Use of the plasma coagulation method for ultrastructural analysis of biological specimens]

Biological particulate specimens, including Saccharomyces cerevisiae yeast, bovine spermatozoa and human blood cells (normal erythrocytes and leukemic cells) were processed for scanning and transmission electron microscopy using the coagulated plasma technique. The specimens were suspended in frozen and thawed plasma; later, coagulation was induced by adding CaCl2. The clot was cut into small pieces and processed as tissue fragments. The technique is an useful tool when processing biological particulate specimens for electron microscopy.     

Use of Low Temperature Scanning Electron Microscopy to Observe Frozen Hydrated Specimens of Nematodes

Frozen hydrated specimens of Pratylenchus agilis and dauer larvae of Steinernema carpocapsae were observed with low-temperature field emission scanning electron microscopy. This new technique provides information about the surface features of nematodes and also allows specimens to be fractured to reveal their internal structure. Furthermore, both halves of fractured specimens can be retained, examined, and photographed either as two-dimensional micrographs or as three-dimensional images for stereo observation (stereology) or quantitative measurements (stereometry). This technique avoids artifacts normally associated with procedures required to prepare nematodes for examination in the transmission and scanning electron microscopes, such as chemical fixation, dehydration, and sectioning or critical point drying.     

Leptosomatides brevicaudatus n. sp. and a Redescription of Leptosomatides marinae Platonova, 1967 (Enoplida: Leptosomatidae)

The free-living marine nematodes Leptosomatides brevicaudatus n. sp. and L. marinae were described and redescribed, respectively, from material collected in the northwest Pacific. Leptosomatides brevicaudatus n. sp. from Simushir Island differs from L. marinae in the ratio c8 (body length divided by tail length measured on the chord) and the length of the spicules. Leptosomatides marinae is redescribed from light microscopy (LM) observations of the type specimens and LM and scanning electron microscopy (SEM) observations of specimens from Hokkaido, Japan. It appears to be impossible to distinguish among some species of Leptosomatides because they are either insufficiently described or known only from females. Secondary sexual characters of males are essential for purposes of identification.     

High-Contrast Observation of Unstained Proteins and Viruses by Scanning Electron Microscopy

Scanning electron microscopy (SEM) is an important tool for the nanometre-scale analysis of the various samples. Imaging of biological specimens can be difficult for two reasons: (1) Samples must often be left unstained to observe detail of the biological structures; however, lack of staining significantly decreases image contrast. (2) Samples are prone to serious radiation damage from electron beam. Herein we report a novel method for sample preparation involving placement on a new metal-coated insulator film. This method enables obtaining high-contrast images from unstained proteins and viruses by scanning electron microscopy with minimal electron radiation damage. These images are similar to those obtained by transmission electron microscopy. In addition, the method can be easily used to observe specimens of proteins, viruses and other organic samples by using SEM.     

Morphological Comparison of Head Shape and Stylet Morphology of Second-stage Juveniles of Meloidogyne Species

Head shape and stylet morphology of second-stage juveniles of one population each of M. incognita, M. javanica, M. arenaria, and M. hapla were compared by light microscopy. Excised stylets of each species were also compared by scanning electron microscopy (SEM). Differences in head morphology were observed only between M. hapla and the other three species. In SEM, differences in stylet size, shape, and relative distance of the dorsal esophageal gland orifice to the base of the stylet were evident. Differences in stylet morphology between M. incognita and M. javanica could not he detected by light microscopy, but M. arenaria and M. hapla could be distinguished from each other and from the other two species. Head shape and styler morphology of second-stage juveniles are considered useful taxonomic characters.     

Gold-FISH: A new approach for the in situ detection of single microbial cells combining fluorescence and scanning electron microscopy

A novel fluorescence in situ hybridisation (FISH) method is presented that allows the combination of epifluorescence and scanning electron microscopy (SEM) to identify single microbial cells. First, the rRNA of whole cells is hybridised with horseradish peroxidase-labelled oligonucleotide probes and this is followed by catalysed reporter deposition (CARD) of biotinylated tyramides. This facilitates an amplification of binding sites for streptavidin conjugates covalently labelled with both fluorophores and nanogold particles. The deposition of Alexa Fluor 488 fluoro-nanogold–streptavidin conjugates was confirmed via epifluorescence microscopy and cells could be quantified in a similar way to standard CARD–FISH approaches. To detect cells by SEM, an autometallographic enhancement of the nanogold particles was essential, and allowed the in situ localisation of the target organisms at resolutions beyond light microscopy. Energy dispersive X-ray spectroscopy (EDS) was used to verify the effects of CARD and autometallography on gold deposition in target cells.     

