Pressure Scanning Electron Microscopy Research Articles

Characterisation of calcium crystals in Abelia spp. using X-ray diffraction and electron microscopy

SummaryThe localisation, chemical composition, and morphology of calcium (Ca) oxalate crystals in the leaves and stems of Abelia mosanensis and of A. × grandiflora were analysed using a variable pressure-scanning electron microscope (VP-SEM) equipped with an X-ray diffraction system, a low temperature SEM (LT-SEM), and a transmission electron microscope (TEM). Foliar analyses of macro- and micro-elements were performed on the leaves and stems of A. mosanensis. A greater number of Ca oxalate crystals were observed in A. mosanensis than in A. × grandiflora. Three morphologically distinguishable types of crystals were observed: the prismatic crystals found inside the chloroplast, multifaceted star-like spherical and bladed aggregate crystals (druses) inside the vacuoles of the mesophyll cells, and small angular crystals (sand crystals) inside the cuticle. Semi-solid crystals that may drip and accumulate to become a solid Ca oxalate crystal were observed by LT-SEM, which indicated the growth of druses of Ca oxalate crystals. The growth of prismatic crystals and of druses were evident through the formation of crystalline lamellae. Micro-analysis indicated that the crystals were Ca oxalate and contained magnesium in A. mosanensis, or silicon in A. × grandiflora. Abelia stems with low foliar calcium concentrations showed no Ca oxalate crystal formation. This is the first report, to our knowledge, on the presence and possible growth of crystals of different morphologies and chemical compositions in Abelia.

Comparison of SEM and VPSEM imaging techniques with respect toStreptococcus mutansbiofilm topography

The study compared images of mature Streptococcus mutans biofilms captured at increasing magnification to determine which microscopy method is most acceptable for imaging the biofilm topography and the extracellular polymeric substance (EPS). In vitro S. mutans biofilms were imaged using (1) scanning electron microscopy (SEM), which requires a dehydration process; (2) SEM and ruthenium red (SEM-RR), which has been shown to support the EPS of biofilms during the SEM dehydration; and (3) variable pressure scanning electron microscopy (VPSEM), which does not require the intensive dehydration process of SEM. The dehydration process and high chamber vacuum of both SEM techniques devastated the biofilm EPS, removed supporting structures, and caused cracking on the biofilm surface. The VPSEM offered the most comprehensive representation of the S. mutans biofilm morphology. VPSEM provides similar contrast and focus as the SEM, but the procedure is far less time-consuming, and the use of hazardous chemicals associated with SEM dehydration protocol is avoided with the VPSEM. The inaccurate representations of the biofilm EPS in SEM experimentation is a possible source of inaccurate data and impediments in the study of S. mutans biofilms.

Non-destructive characterization of oriental porcelain glazes and blue underglaze pigments using μ-EDXRF, μ-Raman and VP-SEM

The study of ancient materials with recognized cultural and economic value is a challenge to scientists and conservators, since it is usually necessary an approach through non-destructive techniques. Difficulties in establishing a correct analytical strategy are often significantly increased by the lack of knowledge on manufacture technologies and raw materials employed combined with the diversity of decay processes that may have acted during the lifetime of the cultural artefacts. A non-destructive characterization was performed on the glaze and underglaze pigments from a group of Chinese porcelain shards dated from the late Ming Dynasty (1368–1644) excavated at the Monastery of Santa Clara-a-Velha in Coimbra (Portugal). Chemical analysis was performed using micro-energy dispersive X-ray fluorescence spectrometry (μ-EDXRF). Mineralogical characterization was achieved by Raman microscopy (μ-Raman) and observation of small-surface crystallization dark spots with a metallic lustre in areas with high pigment concentration was done by variable pressure scanning electron microscopy (VP-SEM). Cobalt aluminate was identified as the blue underglaze pigment and a comparison of blue and dark blue pigments was performed by the ratio of Co, Mn, and Fe oxides, indicating a compositional difference between the two blue tonalities. Manganese oxide compounds were also identified as colouring agents in dark blue areas and surface migration of manganese compounds was verified.

