Microscopic study on the concretion of ceramics in the “Nanhai I” shipwreck of China,Southern Song Dynasty (1,127–1,279 A.D.)

“Nanhai I” shipwreck of China Southern Song Dynasty is the oldest and the most integrally preserved shipwreck in the world. The related conservation and archeological research have caught great attention of different experts all over the world. In this study, different types of concretion covered on the surface of the ceramics in “Nanhai I” shipwreck were analyzed by X‐ray diffractometer, micro‐Raman spectrometer, and scanning electron microscope equipped with energy dispersive spectroscopy. Based on the analyses, we found that the grey concretion was mainly composed of quartz, aragonite, and calcite while the reddish concretion was mainly composed of pyrite and quartz. Our study indicated that the formation process of the grey concretion probably included the crystallization and transformation of aragonite, while the corrosion of iron implements and crystallization of pyrite were highly involved in the formation of reddish concretion.     

Identification of “Huoshan shihu” Fengdou: Comparative authentication of the Daodi herb Dendrobium huoshanense and its related species by macroscopic and microscopic features

“Huoshan shihu,” derived from Dendrobium huoshanense, is a well‐known, valuable, and rare Daodi herb. Because of its higher price and scarce resources, its related species D. officinale and D. moniliforme are usually presented as “Huoshan shihu” for sale. Fengdou, the processed form of D. huoshanense, its identification is particularly important. To effectively identify Fengdou of D. huoshanense and protect the Daodi herb, the morphological and microscopic characteristics of the stems of three Dendrobium species were examined. The results showed that macroscopic and microscopic features helpful for identification of the three species were the total lengths and internode numbers of stems, diameter and length of each internode, number of vascular bundles, and presence or absence of silica masses. The key features useful for distinguishing between D. huoshanense and its related species, and also between their Fengdou, were the stem lengths and the trends of change in diameter in different internodes. The results of the study indicated that a combination of macroscopic and microscopic identification techniques might conveniently and effectively be applied for identification of Dendrobium species. “Longtou Fengwei” is the main feature of the Daodi herb “Huoshan shihu,” (D. huoshanense Fengdou) and reflects the wisdom behind the protection of Daodi herbs in ancient times.     

Three-dimensional analysis of neuronal geometry using HVEM

There is a need for an electron microscopic method for visualization of selectively stained neurons and neuronal processes with higher resolution than can be obtained with the light microscope, but using thick sections that allow visualization of the three-dimensional structure of the neuron. Such a method is required for measurement of the geometry of neurons, and this information is needed to test theoretical predictions on the way in which electrical signals of synaptic origin are processed by the cells. The high voltage electron microscope (HVEM) is well suited to this application, because of its high resolution and ability to form images of thick sections. Use of this instrument requires development of selective stains that can produce diffuse cytoplasmic staining of specific cells or cell populations on the basis of their functional properties. Several such methods currently being employed for light microscopic work can be used directly in the high voltage electron microscope or can be made useful by relatively minor alterations. These include intracellular staining with horseradish peroxidase, axonal tracing with Phaseolus vulgaris leukoagglutinin (PHA-L), and immunocytochemical staining for specific cell markers known to stain the cytoplasm of certain cell populations. Cells stained intracellularly by microinjection of horseradish peroxidase during physiological recording experiments may be stained in thick (ca. 50 μm) sections cut on a vibratome or similar instrument and stained in the standard way, using methods designed for light microscopy. The sections are then postfixed in osmium tetroxide and embedded in epoxy plastic. Sections cut from these blocks at thicknesses of from 1 to 5 μm using a dry glass knife may be examined directly in the HVEM with no further staining. This produces a very clear image of the cell on a relatively unstained background. This method provides more than adequate resolution of the boundary of the neuron, allowing measurement of neuronal processes to better than 10-nm precision. Similar results are obtained when the same method is applied to axonal tracing using PHA-L. In this case, the exogenously applied marker is used to label a small population of nearby neurons and to trace their connections with other cells at a distance. The lectin is detected by immunocytochemistry, but the selective contrast of the image is adjustable because the concentration of antigen in the cell is largely controlled by the experimenter. The lectin is distributed diffusely in the cytoplasm in a pattern identical to that of intracellular staining, so like intracellular staining, it reveals the overall shape of the cell. Immunocytochemical labelling using endogenous antigens known to be distributed in the cytoplasm of specific neurons produced inadequate control of selective contrast when prepared in this manner. Instead, 1–10μm sections cut from blocks of nervous tissue were embedded in polyethylene glycol, stained using a combedded in polyethylene glycol, stained using a combination of immunocytochemistry and histochemical intensification methods, and embedded in plastic on the grid. This method, which is also suited for staining with poorly penetrating markers such as colloidal gold, may also prove useful in a variety of other situations requiring the intensification of selective contrast.     

