Observations of the intestinal mucosa using environmental scanning electron microscopy (ESEM); comparison with conventional scanning electron microscopy (CSEM)

In order to evaluate the potential use of environmental scanning electron microscopy (ESEM) in biology, structural changes of the jejunal villi of rats were studied after periods of fasting and refeeding, using a conventional scanning electron microscope (CSEM) and ESEM. While observation using the CSEM, involves chemical fixation, drying and coating, observation of fresh, unprepared materials can be directly realized with the ESEM. Environmental microscopy provides a relatively new technology for imaging hydrated materials without specimen preparation and conductive coating. Direct observation of biological samples in their native state is therefore possible with an ESEM.

After fasting, the jejunal mucosa is dramatically reduced in size, splits and holes appearing at the tip of the villi. These changes were observed whatever the type of technique used. Artifacts due to the sample preparation for CSEM observation (drying, coating) can therefore be excluded. However, CSEM and ESEM must be used jointly. While, CSEM must be preferred for surface analysis involving high magnifications, ESEM observation, on the other hand, can prove valuable for determining the living aspect of the samples.     

Examination of the transition of cultured neuronal cells from submerged to exposed using an environmental scanning electron microscope (ESEM)

Relatively few studies of fully hydrated live or fixed cultured animal cells viewed by environmental scanning electron microscopy (ESEM) have been published. In some cases there may have been some drying out of the cells. In this study the interface between water and cells is imaged as water is carefully evaporated to expose cells. Technical difficulties associated with the process, including inadvertent rewetting of cells are described. Suggestions are made for optimising operating parameters for viewing fully hydrated cultured cells by ESEM. The prospects for viewing live cultured cells are discussed.     

Field emission scanning electron microscopy (FE-SEM) as an approach for nanoparticle detection inside cells

When developing new nanoparticles for bio-applications, it is important to fully characterize the nanoparticle's behavior in biological systems. The most common techniques employed for mapping nanoparticles inside cells include transmission electron microscopy (TEM) and scanning transmission electron microscopy (STEM). These techniques entail passing an electron beam through a thin specimen. STEM or TEM imaging is often used for the detection of nanoparticles inside cellular organelles. However, lengthy sample preparation is required (i.e., fixation, dehydration, drying, resin embedding, and cutting). In the present work, a new matrix (FTO glass) for biological samples was used and characterized by field emission scanning electron microscopy (FE-SEM) to generate images comparable to those obtained by TEM. Using FE-SEM, nanoparticle images were acquired inside endo/lysosomes without disruption of the cellular shape. Furthermore, the initial steps of nanoparticle incorporation into the cells were captured. In addition, the conductive FTO glass endowed the sample with high stability under the required accelerating voltage. Owing to these features of the sample, further analyses could be performed (material contrast and energy-dispersive X-ray spectroscopy (EDS)), which confirmed the presence of nanoparticles inside the cells. The results showed that FE-SEM can enable detailed characterization of nanoparticles in endosomes without the need for contrast staining or metal coating of the sample. Images showing the intracellular distribution of nanoparticles together with cellular morphology can give important information on the biocompatibility and demonstrate the potential of nanoparticle utilization in medicine.     

Atomic force microscopy (AFM), transmission electron microscopy (TEM) and scanning electron microscopy (SEM) of nanoscale plate-shaped second phase particles

The feasibility of accurately measuring the size and the volume fraction of nanoscale plate-shaped precipitates by atomic force microscopy (AFM) has been explored. For quantitative evaluations their unhandy geometry is conveniently described by superellipsoids. The experimental alloy Ni₆₉Co₉Al₁₈Ti₄ served as a model system: plate-shaped disordered γ-precipitates form in the L1₂ long-range ordered γ′-matrix. The results obtained by AFM are compared with those derived from transmission (TEM) and from high-resolution scanning electron microscopy (SEM). The agreement between the AFM and the TEM results is good. In spite of the low number of SEM images taken, the same holds for the SEM results. In addition, magnetic force microscopy was applied; its results are acceptable. The main advantages of AFM are (i) the numerical output for all three dimensions, (ii) the simplicity of its operation and (iii) the lower cost of the microscope itself. The first point allows the numerical AFM output data to be directly subjected to automated computer-based evaluations. All present experimental and evaluation procedures are also applicable to cube-shaped particles with rounded edges and corners as found, for example, in γ′-strengthened nickel-based superalloys.     