Multifunctional Nanorods Serving as Nanobridges To Modulate T Cell-Mediated Immunity

Electrodeposited nanorods serving as multivalent bridges were fabricated and surface-decorated with ligands for immune cells. Gold and nickel solutions were sequentially electrodeposited on nanoporous anodized disc templates and the template was dissolved to retrieve bisegmented nanorods with different lengths. Gold and nickel segmented nanorods were surface-immobilized with mannose and RGD peptides to prepare immune-cell recruiting nanorods. Surface-functionalization of nanorods were confirmed by fluorescence-labeling of each ligands and confocal microscopy. Dendritic cells and T cells were co-incubated with the surface-functionalized nanorods, and the proximity between the nanorods and the immune cells was visualized by variable pressure scanning electron microscopy and confocal microscopy. The long nanorods were associated with the immune cells, whereas the shorter nanorods were rather endocytosed by cells, suggesting a feasibility of the longer nanorods as bridging for the cells. Cytokine releases from the immune cells were monitored by cultivating lipopolysaccharide-activated dendritic cells with T cells. Interleukine-2 and interferon-γ release profiles showed a strong correlation with the length of the nanorod, where the 4 μm nanorods induced the highest levels of cytokine release compared to 1 or 2 μm nanorods. Thus, we concluded that the proximity of the immune cells increased by bridging the immune cells with the nanobridging system, which subsequently increased cytokine release by facilitating the antigen presentation process.

A novel catalytic method for the synthesis of spherical aragonite nanoparticles from cockle shells

For the first time, we report here a novel top down catalytic approach for the synthesis of aragonite nanoparticles with spherical morphology from cockleshells. Cockle shell is a natural reservoir of aragonite which is a biogenic polymorph of calcium carbonate. Aragonite polymorph is widely used in the repair of fractured bone, development of advanced drug delivery systems, and tissue scaffolds. The method involves an easily performable and low-cost mechanical stirring of the micron-sized cockle shell powders in presence of a nontoxic biomineralization catalyst, dodecyl dimethyl betaine (BS-12). It produces spherical shaped aragonite nanoparticles of 35 ± 5 nm in diameter with a good reproducibility and without any additional impurities at room temperature. The findings were verified with a variable pressure scanning electron microscopy (VPSEM), energy dispersive X-ray spectroscopy (EDX), transmission electron microscopy (TEM),Fourier transmission infrared spectroscopy (FT-IR), X-ray diffractometer (XRD), and thermogravimetric analyzer (TGA).The reproducibility, low-cost and simplicity of the method suggested its potential applications in large scale synthesis of aragonite nanoparticles with spherical morphology in an industrial set up.

A multianalytical approach to investigate stone biodeterioration at a UNESCO world heritage site: the volcanic rock-hewn churches of Lalibela, Northern Ethiopia

A multianalytical approach combining Optical Microscopy (OM), Backscattered Variable Pressure Scanning Electron Microscopy + Energy Dispersive X-ray Spectroscopy (VP-BSEM + EDS), Powder X-ray Diffractometry (PXRD), Raman Spectroscopy, and Microbiological techniques has been applied to characterize decay products and processes occurring at the surface of two rock-hewn churches (Bete Gyorgis and Bete Amanuel) at the UNESCO’s World Heritage site of Lalibela, Northern Ethiopia. The two churches were carved into volcanic scoria deposits of basaltic composition. In their geological history, the Lalibela volcanic rocks underwent late to post-magmatic hydrothermal alteration together with partial laterization and are therefore characterized by a decay-prone highly vesicular microtexture with late stage to post-magmatic precipitation of secondary mineral phases (calcite–zeolite–smectite). The main objective of the study was to gain a better insight into the weathering products and mechanisms affecting the surface of the stone monuments and to assess the relative contribution of natural “geological” weathering processes versus biological/salt attack in stone decay at this unique heritage site. Results indicate that while the main cause of bulk rock deterioration and structural failure could be related to the stone inherited “geological” features, biological attack by micro- (bacteria) and/or macro- (lichens) organisms is currently responsible for severe stone surface physical and chemical weathering leading to significant weakening of the stone texture and to material loss at the surface of the churches walls. A prompt and careful removal of the biological patinas with the correct biocidal treatment is therefore recommended.