Authenticating and distinguishing the eight species of traditional Tibetan medicine “Meiduoluomi” by microscopic technique

Because of the morphological and macroscopic similarity, many species of Erigeron and Aster (Asteraceae) are confusable and usually used under the same name “Meiduoluomi” in traditional Tibetan medicine (TTM). To find an easy, quick, and reliable method to authenticate and distinguish the eight main medicinal plants of these species, the light microscope was used to reveal the morphoanatomic details. The fixed, sectioned, and stained plant materials and epidermis materials were studied by microscopic techniques. The results of the microscopic features are systematically described and illustrated, and comparison parameters are presented. Furthermore, a key to the eight species of “Meiduoluomi” was constructed. Microscopy can be unambiguously used to authenticate and distinguish the eight main species of TTM “Meiduoluomi.” Microsc. Res. Tech., 2009. © 2009 Wiley‐Liss, Inc.     

A technique for studying one and the same section of a cell in sequence with the light and electron microscope

Methods for mounting and staining relatively thin sections on electron microscope grids, in order that one and the same section of a cell can be photographed in sequence with the light and electron microscope are described. Toluidine blue is used as a stain and hexachlorabuta-1,3 diene as a medium which enables the grid carrying the stained sections to be temporarily mounted under a coverslip and examined with an oil-immersion lens. Results obtained with pollen mother cells of Fritillaria lanceolata at zygotene are illustrated.     

Nonmarski differential-interference contrast microscopy in transillumination: its use on unstained or stained sections, smears, and wet mounts, or on fluorochromed sections and cell-culture monolayers

Unstained, lightly stained and conventionally stained microtome tissue sections of two different thicknesses (ca. 4 μm and 1 μm) and also unstained or stained wet mounts of cells were photographed under the microscope using bright-field, positive phase contrast, and Nomarski differential-interference contrast (DIC) in transillumination. The photomicrographs were critically compared. It was found that the density of various stains did not adversely affect the better resolution of the DIC image (as compared to the bright-field image); however, Optical sectioning' of darkly stained objects is not possible. Unstained or stained smears of blood or of epithelial cells of buccal mucosa were examined with DIC in transillumination, then after certain preparatory techniques, the same preparation was examined in the scanning electron microscope, and finally the same areas of the slide were viewed with DIC in epiillumination. Particular attention was given to structures (nuclei and cytoplasm) which appeared in positive or negative relief in the photomicrographs taken by the various techniques. It was concluded that the optically more dense nucleus which always appeared in positive relief by the various methods of examination, was in fact geometrically raised from the surrounding cytoplasm. Acridine orange (AO) stained cell-culture monolayers and H and E stained sections were examined under a fluorescence microscope with DIC optics. By comparing photographs which had been taken with DIC, epi-fluorescence or fluorescence in transillumination, and DIC-fluorescence, it was concluded that the DIC image, which had been superimposed on the fluorescence image, contributed a definite gain in information. Some common errors in the interpretation of the DIC image are discussed; methods of avoiding improper use of equipment are given. The conclusion is drawn that the DIC system is superior to positive and negative phase contrast for the examination of a variety of unstained or stained preparations. Therefore, this method can be used to advantage not only for the examination of unstained preparations, but also on some specimens which have been routinely stained or fluorochromed.     

