Observations of snow crystals using low-temperature scanning electron microscopy

Low-temperature scanning electron microscopy (SEM) was used to observe precipitation particles commonly known as “snowflakes.” The snowflakes were collected in Beltsville, Maryland, at temperatures ranging from ?6° to +1°C, mounted on SEM stubs, frozen in liquid nitrogen (LN₂), and then transferred to a cryosystem mounted on a field-emission SEM. Neither sputter coating with platinum nor irradiation by the electron beam affected their delicate fine structure. SEM observations revealed that snowflakes consisted of aggregations of snow crystals that occurred as hexagonal plates, prismatic columns, needles, and dendrites. In some cases, the snow crystals contained minute surface structures that consisted of rime, microdroplets, short prismatic columns, and amorphous films. Snow crystals from wet snow, which were collected at temperatures of 0° and +1°C, exhibited varying degrees of metamorphism or melting. The discrete crystalline faces and their sharp intersecting angles were gradually replaced by sinuous surfaces that tended to exhibit more spherical shapes. This study indicates that low-temperature SEM is a valuable technique for studying the formation and metamorphosis of snow crystals. The results suggest that combining low-temperature SEM and x-ray analysis could also provide qualitative elemental information on the nucleation particles of snow crystals as well as on the composition of acid snow.     

Combined application of electron backscatter diffraction and stereo-photogrammetry in fractography studies

The main aim of this paper is to report on recent experimental developments that have succeeded in combining electron back-scatter diffraction (EBSD) with stereo-photogrammetry, compared with two other methods for study of fracture surfaces, namely visual fractography analysis in the scanning electron microscope (SEM) and EBSD directly from facets. These approaches will be illustrated with data relating to the cleavage plane orientation analysis in a ferritic and C-Mn steel. It is demonstrated that the combined use of EBSD and stereo-photogrammetry represents a significant advance in the methodology for facet crystallography analysis. The results of point counting from fractograph characterization determined that the proportions of intergranular fracture in C-Mn and ferritic steels were 10.4% and 9.4%, respectively. The crystallographic orientation was determined directly from the fracture surface of a ferritic steel sample and produced an orientation distribution with a clear trend towards the {001} plane. A stereo-photogrammetry technique was validated using the known geometry of a Vickers hardness indent. The technique was then successfully employed to measure the macroscopic orientation of individual cleavage facets in the same reference frame as the EBSD measurements. Correlating the results of these measurements indicated that the actual crystallographic orientation of every cleavage facet identified in the steel specimens is {001}.     

Relocation of minute specimens for scanning electron microscopy

A relocatable stub-holder assembly for SEM that can record the position of small specimens by x-y-coordinates is described. It enables the accurate pin-pointing of objects for further study later.     

Morphological structure of rat tongue using light and scanning electron microscopy

The rat is one of the most commonly used animals in biological research and experimental investigations in medicine. The ultrafine structural components of the epithelium differ depending on the anatomy of the animal and the papilla type. Animal adaptation to food types and environmental circumstances may also be linked to morphological diversity. In the current study, seven male Wistar rat tongues were investigated. For scanning electron microscope (SEM), two rat tongues were immersed in a 10% formalin solution and the other two rat tongues were immersed in a 2.5% glutaraldehyde solution. The tongues of three rats were fixed for regular histological evaluation using triple staining. The three primary components of the Wistar rat tongue are the apex, body and root. The apex had a rounded and bifurcated shape. Filiform papillae and gustatory papillae were easily identified on the dorsal side of the tongue. There were three forms of gustatory papillae; fungiform papillae, vallate papillae and foliate papillae. The purpose of this study was to expose the tongue morphology of the Wistar rat species, which is widely used in investigations. Also, we wanted to show that formalin fixation can be utilized for morphological research in SEM. Finally, the Wistar rat tongue was thoroughly investigated and compared to those of other species.     