Correlative evaluation of variable pressure-scanning electron microscopy to histology methods for vascular tissues

For pathological investigation, vascular tissues implanted with biomaterials or medical devices require microscopic histology and scanning electron microscopy (SEM) assessment to evaluate biocompatibility with the vascular luminal surface. However, these valuable specimens are often susceptible to SEM and histology tissue preparation artifacts. Correlative variable pressure-scanning electron microscopy (VP-SEM) and histology methods were developed and evaluated, using current SEM technology, allowing direct imaging of hydrated samples without lengthy tissue processing, labor, equipment, and their sequela artifacts. Unstained and silver-stained, fixed swine carotid arteries were assessed using a cooling stage and an environmental secondary electron detector. After VP-SEM imaging, arteries were processed for histology evaluation. Assessment of luminal endothelial surface SEM images and tissue histology of the underlying mural wall was made to determine if low vacuum, cryo-freezing, electron beam exposure, and/or staining affected tissue morphology. Conventional SEM requires lengthy tissue processing with dedicated equipment for solvent dehydration, critical point drying, and metallic sputter coating in order to image under high vacuum for secondary electron imaging. This process causes notable artifacts when samples are later processed for microscopic histology. In contrast, silver staining of swine carotid arteries, followed by aldehyde fixation, facilitated VP-SEM imaging and histology preparation with results that were comparable to conventional SEM and had acceptable tissue morphology. VP-SEM with a cooling stage and an environmental secondary electron detector allows efficient SEM imaging and correlative histological assessment of vascular tissue samples. This correlative method may be further developed and implemented for research applications with biomaterial and medical device implanted tissues.

Effect of Packaging Shape and Storage on the Keeping Quality of Mineral Water and a Development of Water-Treatment Device

A notable change of quality and increase in the cost of water-treatment devices has resulted from the current watertreatment and packaging techniques in the food industry. Because of these factors, efforts to discover alternative, less stringent packaging techniques have taken a place of high priority in food science. Pyramidal food packages are capable of altering the crystallization mode of water’s mineral content. The effects of pyramidal and square packaging shapes on mineral water were studied by examining ice surface morphology. Ice surface morphology was examined by variable pressure scanning electron microscopy VP-SEM. The pyramid shaped package promotes the formation of filament-shaped crystals, and reduction of solid substrate-bound crystals. These results provide evidence that a pyramidal package acts to structure water and mineral particles in an ordered manner.

A novel method for the synthesis of calcium carbonate (aragonite) nanoparticles from cockle shells

We report a simple, novel and low-cost method for the synthesis of aragonite nanoparticles from cockle shells. Aragonite is one of the least abundant biogenic polymorphs of calcium carbonate and is widely used as a biomaterial for the repair of fractured bone, development of advanced drug delivery systems, and tissue scaffolds. The method involves a simple mechanical grinding of the micron-sized cockle shell powders in the presence of a non-toxic and non-hazardous biomineralization catalyst, dodecyl dimethyl betaine (BS-12). The method produces rod-shaped aragonite particles with a diameter of 20 ± 5 nm with good reproducibility and without any additional impurities. This was confirmed by a combined analysis of variable pressure scanning electron microscopy (VPSEM), transmission electron microscopy (TEM), Fourier transmission infrared spectroscopy (FTIR), thermogravimetric analyzer (TGA), X-ray diffraction spectroscopy (XRD) and energy dispersive X-ray analyser (EDX). The method should find potential applications in industry for the large scale synthesis of aragonite nanoparticles from a low cost but abundant natural resource such as cockle shells.

