Scanning electron microscopy with an ionic liquid reveals the loss of mitotic protrusions of cells during the epithelial-mesenchymal transition

Epithelial-mesenchymal transition (EMT) is a key event in cancer metastasis and is characterized by increase in cell motility, increase in expression of mesenchymal cell markers, loss of proteins from cell-to-cell junction complexes, and changes in cell morphology. Here, the morphological effects of a representative EMT inducer, transforming growth factor (TGF)-β1, were investigated in human lung adenocarcinoma (A549) cells and pancreatic carcinoma (Panc-1) cells. TGF-β1 caused morphological changes characteristic of EMT, and immunostaining showed loss of E-cadherin from cell-to-cell junction complexes in addition to the upregulation of the mesenchymal marker vimentin. During scanning electron microscopy (SEM) with an ionic liquid, we observed EMT-specific morphological changes, including the formation of various cell protrusions. Interestingly, filopodia in mitotic cells were clearly observed by SEM, and the number of these filopodia in TFG-β1-treated mitotic cells was reduced significantly. We conclude that this reduction in such mitotic protrusions is a novel effect of TGF-β1 and may contribute to EMT.     

Ionic liquid enables simple and rapid sample preparation of human culturing cells for scanning electron microscope analysis

Ionic liquid is a kind of salt that stays in a molten state even at room temperature. It does not vaporize at all in vacuum and facilitates electrical conductivity to the sample surfaces for observations with a scanning electron microscope (SEM). In this study, we used an ionic liquid in SEM for the first time to observe fixed human culture cells. The condition for the cell culture using wrapping sheets and SEM settings were varied to elucidate the optimized protocol. Compared to samples prepared by the conventional way, the ionic liquid‐treatment of samples gave SEM images of the cellular ultra structures in more detail, enabling observation of microvilli that made bridges between separated cells. In addition, the ionic liquid treatment is less time consuming as well as less laborious compared with the conventional way that includes dehydration, drying, and conductivity treatments. Totally, we concluded the ionic liquid is a useful reagent for SEM sample preparation. Microsc. Res. Tech., 2011. © 2010 Wiley‐Liss, Inc.     

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.     

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.     

Comparative study of hydrophilic and hydrophobic ionic liquids for observing cultured human cells by scanning electron microscopy

An ionic liquid (IL) is a salt that remains in the liquid state at room temperature. It does not vaporize under vacuum and imparts electrical conductivity to samples for observation by scanning electron microscopy (SEM). Recently, the usefulness of ILs has been widely recognized. In our previous study, one of the ILs 1-ethyl-3-methylimidazolium tetrafluoroborate (EtMelm(+) BF(4)(-)) was used for SEM analysis of biological samples. In comparison with the conventional method, samples prepared using EtMelm(+) BF(4)(-) provided more detailed SEM images of the cell ultrastructure, enabling the observation of protrusions. In addition, the IL treatment is a less time consuming and simple method that does not include dehydration, drying, and conductivity treatments, which are an essential parts of the conventional method. In this study, we compared the usefulness of four hydrophobic and three hydrophilic ILs for SEM to observe fixed cultured human A549 cells. All ILs worked well to prevent "charge-up" effect for SEM observation. However, the hydrophilic ILs tended to provide clearer images than the hydrophobic ILs. We concluded that various ILs can be used for SEM sample preparation and their application to a wide range of fields is anticipated in future.     

Morphological analyses of minute crystals by using stereo-photogrammetric scanning electron microscopy and electron back-scattered diffraction

We present a new method for the morphological analyses of minute faceted crystals by combining stereo-photogrammetric analysis of scanning electron microscope images and electron back-scattered diffraction. Two scanning electron microscope images of the same crystal, recorded at different tilt angles of the specimen stage, are used to determine the orientations of crystal edges in a specimen-fixed coordinate system. The edge orientations are converted to the indices [ uvw ] in the crystal system using the crystal orientation determined by electron back-scattered diffraction analysis. The Miller indices of crystal facets are derived from the indices of the edges surrounding the facets. The method is applicable to very small crystal facets. The angular error, as derived from tests using a calcite crystal of known morphology, is a few degrees.
To demonstrate the applicability of the method, the morphology of boehmite (γ-AlOOH) precipitated from solution during the dissolution of anorthite was analyzed. The micrometre-sized boehmite crystals are surrounded by two {010} basal facets and eight equivalent side facets that can be indexed equally well as {323}, {434} or {545}. We suggest that these side facets are in fact {111}, the morphology having been modified slightly (by a few degrees) by a small extension associated with opening along (010) microcleavage planes. Tiny {140} facets are also commonly observed.     