Composition, speciation and distribution of iron minerals in Imperata cylindrica.

A comparative study of the roots, rhizomes and leaves of an iron hyperaccumulator plant, Imperata cylindrica, isolated from the banks of an extreme acidic environment, using complementary techniques: Mösbauer spectroscopy (MS), X-ray diffraction (XRD), scanning electron microscopy (SEM) coupled to energy-dispersive X-ray microanalysis (EDAX) and transmission electron microscopy (TEM), has shown that two main biominerals, jarosite and ferrihydrate-ferritin, accumulate in the different tissues. Jarosite accumulates mainly in roots and rhizomes, while ferritin has been detected in all the structures. A model of iron management in I. cylindrica is presented.     

Scanning Electron Microscopy of Pine Seedling Wood Tissue Sections Inoculated with the Pinewood Nematode Bursaphelenchus xylophilus Previously Prepared for Light Microscopy

Scanning electron microscopy (SEM) was applied to paraffin-embedded wood sections to study the histopathology of pine seedlings inoculated with the pinewood nematode (PWN), Bursaphelenchus xylophilus. The sections, which had been previously prepared and observed by light microscopy (LM) on glass slides, were originally obtained from experiments in which pine seedlings had been inoculated with PWN. The cover glass was removed by soaking the glass slide in xylene for 3 to 5 days. The glass slides were cut into small pieces so that each piece contained one wood section. Each piece of the glass slide was attached with double adhesive tape to an aluminum stub. The specimens were sputter-coated with gold and examined with a scanning electron microscope (JEOL-JSM 5200). Compared to LM (as documented in previous reports) SEM provided greater depth of focus and resolution of the damaged wood tissues, nematodes and associated bacteria. SEM made it possible to observe the relationship between bacterial distribution and nematode distribution in wood tissues. SEM observations also suggested the possibility of documenting the death of ray cells and other parenchyma cells in relation to disease development. Finally, the current study of PWN in pine seedlings demonstrated that glass slides prepared for LM observations more than 25 years earlier could be successfully processed for examination by SEM.     

Use of confocal laser scanning microscopy on soil thin-sections for improved characterization of microbial growth in unconsolidated soils and aquifer materials

Confocal laser scanning microscopy (CLSM) was utilized to examine samples from an aquifer microcosm that was used to investigate microbially mediated losses in hydraulic conductivity. Samples were fixed, dehydrated and dried to prepare the biological material in a fashion similar to that used previously for viewing under the scanning electron microscope. Then, samples were prepared as thin-sections by employing soil micromorphological techniques. Serial images generated by the CLSM technique were visualized using computer three-dimensional rendering software. Results from the CLSM technique were compared with simple fluorescence microscopy of thin-sections and scanning electron microscopy (SEM) of samples from the microcosm. Computer visualization of serial sections with the CLSM technique provided images on a submicron scale in three dimensions. SEM has a much higher resolution, on a nanometer scale, but the results are not three dimensional. Artifacts associated with thin-section preparation are minimal for natural porous media composed mostly of sand, such as aquifer materials. Also, CLSM images are affected minimally by changes to biological material due to sample preparation, whereas artifacts associated with SEM images are very prominent, due to the higher magnification and resolution. CLSM of thin-sections and SEM are very powerful methods for viewing microbial growth in natural porous media, but CLSM is preferable because it allows three-dimensional visualization and measurements of cells and aggregates with few artifacts.     

A physico-chemical and biological study of novel chitosan-chloroquinoline derivative for biomedical applications

This paper describes an elegant cross-linking technique for the preparation of chitosan-chloroquinoline derivative by using a greener technique. Chitosan solution in aqueous acetic acid was treated with 2-chloroquinoline-3-carbaldehyde solution to form hydrogel; the resulting hydrogel was subjected to solvent exchange. Combining the results of FTIR and XRD confirmed that 2-chloroquinoline-3-carbaldehyde have been reacted to chitosan. The morphology of the derivative was investigated by X-ray diffraction (XRD) and scanning electron microscopy (SEM). The thermal stability of the derivative was examined by thermogravimetric analysis (TGA). The photoluminescence (PL) spectra of chitosan-chloroquinoline derivative show red-shifted emission maximum. The microbiological screening has demonstrated the antimicrobial activity of the derivative against bacteria viz. Staphylococcus aureus, Escherichia coli and Candida albicans. The obtained results showed that the chitosan-chloroquinoline derivative might be a promising candidate for novel antimicrobial agents for biomedical applications.     