Fungal biosorption of silver particles on 20th-century photographic documents

Microbial deterioration is a common problem in photographic collections and is considered a major cause of loss of documents. However, few studies so far have been addressed to biological damage on these materials. Several species of naturally occurring fungi can cause infections on the gelatin-silver emulsion of both positive and negative photographic material, producing defacement and loss of mechanical and aesthetical properties of the objects. In this study a particular phenomenon, spontaneously caused by fungi on 20th-century photographic films and positive supports, was documented by means of variable pressure scanning electron microscopy (VP-SEM) combined with electronic dispersion spectroscopy (EDS). This technique allowed the observation of entire, unaltered films without metallisation thus with a not invasive approach. The ability of some fungi to alter the distribution of silver crystals in the gelatin emulsion was described thanks to a backscattered electrons detector that showed differences in the atomic number of the visualised objects, giving rise to an appreciable contrast in case of different chemical composition.

Rapid micropropagation of monopodial orchid hybrid (Aranda Wan Chark Kuan ‘Blue’ × Vanda coerulea Grifft. ex. Lindl.) through direct induction of protocorm-like bodies from leaf segments

A competent protocol for accelerated plant regeneration system via direct induction of protocorm-like bodies (PLBs) from leaf of orchid hybrid Aranda Wan Chark Kuan ‘Blue’ × Vanda coerulea Grifft. ex. Lindl. was developed for the first time to establish a basis for mass production. Using tissue culture technique, the conditions for PLB induction from leaf explants and conversion of PLBs into plantlets were investigated. Leaves were transferred to MS medium containing different types and concentrations of cytokinins (namely, N6-benzyladenine, 6-furfurylaminopurine, N-phenyl-N′-(1,2,3-thidiazol-5-yl)urea/TDZ and zeatin) for PLB induction. By means of exploring the effects of cytokinins, it was determined that the optimum PLB induction occurred on MS media supplemented with 1.5 mg l−1 TDZ; whereby accordingly, PLB induction (with a frequency of 94.8 %) was observed as early as 8 days of culture and an average of 25 PLBs was obtained from 1 cm2 leaf segment after 40 days of culture. Variable pressure scanning electron microscopy indicated the different developmental stages of PLBs in detail. Light/stereo microscopic observation showed the maturation of PLBs and gradual formation of shoot and leaf primordia. More than 96 % conversion (with well-developed shoots and roots) was achieved within the next 30 days of culture, when well developed PLBs were transferred in MS medium supplemented with 1 mg l−1 BA, 0.5 mg l−1 IBA plus 60 mg l−1 adenine sulphate. After 60 days of transfer in plastic pots filled with sand and perlite (2:1; v/v) and with charcoal and coconut fibre (1:1; v/v), subsequently, 90 % well-acclimatized plantlets were recovered.

Biofouling of various metal oxides in marine environment

Biofouling has induced serious problems in various industrial fields such as marine structures, bio materials, microbially induced corrosion (MIC) etc. The effects of various metals on biofouling have been investigated so far and the mechanism has been clarified to some extent(1,2), and we proposed that Fe ion attracted lots of bacteria and formed biofilm very easily(3). In this study, we investigated the possibility for biofouling of Pseudomonas aeruginosa on various metal oxides such as Fe2O3, TiO2, WO3, AgO, Cr2O3 etc. And in addition of such a model experiment on laboratory scale, they were immersed into actual sea water as well as artificial sea water. As for the preparation of metal oxides, commercial oxide powders were used as starting material and those whose particle sizes were under 100 micrometers were formed into pellets by a press. Some of them were heated to 700 °C and sintered for 10 hours at the temperatures. After the calcinations, they were immersed into the culture of P. aeruginosa at 35 °C in about one week. After the immersion, they were taken out of the culture and the biofouling behaviors were observed by optical microscopy, low pressure scanning electron microscopy (low pressure SEM) etc. Biofouling is generally classified into several steps. Firstly, conditioning films composed of organic matters were formed on specimens. Then bacterial were attached to the specimen's surfaces, seeking for conditioning films as nutrition. Then bacteria formed biofilm on the specimens. In marine environment, more larger living matters such as shells etc would be attached to biofilms. However, in the culture media, only biofilms were formed.