In vivo and in vitro study of porcine oviductal epithelial cells, cumulus oocyte complexes and granulosa cells: a scanning electron microscopy and inverted microscopy study

The morphology and structure of porcine oviductal epithelial cells (POEC), cumulus-oocyte complexes (COCs) and granulosa cells (GC) were investigated in vivo and in vitro conditions using scanning electron microscopy (SEM) and inverted microscopy. The POEC contained columnar ciliated cells and spherical shaped non-ciliated cells. Both non- and ciliated cells appeared either in groups or distributing among each other. However, the isolation of cells was observed after culture for 48 h. A total of 921 oocytes from 20 ovaries was isolated resulting in an average of 46 oocytes per ovary. They were round in shape, surrounded by zona pellucida with layers of cumulus cells ranging between 89.16 and 144.68 μm in size. As for COCs, they were classified into 4 types; intact-, multi-, partial-cumulus cell layers and completely denuded oocyte. Interestingly, changes in morphology of COCs with intact and multi-cumulus cell layers were observed in the in vitro study. The GCs in the follicular fluid were also round in shape and found as clusters. After culturing in in vitro for 48 h, no change in morphology was observed. The GC appeared in smaller clusters or were present as single cells and their sizes ranged from 6 to 8 μm. The results obtained from this study allow us to have a better understanding of the morphology and nature of cells under both in vivo and in vitro conditions. This information is also important for the study of their secretions and biochemical compositions, which is of great importance to the use of cells as feeder cells in in vitro fertilization in current studies.     

STEM (scanning transmission electron microscopy) analysis of femtosecond laser pulse induced damage to bulk silicon

This work reports on the structural changes that take place in wafer grade silicon when it is micro-machined with ultra-short laser pulses of 150 fs duration. A Chirped Pulse Amplification (CPA) Ti:Sapphire laser was used, with an operating wavelength centered on 775 nm and a maximum repetition rate of 1 KHz. The laser induced damage was characterized over the fluence range 0.43–14 Jcm^-2, and for each fluence a progressively increasing number of pulses was used. The analytical tools used to characterize the samples were all based upon electron microscopy. A 30 KeV scanning transmission electron microscope (STEM) imaging technique was developed to observe defects in the crystal lattice and the thermal-mechanical damage in the area surrounding the laser machined region. Mechanical cross sectioning (in conjunction with Scanning Electron Microscope (SEM) surface imaging) was also used to reveal the internal structure, composition, and dimensions of the laser machined structures. Based on this analysis, it will be shown that laser machining of silicon with femtosecond pulses can produce features with minimal thermal damage, although lattice damage created by mechanical stresses and the deposition of ablated material both limit the extent to which this can be achieved, particularly with high aspect ratios. A key feature of the work presented here is the high-resolution STEM images of the laser-machined structures. PACS  42.65.Re; 42.62.Cf; 61.80.Ba; 61.82.Fk; 68.37.Hk; 68.37.Lp     

External morphology of Chrysomya pinguis (Walker) (Diptera: Calliphoridae) revealed by scanning electron microscopy

The external morphology of the adult female blow fly, Chrysomya pinguis (Walker, 1858), was examined using scanning electron microscopy. Ultrastructure of the compound eye, ocellus, antenna, palpus, spiracles and portions of the thorax was highlighted. A great difference was observed in sculpture patterns of the corneal lens surface of the ommatidia of the compound eye and that of the ocelli, with the former being densely pustulate and the latter extremely sinuous. Several sensilla types were observed in this study including: sensilla chaetica, sensilla trichodea, sensilla basiconica and sensilla coeloconica. Their probable functions are discussed with reference to other literature. Morphological information about C. pinguis that was revealed in this study allows us to know such types of structures in much better detail and may eventually provide a basis for understanding some of the biological behavior of this fly species in the future.     

Experimental study of annular bright field (ABF) imaging using aberration-corrected scanning transmission electron microscopy (STEM)

Experimental parameters used in the annular bright field (ABF) imaging method were tested using images simulated with the multislice method. Images simulated under identical conditions were found to agree well with experimental images. The ABF technique was shown to be relatively insensitive to the sample thickness and the defocus. In experimental ABF images, atomic columns exhibited dark contrast over a wide range of specimen thickness and defocus values, from 10 to 70 nm and ?20 to +20 nm, respectively. A series of diffraction patterns at atomic columns, obtained using the diffraction imaging method, exhibited higher intensities in their central regions (0–11 mrad) for light elements and in their peripheral regions (11–22 mrad) for heavy elements. The results indicated that the contrast of light elements is enhanced by subtraction of the central region of the transmitted beam, since this is blocked by a circular mask in the ABF-STEM technique. Thus, the overall contrast of light elements is greatly improved, allowing them to be clearly visualized.     

Analysis of the spatial orientation distribution of building blocks in polycrystals as determined using transmission electron microscopy and a backscattered electron beam in a scanning electron microscope

The advantages and disadvantages of the method of automatic analysis of electron backscattering diffraction (EBSD) patterns for studying spatial orientation distributions are considered as compared to transmission electron microscopy (TEM). A misorientation spectrum in a test alloy (Kh20N80 alloy) having a high content of annealing twins is experimentally studied using both TEM and EBSD, and the results obtained are compared.     

