) study, the undersurfaces of Balb/c 3T3 fibroblastic cells that have been cultured on Thermanox discs and embedded in LR White resin. Ten to twenty-minute ionization times were found to reveal the largest area of the undersurface without causing damage to the cells. Chemical etching of the resin was also shown to reveal the undersurface of the cells, but caused some damage, preventing successful re-embedding for transmission electron microscopy, and at higher magnifications revealed less detail. A circular impression within the main outline of the cells was observed in many cells, which is considered to reflect the presence of a nucleus. The undersurfaces of most cells, after applying both methods of etching, displayed a number of very short processes. Subsequent transmission electron microscopy of ultrathin sectioned, re-embedded, areas of the gold sputter-coated blocks revealed the depth of ionization that had occurred and confirmed that the specimens observed in SEM were the undersurfaces of cells. This method can be modified to study the attaching surface of any organism to a substratum.     

扫描电子显微镜探头新进展

介绍了扫描电子显微镜（SEM）信号探头研究的最新成果。针对常见扫描电子显微镜缺陷而开发的新型探头不仅改善了仪器成像质量,也极大地扩展了仪器的使用范围,简化了样品的准备工艺过程。     

New technique for in-situ measurement of backscattered and secondary electron yields for the calculation of signal-to-noise ratio in a SEM

The quality of an image generated by a scanning electron microscope is dependent on secondary emission, which is a strong function of surface condition. Thus, empirical formulae and available databases are unable to take into account actual metrology conditions. This paper introduces a simple and reliable measurement technique to measure secondary electron yield (δ) and backscattered electron yield (η) that is suitable for in-situ measurements on a specimen immediately prior to imaging. The reliability of this technique is validated on a number of homogenous surfaces. The measured electron yields are shown to be within the range of published data and the calculated signal-to-noise ratio compares favourably with that estimated from the image.     

An improved model for gaseous amplification in the environmental SEM

We present a new model for the gas amplification effect used in many environmental scanning electron microscopes, wherein molecular complexity is shown to be the critical factor. Monte Carlo simulations, based on experimental electron scattering cross-sections, are used to deduce a predictive model for the amplification process that is superior to the Townsend gas capacitor model. These predictions are compared with experimentally obtained amplification curves. Significantly, it is shown that the ionization efficiency of the electrons changes dramatically over the gap distance, and a constant value cannot be assumed. Atomic and molecular excitations affect the amplification process in two ways: first, they serve to lower the average kinetic energy of the imaging electrons, thereby keeping a greater fraction near the ionization threshold energy. Second, molecular normal modes determine the effectiveness of positive gas ions in producing additional secondaries upon surface impact. Practical implications such as signal gain and fraction of useful signal as a function of operating conditions are discussed in the light of the new model. Finally, we speculate on potential new contrast mechanisms brought about by the presence of an imaging gas.     

A comparison of two models for the characteristic X-ray fluorescence correction in thin foil analysis

It is shown that Philibert & Tixier's calculation for the contribution of characteristic fluorescence in thin foil X-ray microanalysis is in error. The calculation of Nockolds et al. for this contribution is shown to be correct.