Pollen morphological investigations of family Cactaceae and its taxonomic implication by light microscopy and scanning electron microscopy

The family Cactaceae is the diversified group of angiosperm plants whose pollen statistics has been used for taxonomic identification. In this article, we present the pollen morphology of eight species belong to seven taxonomically complex genera of Cactaceae including Astrophytum, Cylindropuntia, Echinocereus, Echinopsis, Mammillaria, Opuntia, and Thelocactus using light and scanning electron microscopy. The pollen grains were acetolyzed, measured, described, and electron photomicrographs were taken. Cactaceae can be characterized by presenting different palynomorphological features including pollen type, sculpturing, polar and equatorial diameter, aperture orientation, exine thickness, P/E ratio, and echini features. Four types of pollen shapes, that is, prolate spheroidal (three species), subprolate (two species), prolate (two species), and oblate spheroidal in Echinocereus reichenbachii were observed. The polar and equatorial diameter observed maximum in O. ficus indica 116.95 and 112.27 μm while minimum in M. compressa 38.42 and 21.05 μm. Pollen of two types, tricolpate in members of subfamily Cactioideae and pantoporate in the Opuntioideae were examined. The fertility percentage has been observed maximum in Opuntia macrocentra (83.84%) and minimum in Opuntia ficus‐indica (57.89%). Exine sculpturing showing great variations such as granulate, reticulate, granulate perforate and micro‐echinate foveolate ornamentation was examined only in Echinopsis eyriesii. A key to species, based on pollen micromorphological attributes, has been constructed for correct identification of complex cactus species.     

扫描电镜电子背散射衍射系统的研制

本文介绍了扫描电镜电子背散射衍射接收系统,并将该系统成功地安装在JSM-840扫描电镜上。     

Charging control using pulsed scanning electron microscopy

This paper describes a novel method to observe highly charging specimens at high-beam voltages without specimen preparation. It is found that the technique greatly reduces charging artifacts such as image shift, astigmatism, and defocussing without sacrificing image quality. Images obtained of uncoated specimens are found to be comparable to gold-coated specimens and without exhibiting charging effects. The technique also allows the study of charge distribution effects in specimen charging of which very little understanding exists, particularly as far as the spatial and time-dependent properties of charging are concerned.     

Chromosome observation by scanning electron microscopy using ionic liquid

Electron microscopy has been used to visualize chromosome since it has high resolution and magnification. However, biological samples need to be dehydrated and coated with metal or carbon before observation. Ionic liquid is a class of ionic solvent that possesses advantageous properties of current interest in a variety of interdisciplinary areas of science. By using ionic liquid, biological samples need not be dehydrated or metal-coated, because ionic liquid behaves as the electronically conducting material for electron microscopy. The authors have investigated chromosome using ionic liquid in conjunction with electron microscopy and evaluated the factors that affect chromosome visualization. Experimental conditions used in the previous studies were further optimized. As a result, prewarmed, well-mixed, and low concentration (0.5～1.0%) ionic liquid provides well-contrasted images, especially when the more hydrophilic and the higher purity ionic liquid is used. Image contrast and resolution are enhanced by the combination of ionic liquid and platinum blue staining, the use of an indium tin oxide membrane, osmium tetroxide-coated coverslip, or aluminum foil as substrate, and the adjustment of electron acceleration voltage. The authors conclude that the ionic-liquid method is useful for the visualization of chromosome by scanning electron microscopy without dehydration or metal coating.     

Phase identification of individual crystalline particles by electron backscatter diffraction

Recently, an electron backscatter diffraction (EBSD) system was developed that uses a 1024 × 1024 CCD camera coupled to a thin phosphor. This camera has been shown to produce excellent EBSD patterns. In this system, crystallographic information is determined from the EBSD pattern and coupled with the elemental information from energy or wavelength dispersive X-ray spectrometry. Identification of the crystalline phase of a sample is then made through a link to a commercial diffraction database. To date, this system has been applied almost exclusively to conventional, bulk samples that have been polished to a flat surface. In this investigation, we report on the application of the EBSD system to the phase identification analysis of individual micrometre and submicrometre particles rather than flat surfaces.     