Initial attachment ofCandida albicans cells to buccal epithelial cells

The rapid-freezing technique was applied in association with scanning and transmission electron microscopy to observe the initial attachment (or contact) ofCandida albicans cells to exfoliated human buccal epithelial cells. Low temperature scanning electron microscopy provided detailed three-dimensional morphological features of the yeast-epithelial cell association; adhesion ofC. albicans cells to host cells was primarily owing to an interaction between fibrillar layer of the yeast cell wall and the membrane interdigitations of the epithelial cells. Such a particular interconnection between the two cells was confirmed by the freeze-substitution fixation for transmission electron microscopy. These results clearly demonstrate the outermost fibrillar cell wall layer ofC. albicans responsible for adhesion to host cells.     

Cryofracturing for Scanning Electron Microscope Observations of Internal Structures of Nematodes

Nematodes were prepared for scanning electron microscopy by cryofracturing in ethanol and then by critical-point drying in carbon dioxide. Cross sections of Caenorhabditis briggsae and Xiphinema americanum showed the arrangement of the intestine, ovaries, muscle cells, and some layers of the cuticle. The technique is complementary to transmission electron microscopy and facilitates the interpretation of results from thin sections.     

Extracellular matrix of plant callus tissue visualized by ESEM and SEM

Actinidia deliciosa endosperm-derived callus culture is stable over a long period of culture. This system was used to investigate the ultrastructure of extracellular matrix occurring in morphogenic tissue. Specimens were prepared by different biological techniques (chemical fixation, liquid nitrogen fixation, glycerol substitution, critical-point drying, lyophilization) and observed by scanning electron microscopy (SEM). Fresh and wet samples were analyzed with the use of environmental scanning electron microscopy (ESEM). Extracellular matrix was observed on the surface of cell clusters as a membranous layer or reticulated network, shrunken or wrinkled, depending on the procedure. Generally, shrunken membranous layers with a globular appearance and fibrils were noted after critical-point drying and liquid nitrogen fixation. Smoother surface layers without visible fibrils and showing porosity were typically seen by environmental scanning electron microscopy. Preservation with glycerol substitution caused wrinkled appearance of examined layer. Analysis of fresh samples yielded images closer to their natural state than did critical-point drying or fixation in liquid nitrogen, but it seems best to compare the results of different visualization methods. This is the first report of ESEM observations of plant extracellular matrix and comparison with SEM images from fixed material.     

Precipitation of calcite induced by Synechocystis sp. PCC6803

Calcite with laminate structure was successfully prepared by culturing Synechocystis sp. PCC6803 with different concentrations of calcium chloride (CaCl₂) in BG11 media. S. PCC6803 was examined by scanning electron microscopy (SEM), transmission electron microscopy (TEM), laser confocal scanning microscope (LCSM) and energy dispersive spectroscopy (EDS). The effects of Ca²⁺ concentrations and pH values on calcification were investigated and the micro morphs of the CaCO₃ crystals were observed by means of SEM. These results showed that CaCO₃ crystals could be more easily formed with increasing the concentration of CaCl₂ in S. PCC6803 culture solution. S. PCC6803 could largely bind calcium ions, most of which were present in extracellular polymeric substances and on the cell wall. Inside the cells there were a lot of circular areas rich in calcium ions without the crystallization of calcium. Some cells produced a thicker gelatinous sheath outside of the translucent organic thin layer. And the cells inside also produced major changes that the original chloroplasts were almost transformed into starch grains whose sizes were from 0.5 to 1 μm with relatively uniform in sizes. At the same time the cell sizes significantly reduced to only about 8–9 μm almost changing to half of its original diameters. The calcite crystals with a highly preferred orientation induced by S. PCC6803 were observed with X-ray diffraction (XRD). A critical implication was that S. PCC6803 could induce bio-calcification and then mediate the further growth of CaCO₃ crystals in the biological system.     