Facile Synthesis of Calcium Carbonate Nanoparticles from Cockle Shells

A simple and low‐cost method for the synthesis of calcium carbonate nanoparticles from cockle shells was described. Polymorphically, the synthesized nanoparticles were aragonites which are biocompatible and thus frequently used in the repair of fractured bone and development of advanced drug delivery systems, tissue scaffolds and anticarcinogenic drugs. The rod‐shaped and pure aragonite particles of 30 ± 5 nm in diameter were reproducibly synthesized when micron‐sized cockle shells powders were mechanically stirred for 90 min at room temperature in presence of a nontoxic and nonhazardous biomineralization catalyst, dodecyl dimethyl betaine (BS‐12). The findings were verified using a combination of analytical techniques such as variable pressure scanning electron microscopy (VPSEM), transmission electron microscopy (TEM), Fourier transmission infrared spectroscopy (FT‐IR), X‐ray diffraction spectroscopy (XRD), and energy dispersive X‐ray analyser (EDX). The reproducibility and low cost of the method suggested that it could be used in industry for the large scale synthesis of aragonite nanoparticles from cockle shells, a low cost and easily available natural resource.

Non-Destructive Investigation of Pre-Columbian Goldwork from Panama with Variable Pressure Scanning Electron Microscopy

Extended abstract of a paper presented at Microscopy and Microanalysis 2011 in Nashville, Tennessee, USA, August 7–August 11, 2011.

Structure of the Empty Ornamental Nuptial Gift of Empis snoddyi Steyskal (Diptera, Empididae) as Revealed by Variable Pressure Scanning Electron Microscopy

Extended abstract of a paper presented at Microscopy and Microanalysis 2011 in Nashville, Tennessee, USA, August 7–August 11, 2011.

The formation and removal characteristics of aerosols in ammonia-based wet flue gas desulfurization

The characteristics of aerosol generation were studied experimentally in an ammonia-based wet flue gas desulfurization process. Particle size distributions and concentrations, morphologies and compositions before and after desulfurization were measured using an electrical low pressure impactor and scanning electron microscopy, respectively. The results show that aerosols can be generated between ammonia and sulfur dioxide resulting in gas-phase reaction and the aerosol concentration at the outlet of scrubber is significantly higher than at the inlet. Before desulfurization the particles are primarily silica-alumina minerals including O, Al, Si and C, while after ammonia-based desulfurization aerosol particles have smooth surfaces with regular structures, such as cubic and prismatic crystals, and contain principally O, S and N. Particle sizes up to 10 μm were measured, but the majority of the aerosol particles are in the submicron range. Separation of submicron particles from flue gas is difficult by conventional desulfurization scrubber; however high removal efficiency can be achieved based on the enlargement of the particles by heterogeneous condensation. The influence of the supersaturation degree and liquid–gas ratio on the removal efficiencies of the particles are demonstrated additionally. It indicates that aerosols generated in ammonia-based desulfurization process can be deliquesced and gradually enlarged in a high humidity flue gas, but the supersaturation degree higher than 1.2 is required for heterogeneous nucleation of water vapor with ash particles from coal combustion.

History and Surface Condition of the Lewis Chessmen in the Collection of the National Museums Scotland (Hebrides, late 12th-early 13th centuries)

This paper summarises on-going work to examine the surface condition of the 11 chess pieces in the collection of the National Museums Scotland. The chess pieces are part of the hoard found on the isle of Lewis on the west coast of Scotland and are remarkable examples of medieval ivory carving. The aim of the work is to characterise surface features that can be used to draw new information about the materials from which the pieces were made, their history from the period of their manufacture until they were found in the first part of the 19th century, and whether they were originally coloured. The chess pieces were examined using low-powered optical microscopy and non-destructive X-ray Fluorescence spectroscopy. Modern samples of ivory were exposed to fungi under controlled conditions and the surface damage investigated using optical microscopy and controlled pressure scanning electron microscopy with energy-dispersive X-ray analysis.