Tridimensional ultrastructure of perfusion fixed gastrointestinal epithelial cells by high resolution scanning electron microscopy

Improvements in the design of modern scanning electron microscopes (SEM) and new methods of specimen preparation incorporating chemical removal of the cytosol and cytoskeleton, now make it possible to view cells and their organelles in three dimensions (3D) at high magnification. In this experiment, high resolution SEM (HRSEM) utilizing new methods of tissue preparation was used to study the intracellular structures of the mouse ileum. In addition, in vivo intestinal perfusion was used to further enhance cellular preservation. Using these modifications it was possible to visualize, in 3D, the fine structure of intestinal epithelial cells and intracellular organelles such as the nucleus, mitochondria, endoplasmic reticulum, and Golgi complex, as well as microvilli and cell membrane. Whole mitochondria appeared as irregularly shaped organelles which contained tubular cristae. Plate-like cristae were not observed. The brush border was found to be a closely packed array of cylindrical projections. The extensive folding and structural intricacy of lateral cell membranes between absorptive cells could only be appreciated by viewing this tissue with 3D HRSEM. The use of HRSEM to study 3D ultrastructure of cells and their organelles will improve our understanding of the structure-function relationships in both the healthy and diseased gastrointestinal tract.     

Polyethylene glycol (PEG) embedding and subsequent de-embedding as a method for the correlation of light microscopy,scanning electron microscopy,and transmission electron microscopy

The polyethylene glycol (PEG) embedding and subsequent deembedding method was applied to the observation of general tissues in scanning electron microscopy (SEM). Resulting SEM images were of high quality. It was demonstrated that intermicroscopic correlation of images between light microscopy (LM), scanning electron microscopy (SEM), and transmission electron microscopy (TEM) is easily and reliably done by means of the PEG method. In particular, the exact correlation of immuno-LM with SEM is shown to be of potential value.     

Quantitative metallography by electron backscattered diffraction

Although electron backscattered diffraction (EBSD) in the scanning electron microscope is used mainly to investigate the relationship between local textures and microstructures, the technique has now developed to the stage where it requires serious consideration as a tool for routine quantitative characterization of microstructures. This paper examines the application of EBSD to the characterization of phase distributions, grain and subgrain structures and also textures. Comparisons are made with the standard methods of quantitative metallography and it is shown that in many cases EBSD can produce more accurate and detailed measurements than the standard methods and that the data may sometimes be obtained more rapidly. The factors which currently limit the use of EBSD for quantitative microstructural characterization, including the speed of data acquisition and the angular and spatial resolutions, are discussed, and future developments are considered.     

New application of air-drying techniques for studying Ephemeroptera and Plecoptera eggs by scanning electron microscopy

Hexamethyldisilizane (HMDS) and tetramethylsilane are organic compounds that are volatile at ambient temperature and which can therefore be used for air-drying biological samples for SEM studies. The techniques using these compounds provide results that are comparable with those obtained by critical point drying, but which involve a very simple process that saves time and money. Both techniques were applied to SEM studies of Ephemeroptera and Plecoptera eggs in order to assess their suitability as alternative methods to critical point drying for these kinds of biological material. The results show no morphological differences between eggs HMDS air-dried and critical point-dried.     

Surface investigations on HgBr2 single crystals by using confocal scanning laser microscopy

A confocal scanning laser microscope operating at 514 and 488 nm has been used to obtain two-dimensional (2-D) images of the mercuric bromide (HgBr2) crystal surface by photoluminescence, reflection, and transmission phenomena. Our measurements indicate that regions showing a strong photoluminescence may appear on the surface. By processing the 2-D images. we obtained the three-dimensional images, which offer a better possibility for the investigation. The analysis of spectral lines may be correlated with the presence of the Hg impurities.     