Correcting for 3D distortion when using backscattered electron detectors in a scanning electron microscope

A variable pressure scanning electron microscope (VPSEM) can produce a topographic surface relief of a physical object under examination, in addition to its two‐dimensional (2D) image. This topographic surface relief is especially helpful when dealing with porous rock because it may elucidate the pore‐space structure as well as grain shape and size. Whether the image accurately reproduces the physical object depends on the management of the hardware, acquisition, and postprocessing. Two problems become apparent during testing: (a) a topographic surface relief of a precision ball bearing is distorted and does not correspond to the physical dimensions of the actual sphere and (b) an image of a topographic surface relief of a Berea sandstone is geometrically tilted and topographically distorted even after standard corrections are applied. The procedure presented here is to ensure the veracity of the image, and includes: (a) adjusting the brightness and contrast levels originally provided by the manufacturer and (b) tuning the amplifiers of the backscatter detector plates to be equal to each other, and producing zero voltage when VPSEM is idle. This procedure is tested and verified on the said two physical samples. SCANNING 31: 59–64, 2009. © 2009 Wiley Periodicals, Inc.     

Focused ion beam scanning electron microscopy in biology

Since the end of the last millennium, the focused ion beam scanning electron microscopy (FIB‐SEM) has progressively found use in biological research. This instrument is a scanning electron microscope (SEM) with an attached gallium ion column and the 2 beams, electrons and ions (FIB) are focused on one coincident point. The main application is the acquisition of three‐dimensional data, FIB‐SEM tomography. With the ion beam, some nanometres of the surface are removed and the remaining block‐face is imaged with the electron beam in a repetitive manner. The instrument can also be used to cut open biological structures to get access to internal structures or to prepare thin lamella for imaging by (cryo‐) transmission electron microscopy. Here, we will present an overview of the development of FIB‐SEM and discuss a few points about sample preparation and imaging.     

Determination of diffusion controlled reaction rates at a solid/liquid interface using scanning electron microscopy

A high‐resolution method has been developed for the determination of localized values of interfacial reaction rate and mass transfer coefficient in aqueous solution. Scanning electron microscopy has been successfully applied to this problem through the measurement of electroplated film thickness formed under limiting current conditions. The method involves the calculation of local values of reaction rate via Faraday's laws and subsequent conversion of the data to absolute values of mass transfer coefficient. The technique has been verified in an undisturbed, turbulent flow regime (rotating cylinder electrode) through the use of Sherwood group dimensionless analysis. The resulting relationship shows comparable accuracy relative to electrochemical measurements. Favourable comparison has also been made with the generally accepted rotating cylinder correlation of Eisenberg, Tobias and Wilke. Differential rates of mass transfer to a single surface under conditions of disturbed flow have also been examined at a high spatial resolution using the stepped rotating cylinder electrode geometry. In this case, reaction rates have been measured as a function of circumferential distance within a recirculation zone situated immediately downstream of a backward‐facing step.     

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”.     

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.     

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.     

Interfacial ultramorphology evaluation of resin luting cements to dentin: A correlative scanning electron microscopy and transmission electron microscopy analysis