Electron microscopy of semithick sections: Advantages for biomedical research

Many transmission electron microscopes are available which can be used to examine biological material in 0.25–0.50-μm-thick sections. When compared to the traditional thin section, these “semithick” sections possess a number of inherent advantages: They can be screened for content with the phase contrast light microscope, they facilitate many types of studies requiring an analysis of serial sections, and they are frequently the optimum thickness for stereomicroscopy. Structures such as microtubule-associated components, as well as structural relationships between cellular constituents, may also be clearly visible in semithick sections which are not visible, or go unnoticed, in thin sections. Together these advantages enable an investigator to obtain a more complete three-dimensional picture of a cell or cell component in a significantly (i.e., up to 90%) shorter period of time than would be required if thin sections were used. Semithick sections may, therefore, make a study feasible which is not approachable, or which is approachable only with great difficulty, by conventional thin sectioning techniques.     

A simple method of preparing 1–2 μm sections of large tissue blocks using glycol methacrylate

The method described allows the production of 1–2 μm sections of large tissue blocks. Sections can be obtained from conventional routine histological microtomes. The hydroxyethyl methacrylate is compatible with a wide range of fixatives and a variety of standard histological staining techniques may be applied. Small biopsies, e.g. renal, liver, jejunal etc., can be processed cut and stained within 24 h of receipt. Blocks and stained sections show no apparent deterioration over the 4 years in which the technique has been employed in this laboratory.     

Correlated light optical and scanning electron microscopy of Gram smears of bacteria and paraffin sections of cardiac muscle

Light-microscope slides (3 in. × 1 in.) bearing Gram smears of Erysipelothrix rhusiopathiæ, or Staphylococcus aureus, after preliminary examination under the light-optical microscope (LM), were cut down in size, glued onto specimen stubs, coated with gold and examined in the scanning electron microscope (SEM). These preparations served as a control for investigations into bacteria-cell junctions in tissue. Cover-slips from stained sections of staphylococcal or swine erysipelas endocarditis mounted on 3 in. × 1 in. microscope slides (which had been intensively studied previously with conventional light microscopy) were floated off by immersing the slides in xylol. After dehydration of the tissues on the slides, the preparations were treated similarly to the Gram smears, and were examined with the SEM. Lesions of endocarditis were thus examined, and the information gained from these preliminary examinations shed new light on the pathogenesis of the disease. This information had not previously been available by any other technique. Because of this, and in view of the simplicity of preparing sections for scanning electron microscopy, it is suggested that the SEM might be a useful tool to be applied to routine histological sections.     

Effect of specimen preparation and section transfer techniques on the preservation of ultrastructure,lipids and elements in cryosections

Cryofixation, cryoultramicrotomy, and proper transfer of the cryosections into the electron microscope are important for the preservation of good ultrastructure and the measurement of subcellular elemental distributions. These techniques are applicable to tissue systems which can be rapidly frozen so that minimal to no ice damage occurs during the cryofixation step. For the transfer step we have compared the cryotransfer of hydrated sections and subsequent freeze-drying in the electron microscope with the transfer of sections into an external freeze-dryer, followed by exposure to room temperature and humidity before introduction into the electron microscope. The use of a cryotransfer stage for section transfer from the cryoultramicrotome to the electron microscope and low temperature observation of the thin sections avoids the potential problem of rehydration damage to freeze-dried sections as well as provides protection from the possibility of melting of the lipids in the sections. Both of these problems may lead to loss of in situ elemental distribution and morphology. In this report, observations are presented which show the damaging effects of temperatures above 273 K on ultrastructure due to lipid melting in tissues with high lipid content and the redistribution of elements which can be encountered when thin sections become inadvertantly rehydrated.     