Deconvolution of scanning electron microscopy images

This paper describes a method of removing blurs in scanning electron microscopy (SEM) images caused by the existence of a finite beam size. Although the resolution of electron microscopy images has been dramatically improved by the use of high-brightness electron guns and low-aberration electron lenses, it is still limited by lens aberration and electron diffraction. Both are inevitable in practical electron optics. Therefore, a further reduction in resolution by improving SEM hardware seems difficult. In order to overcome this difficulty, computer deconvolution has been proposed for SEM images. In the present work, the SEM image is deconvoluted using the electron beam profile estimated from beam optics calculation. The results show that the resolution of the deconvoluted image is improved to one half of the resolution of the original SEM image.     

Taxonomic significance of caryopsis in subfamily Panicoideae (Poaceae) using scanning electron microscopy and light microscopy

In recent study, 13 taxa of subfamily Panicoideae were investigated for morphological characterization of caryopsis. Light microscope (LM) and scanning electron microscope (SEM) were utilized to study macro‐ and micro‐morphological caryopsis features respectively. Caryopsis size in studied taxa was recorded as 1.5–10 mm long and 1–4 mm wide. Caryopsis color was brown, green, yellow, and whitish‐brown. Caryopsis shape studied was obovate, elliptic, linear oblate, and round shallowly obtriangular. Hilum position is grooved and depressed. Caryopsis compression type was lateral and dorsiventral. Major variations among studied taxa were observed in terms of caryopsis surface pattern and epicuticular projection types. Six types of caryopsis surface pattern were observed viz. scabrate, rugose, striate, reticulate, papillate, and scabridulous. Structures such as silica cells, bulges, spines, prickles, granules, and bicellular microhair were studied as epicuticular projections. Major variations were observed among Cenchrus pennisetiformis and Cenchrus ciliaris as both has entirely two different types of surface patterns and epicuticular projections. Anticlinal wall thickness and pattern as well as periclinal wall texture and level were investigated. The present research work emphasized on caryopsis characterization of subfamily Panicoideae and it is recommended to establish phylogeny within subfamily Panicoideae and with other subfamilies of Poaceae.     

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”.     

Identification of monocot flora using pollen features through scanning electron microscopy

Pollen used to track structural and functional evolution in plants as well as to investigate the problems relative to plant classification. Pollen characters including ornamentation, shape, apertural pattern, pollen symmetry, colpus length, width, and margins used to detect the similarities and dissimilarities between genera and also species of the same genus. In this study pollen features of 20 monocot species belonging to 15 genera of the Amaryllidaceae, Asparagaceae, Iridaceae, Ixioliriaceae, Liliaceae, and Xanthorrhoeaceae were studied using scanning electron microscopy (SEM) and light microscopy (LM). In this study two species that is Zephyranthes citrina and Tulbaghia violacea were reported for the first time from Pakistan. Pollen grains were visualized with LM. Non‐acetolyzed and acetolyzed pollen were examined using SEM. A taxonomic key was developed to highlight the variation in pollen features in order to make their systematic application for correct species identification.     

A simple method to 'point count' silt using scanning electron microscopy aided by image analysis

This study presents a simple method to ‘point count’ silt‐sized grains using backscattered scanning electron microscopy together with image analysis. The work materialized out of the need to determine the heavy mineral abundance within silt obtained from coastal dunes to aid in the interpretation of dune weathering. This technique allows two broad mineral groups to be quantified according to their modal abundance. The groups are characterized by their dominant atomic elements present; atomic numbers > 20 are classified as ‘high’ (metal oxides, zircon, monazite, carbonates, pyroxenes and amphiboles) and those < 20 as ‘low’ (quartz, feldspars and organics). As a check on this technique, X‐ray fluorescence was used. This showed a strong positive correlation (r² = 0.85) with the developed point counting technique.