The objective of this study was to analyze the dentin‐resin cements interfacial ultramorphologies using two different methods: scanning (SEM) and transmission electron microscopy (TEM). Four commercial products were evaluated: two conventional cementing system (RelyX ARC/Adper? Scotchbond? Multi‐Purpose Plus, 3M ESPE and Clearfil Esthetic Cement/DC Bond, Kuraray) and two self‐adhesive resin cements (RelyX Unicem, 3M ESPE and Clearfil SA Cement, Kuraray). Prepolymerized resin disks (Sinfony, 3M ESPE) were cemented on oclusal dentin surfaces of 24 third human molars, simulating the indirect restorations. After 24 h, teeth were sectioned into 0.9‐mm thick slabs and processed for microscopy analyses (SEM or TEM/ n = 3). Qualitative characterization of dentin‐resin cement interface was performed. Hybrid layer formation with long and dense resin tags was observed only for RelyX ARC cementing system. Clearfil Esthetic Cement/DC Bond system revealed few and short resin tags formation, whereas no hybridization and resin tags were detected for self‐adhesive resin cements. Some interfacial regions exhibited that the self‐adhesive resin cements were not bonded to dentin, presenting bubbles or voids at the interfaces. In conclusion, TEM and SEM bonding interface analyses showed ultramorphological variations among resin cements, which are directly related to dental bonding strategies used for each resin cement tested. Microsc. Res. Tech. 76:1234–1239, 2013. © 2013 Wiley Periodicals, Inc.     

Tribological performance of an ionic liquid as a lubricant for steel/aluminium contacts

The wear and friction behaviour of an ionic liquid 1‐ethyl‐3‐hexylimidazolium tetrafluoroborate (L206) was investigated as a lubricant for steel/aluminium contacts using an Optimol SRV® oscillating friction and wear tester. The elemental composition and chemical nature of the antiwear films generated on the aluminium surface were analysed using a scanning electron microscope with a Kevex energy dispersive X‐ray analyser attachment (SEM/EDS) and X‐ray photoelectron spectroscopy (XPS). A low friction coefficient (˜0.05) was recorded when lubricating with L206; a small amount of water (5 wt. %) in L206 effectively reduced the wear volume and greatly increased the microhardness of the aluminium alloy, but had little effect on the friction coefficient. The SEM/EDS results showed that severe corrosive wear occurred on the aluminium alloy when lubricating with neat L206, which could be avoided by the addition of water in L206. The XPS results indicated that the species AlF₃, Al₂O₃, AlO(OH), and Al(OH)₃ formed during friction; there was no indication of boron on the worn surfaces.     

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.     

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.     

Intraspecific variation in spermoderm pattern of tribe Acacieae (Mimosoideae) using scanning electron microscopy techniques

Current research carried out in Pakistan is the first report on spermoderm ornamentation of eight species of tribe Acacieae (Mimosoidae) by using scanning electron microscopic techniques representing two genera, Fedherbia and Acacia were examined. Different spermoderm ornamentation were observed, described and discussed for their taxonomic importance. Seeds surfaces of the studied tribe possess novel variations in macro and micro morphology. Great variations were observed in both qualitative and quantitative characters of seeds. Seeds shape was oblong, ovate to elliptical and spermoderm ornamentation was levigate, rugose, polygonal and discoid, colliculate, and papillose type. These variations in the spermoderm ornamentation can be used as an aid in identification and classification of the members of tribe Acacieae.     

Comparative study of dental enamel loss after debonding braces by analytical scanning electron microscopy (SEM)

Clinical procedures when shear forces are applied to brackets suggest adhesion forces between 2.8 and 10.0 MPa as appropriate. In this study dental enamel was evaluated by scanning electron microscopy (SEM) before and after removing the brackets. Thirty bicuspids (previous prophylaxis) with metallic brackets (Roth Inovation 0.022 GAC), Transbond Plus SEP 3M Unitek adhesive and Transbond XT 3M resin were used. The samples were preserved to 37°C during 24 hr and submited to tangential forces with the Instron Universal machine 1.0 mm/min speed load strength resistance debonding. Also the Adhesive Remanent Index (ARI) test was made, evaluating the bracket base and the bicuspid surface. All the bracket SEM images were processed with AutoCAD to determine the enamel detached area. The average value was 6.86 MPa (SD ± 3.2 MPa). ARI value 1= 63.3%, value 2= 20%, value 3= 13.3% and 33% presented value 0. All those samples with dental enamel loss, presented different situations as fractures, ledges, horizontal, and vertical loss in some cases, and some scratch lines. There is no association between the debonding resistance and enamel presence. Less than half of the remanent adhesive on the dental enamel was present in most of the samples when the ARI test was applied. When the resin area increases, the debonding resistance also increases, and when the enamel loss increases, the resin free metallic area of the bracket base decreases in the debonding.