Acridine orange--its use in the specific staining of DNA in mammalian tissue sections

This paper reports on a new method for the use of acridine orange (AO) in an aqueous solution at pH 4.5 for staining DNA of rat tissue sections from which RNA has been extracted selectively with cold phosphoric acid. Not only this, AO can also be used as dye-SO2 reagent, prepared with NHCl and potassium metabisulphite, for staining DNA-aldehyde molecules of acid-hydrolysed tissue sections. AO samples, manufactured by the National Aniline Division as well as by G. T. Gurr have been used with equal success. Studies of stained sections under light microscope reveal the presence of specifically stained yellowish-orange nuclei. Those sections under fluorescent microscope with proper exciter and barrier filters reveal nuclei of maroon colour. The in situ absorption spectra of nuclei stained with AO-SO2 following acid-hydrolysis of tissue sections as well as those of nuclei stained with an aqueous solution of the dye following extraction of RNA have been presented herein. The mode of binding in the former case has been considered to be due to binding of the teritary amino group of the dye molecules with the DNA-aldehyde molecules and in the latter case to be due to electrostatic binding between the positively charged dye molecules with negatively charged phosphate groups of DNA. Implications of all these findings have been discussed.     

Using the modified PSO method to identify a Scott-Russell mechanism actuated by a piezoelectric element

This paper mainly proposes an efficient modified particle swarm optimization (MPSO) method, to identify a Scott-Russell (SR) magnification mechanism driven by a piezoelectric actuator (PA), in which Bouc-Wen model is employed to describe the hysteresis phenomenon. In system identification, we adopt the MPSO to find parameters of the SR mechanism and the PA. This new algorithm is added with “distance” term in the traditional PSO's fitness function to avoid converging to a local optimum. It is found that the MPSO method can obtain optimal high-quality solution, high calculation efficiency, and its feasibility and effectiveness are demonstrated for the modified IEEE 30-bus system. Finally, the comparisons of numerical simulations and experimental results prove that the MPSO identification method for the SR magnification mechanism is feasible.     

Serial sectioning,electron microscopy,and three-dimensional reconstruction of fine nerve fibres and other extended objects

Two different techniques for three-dimensional reconstructions of nerve fibres or other extended fine tissue structures were developed to study the afferent innervation of the cat's knee-joint capsule. The re-embedding technique starts with series of semithin sections for light microscopic reconstructions. In a second step, selected semithin sections can be re-embedded for ultramicrotomy to examine ultrastructural details. This method offers the possibility to investigate fine structures over a distance of several hundred micrometres without any loss of ultrastructural information. The serial section-ESI technique is based on the electron spectroscopic imaging of semithin sections. Small tissue blocks are cut into series of either semithin or alternate semi- and ultrathin sections which can be directly used for a complete ultrastructural investigation. Finally, true-to-scale three-dimensional reconstructions are performed by graphical techniques or computer-aided methods.     

Electron crystallography at atomic resolution: The structure of the odd-chain paraffin n-tritriacontane

The crystal structure of the odd-chain paraffin, n-tritriacontane, nC₃₃H₆₈, is determined directly by using low-dose electron microscope images and electron diffraction intensity data from epitaxially grown microcrystals. Phases of the most intense “polyethylene” reflections are determined from triplet structure-invariant relationships often used in X-ray crystallography. Low-dose electron microscopic images provide phases of the low-angle “lamellar” reflections and these can be used with one-dimensional structure-invariant relationships to determine other phases on the 00? reciprocal row. The phase set is sufficient to calculate an electrostatic potential map which is directly interpretable as a structure image at atomic resolution.     

Microstructure and material properties of hind wings of a bamboo weevil Cyrtotrachelus buqueti (Coleoptera: Curculionidae)

Wings of flying insects, as representative biomaterials, are composed of a flexible membrane and a stiff vein structure that are prone to bending and deformation under aerodynamic forces. Therefore, we must investigate the application value of insect wings in the field of engineering design from the perspective of bionics, which is a new challenge. In this study, we measured the mechanical properties of the hind wings of Cyrtotrachelus buqueti including “dried” and “fresh” samples. The wing membrane samples were prepared by carefully cutting the hind wings into 2.0 mm by 8.0 mm rectangular segments using a gauge. As the major wing veins are the main loading units under aerodynamic forces, we also separated them from the wings as a kind of investigative specimen. The wing membranes were adhered to a specially designed paper fixture and the mechanical properties of the wing veins and membranes were evaluated using a tensile testing machine. We observed the microstructure of the samples using a scanning electron microscope and accurately measured the thicknesses of desired the wing membranes and veins. The results show that there is a difference in the mechanical properties of the two samples. The elastic modulus and Poisson's ratios vary over the region in hind wing, so we can conclude that the wing membrane is an anisotropic and non‐uniform material.     