Testing meat tenderness using an in situ straining stage with variable pressure scanning electron microscopy

This project developed analytical protocols allowing in situ tensile testing of beef M.semitendinosus in a variable pressure scanning electron microscope (VP-SEM) fitted with a straining stage. The objective of this project was to develop an appropriate testing regime for the tensile testing of meat following various pre-treatments (marinaded, cooked, frozen). The in situ testing, at 100% relative humidity, was validated with ex situ testing using the same tensile stage. From the ex situ tensile tests the apparent elastic modulus ranged from 0.1MPa to 0.3MPa with the cooked, non-marinaded meats being stiffest. The low strain rates imposed by mechanical considerations of the straining stage resulted in lower values of fracture toughness and UTS than those reported in the literature. Fracture behaviour observed during the in situ tensile tests revealed that fracture propagated across the perimysial connective tissue. For the cooked non-marinaded steak, catastrophic rupture occurred in the crosslinks of the connective tissue. The cooked marinaded steak with more gelatinized connective tissue showed a less catastrophic rupture. Limitations of the testing geometry of an in situ straining stage are critical for their impact on image quality. The working distance for VP-SEM is necessarily short meaning that a sample needs to be close to the final lens of the SEM for high quality images. This is hard to achieve within the geometrical constraints of most sample chambers. However, it was possible to capture real time images of muscle failure during tensile testing of meat and relate that to the impact of meat processing treatments. Such information is vital for determining the structural factors affecting meat tenderness.

Study of Inorganic Particles, Fibers, and Asbestos Bodies by Variable Pressure Scanning Electron Microscopy with Annexed Energy Dispersive Spectroscopy and Micro-Raman Spectroscopy in Thin Sections of Lung and Pleural Plaque

In a previous work it has been demonstrated that micro-Raman spectroscopy is a technique able to recognize crystalline phases on untreated samples. In that case, inorganic particles and uncoated fibers from bronchoalveolar lavage (BAL) of a patient affected by pneumoconiosis were identified and characterized. In this work the technique is applied to asbestos bodies, that is, to coated fibers, and on crystallizations and fibrous phases observed in the plural plaque from patients affected by mesothelioma. From the Raman analysis the abundant fibrous material observed in the pleural area is talc, whereas rounded grains in the pleural tissue show the Raman spectrum of apatite, a calcium phosphate mineral particular to bones. In the pulmonary tissue many asbestos bodies, consisting of the incorporated fibers coated by iron-rich proteins, were observed. Under the 632.8 nm laser beam of the spectrometer, photo-crystallization of hematite in the iron-rich material forming the asbestos bodies can be proposed by the changes in the Raman spectra acquired during subsequent acquisitions. Nevertheless, the identification of the mineral phase constituting the incorporated fiber was possible by analyzing the Raman spectra; the results were confirmed by variable pressure scanning electron microscopy with annexed energy dispersive spectroscopy (VP-SEM-EDS) analyses.

Electron detection in the intermediate chamber of the variable pressure SEM

Two concepts of the secondary electron detection inside the intermediate chamber of the variable pressure scanning electron microscopy have been discussed. One of them, the two-stage secondary electron detector has been the subject of previous publications. The other one, the intermediate secondary electron detector is an improved solution in respect of its complexity, vacuum demands and dimensions. Both detector systems apply some kinds of scintillators, so they present advantages specific for scintillator detectors but extended to a wide range of gas pressures, from high vacuum to pressures exceeding 10 mbar. The detector functioning is illustrated with computer simulations of electron and ion flow in the input region of the detectors made with use of the MC-SIMION program. This software combines a commercially available program SIMION 3D v.7.0, destined for computations of charged particles trajectories in electric and magnetic fields, and the Monte Carlo one written in the SIMION internal language.