Entrapment and lysis of the cyanobacterium Phormidium luridum by aqueous colonies of Myxococcus xanthus PCO2

A Myxococcus xanthus isolate from a farm drainage ditch, designated strain PCO2, is capable of rapidly inducing lysis of both agar and liquid-grown cultures of the cyanobacterium, Phormidium luridum, var. olivacea. Microscopic studies of the predator-prey interaction demonstrate that lysis of the cyanobacterium occurs within clumps and spherules formed by the cells of M. xanthus PCO2. In the earliest stage, one sees the formation of irregular microclumps of bacteria and cyanobacterial filaments. As these clumps mature, colonies 1 to 6 mm in diameter develops. The center of these densely green colonies contains cyanohacteria in various stages of degradation, while the periphery is almost exclusively a tightly woven mass of myxobacterial cells. Electron microscopy shows that long extrusions from the outer membrane of the M. xanthus PCO2 cells are involved in the formation both of initial clumps and of mature colonial spherules. These extrusions appear to efficiently entangle the cyanobacterial filaments in the culture environment. Predator-to-prey ratios of 1/10, 1/100 and 1/1,000 have resulted in cyanobacterial lysis. Because the entrapment and lysis of P. luridum filaments by M. xanthus PCO2 appears to be independent of any other heterotrophic nutritional requirement, as well as of environmental agitation, this system has potential as a biological control technique for undesirable aquatic cyanobacteria.Abbreviations TEM transmission electron microscopy - SEM scanning electron microscopy - AB algae broth - ABT algae broth plus 0.2% tryptone     

有机荧光团及荧光标记细胞的阴极荧光成像研究

目的 阴极荧光（CL）成像是一种以电子束为激发源的高分辨荧光成像技术,但生物材料对电子束的敏感性限制了CL技术在生命科学中的广泛应用。为了研究和发展CL技术在生物样品中的应用,本文旨在通过探究电子辐照引起碳基材料的结构损伤、有机基团的降解及荧光猝灭等问题,深入理解电子源对有机荧光团的激发特性。方法 本研究应用扫描电镜（SEM）和阴极荧光谱仪系统（SEM-CL）,研究电子源对有机荧光团及荧光探针标记细胞的激发特性,观测了有机物的CL信号的发射特性、强度衰减、成像方式及特点。结果 实验结果显示,在低能量（2.5～5 keV）和低束流（～10 pA）电子辐照下,有机荧光微珠发射出较强的荧光,CL像分辨率达到～30 nm。荧光微珠经过12 min辐照,信号强度衰减了25%,CL像仍保持了可接受的发光强度和足够的信噪比。此外,还获得了从细胞表面到内部一定深度内,荧光标记的亚细胞结构信息。结论 在SEM-CL系统中,可以同时获得由电子束激发产生的电子像和CL像,实现阴极荧光与电子显微镜关联（CCLEM）成像。本实验的研究结果为CCLEM技术应用于生物结构研究提供了数据及技术支持。     

Coupling scanning electron microscopy with DNA bar coding: a novel approach for thrips identification

The small size and cryptic nature of thrips pests often make their monitoring and the identification process difficult. With the aim to obtain accurate identification of various pest species in the order Thysanoptera, a pilot study was conducted on an invasive thrips species Scirtothrips dorsalis Hood in Florida. The specific objective of this pilot study was to assess if the thrips specimens processed to be observed under scanning electron microscopy (SEM) can be further utilized for DNA based assays. Larvae and adults of S. dorsalis were subjected to traditional morphological identification using high resolution SEM prior to their DNA extraction. Sequence results of both mtCO1 and ITS rDNA of individual larva and adult S. dorsalis were found to be in agreement with the taxonomic identification conducted using SEM and each result confirmed the other technique. Our results suggest that steps involved during specimen preparation and observation under SEM does not impact DNA analysis of the sample. The two techniques together could be used for the correct identification of various thrips species using the same specimen. The proof of concept was further tested on three other important thrips species Frankliniella occidentalis (Pergande), F. schultzei (Trybom) and Thrips palmi Karny, which confirmed the robustness of the technique. The technique has broader significance for different areas under entomological sciences especially medical, forensic and taxonomic studies where the samples put under a high beam of ions for investigation are often required for DNA analysis.     

Use of the Scanning Electron Microscope for Viewing Bacteria in Soil

Scanning electron microscopy was used for viewing Bacillus cereus and Staphylococcus aureus in three different soils. Both organisms were detected in the test soils at an approximate concentration of 10⁷ cells per gram of soil; theoretically, the minimal number of microorganisms required for detection with the scanning electron microscope technique was between 10⁷ and 10¹⁰ cells per gram of soil. Because the concentration of cells was critical, the use of scanning electron microscopy as an extraterrestrial life detection instrument would be limited with soils containing more than 10⁷ bacteria per gram of soil.