Strategies and results of field emission scanning electron microscopy (FE-SEM) in the study of parasitic protozoa

Field emission scanning electron microscopy (FE-SEM) provides a range of strategies for investigating the structural organization of biological systems, varying from isolated macromolecules to tissue organization and whole organisms. This review covers some of the results so far obtained using FE-SEM observation and various protocols of sample fixation to analyze the structural organization of parasitic protozoa and their interaction with host cells. The employment of FE-SEM can be broadened through the use of gold-labeled molecules or tracers, gradual extraction by detergents, and cleavage techniques. These analyses provide significant contributions to the characterization of these organisms concerning ultrastructure, cytoskeleton, motility and intracellular behavior.     

Adsorption geometry of glycine on Cu（001） determined with low—energy electron diffraction and scanning tunnelling microscopy

Using low-energy electron diffraction (LEED) and scanning tunnelling microscopy (STM) it has been found that glycine molecules adsorbed on Cu(001) can form but only the (2×4) and c(2×4) superstructures. On the basis of the missing LEED spots of the surface, it has been concluded that: each (2×4) unit cell consists of two molecules, one being the mirror image of the other; the C－C axis of both molecules lies in the mirror plane of the Cu substrate without a significant shift and twist from the plane; and the two O atoms of the carboxylate group of both molecules locate at the same height level without significant buckling. According to these conclusions, a structural model has been proposed for the (2×4) superstructure (a model for the c(2×4) superstructure already exists). We argue that the (2×4) and c(2×4) superstructures must have similar specific surface free energy, that their hydrogen bonds must be of N-H-O_II type, and that their local adsorption geometry must be similar or even the same. The advantage of combining STM with LEED to determine surface structures is clearly demonstrated.     

Structure of AuSi nanoparticles on Si(111) from reflection high-energy electron diffraction and scanning tunneling microscopy

Gold-rich Au _x Si_1−x particles grown on Si(111)7 × 7 are studied by reflection high-energy electron diffraction (RHEED) and scanning tunneling microscopy (STM). The diffraction patterns reveal that (1) at least two different crystal structures coexist on the substrate; (2) the most prominent data correspond to a rhombohedral or quasi closed-packed structure; and (3) the particles show formation of an unusual contact facet to the substrate. Complete crystal alignment of the particles to the substrate lattice is found with no hints of random orientation. The findings are compared to STM images in terms of their structure, orientation, and morphology.     

Dissociative electron attachment to the explosive detection taggant 2,3-dimethyl-2,3-dinitrobutane (DMNB)

A detailed study on dissociative electron attachment (DEA) to 2,3-dimethyl-2,3-dinitrobutane (DMNB) in the gas phase is presented. Ion yields as a function of the incident electron energy from about 0 to 14 eV have been measured for the most dominant fragments including anions such as NO₂ ⁻, [M-NO₂]⁻ or N₂O₄ ⁻. To help identifying which anion and neutral fragments are formed upon electron attachment we calculated the thermodynamic thresholds using the G4(MP2) method.     

Characterization of the response to moisture of talc and perlite in the environmental scanning electron microscope

This article focuses on the application of ESEM to study the dynamic interaction of hydrophobic and hydrophilic filler materials of interest for the cosmetic and pharmaceutical industries. The ability of ESEM to attain different thermodynamic stages was used to record real-time information of hydration and dehydration processes of those materials in the presence of water and sweat (used as a model physiological fluid). This information is of great importance to understand the behavior of a product containing those fillers as well as to identify potential processing issues related to the interaction of the filler with moisture.     

3D reconstruction and scanning electron microscopy of salivary glands of the millipede Rhinocricus padbergi (Verhoef, 1938) (Diplopoda: Spirobolida)

The three-dimensional technique applied to the salivary gland of the millipides Rhinocricus padbergi showed that it is a single structure, dorsolaterally located to the animal gut and composed of two distinct portion: an acinar and a tubular one. The last one opening onto the oral cavity. This work shows that the salivary gland in R. padbergi, although is a single body, it displays two excretory ducts (one in each side of the anterior end of the gut) which has the function of carrying out the secretion produced toward to the oral cavity, contrary to the anterior data registered by other authors who suggested that the salivary gland in diplopod animals would be a paired structures.     

Using the perturbation method to compute the electron flux behind a two-layer barrier

The perturbation method is used to compute the electron flux behind a flat two-layer absorber one of whose layers is thin. Analytical expressions relating the fluxes behind two- and one-layer barriers are derived. The theoretical results are compared with experimental data.Translated from Izvestiya VUZ. Fizika, Vol. 11, No. 8, pp. 49–53, August, 1968.     

The multi-functional aspect of scanning holographic microscopy: a review(Invited Paper)

Recent developments in scanning holographic microscopy that offer the prospects of new quantitative tools and imaging modalities in bio, micro, and nano sciences are reviewed. The versatility of the method is emphasized. Scanning holography can operate in an incoherent mode for fluorescence imaging, in a coherent mode for quantitative phase imaging, or in a tomographic mode for axial sectioning and rejection of the out-of-focus haze. Possible applications are illustrated with examples, and future prospects ...