Correlation averaging of a badly distorted lattice: The surface protein of Pyrodictium occultum

The surface protein of the archaebacterium Pyrodictium occultum forms two-dimensional periodic arrays of extremely poor order. Two variants of correlation averaging have been applied in order to retrieve the unit cell structure from electron micrographs of negatively stained samples: straightforward correlation averaging correcting for lateral displacements only and a more elaborate approach, including a partial compensation for rotational disorder. Surprisingly, both routes yield virtually identical structures. Inclusion of molecular motifs from highly disordered domains, which are rejected in the “straightforward” approach, appears not to improve resolution, possibly because the high local strain tends to distort the individual molecules.     

A simple “hands‐off” apparatus to inflate concealed soft parts of the genitalia of small insect specimens

We describe a simple, effective and relatively cheap instrument for the inflation of soft parts of insect genitalia, particularly the vesica (endophallus) of small male lepidopteran specimens. It can also be applied in female genitalia and for the hyperextension of postabdominal segments of the telescoping type. A fine glass capillary is mounted on a self‐constructed micromanipulator system with capillary holder, moveably placed aside a microscope. The micromanipulator is constructed from the x‐ and y‐pinions of a discarded microscope table and the z‐pinion of a discarded microscope. Preparation is carried out as usual on a microscopic slide. The inflatable soft parts are pushed out mechanically and then fully inflated and subsequently hardened by pumping absolute alcohol with a medical syringe via a flexible PVC tube, the capillary holder and the glass capillary into the phallus. With affordable effort, our “hands‐off”apparatus might considerably ease preparation. Microsc. Res. Tech. 76:258–262, 2013. © 2013 Wiley Periodicals, Inc.     

Zone-axis diffraction patterns by the “Tanaka” method

The “Tanaka” method is one of several techniques that make it possible to obtain zone-axis electron diffraction patterns in a transmission electron microscope without the restriction in the field of view that limits normal convergent-beam diffraction patterns. The method employs a convergent-beam of electrons focused to a probe in a plane that does not coincide with the specimen. The selected area aperture can then be used to eliminate all but one of the diffracted beams to obtain the desired pattern. Practical details of operation and values of operating parameters are discussed. The Tanaka method is a useful addition to the techniques available to the electron microscopist, especially since no instrumental modification is required.     

A low-temperature scanning tunneling microscopy investigation of single electron effects

The design and the performance of a low-temperature scanning tunneling microscope (STM) operating in ultrahigh vacuum (UHV) is described. The sample and the STM are cooled by means of a helium bath cryostate. The STM consists of a “beetle”-type microscope, which is mounted on a spring suspension stage that can be operated between room temperature and 30 K. We present the results of tunneling spectroscopy on ligand-stabilized Au₅₅-clusters on a thiol-covered gold substrate. I(V)-curves taken at 100 K clearly show coulomb staircase structures, which agree well with theoretical predictions for these structures.     

Freezing and drying of biological tissues with a toggle-link helium freezer and an improved freeze-drying apparatus: Application to neuropeptide immunocytochemistry

A freezing apparatus has been developed for bringing blocks of tissue into contact with a block of sapphire chilled to 17°K. A toggle linkage minimizes rebound by slowing the rate of approach of the tissue to the cold surface to a velocity of zero. A glove box limits condensation on the surface of the sapphire, and a miniature moist chamber protects the specimen from drying and premature freezing. About 50 blocks of tissue can be frozen in an hour and a half by using 5 liters of liquid helium. The tissue is then frozendried at controlled temperature, fixed with OsO₄ vapor, and infiltrated with epoxy resin in a simple bench-top freeze-drier without breaking vacuum. About two-thirds of the blocks are useful for electron microscopy. Brain tissue frozen and dried by using these methods retains enough immunoreactivity for enkephalin in plastic sections to permit its detection with immunohistochemistry by using both the light microscope (with immunofluorescence) and the electron microscope (with colloidal gold).