Ordinary Microscope Research Articles

LAYERED SYNTHETIC MICROSTRUCTURES AS OPTICAL ELEMENTS FOR THE EXTREME ULTRAVIOLET AND SOFT X-RAYS

In recent years, the availability of intense continuous radiation sources, such as electron synchrotrons and laser-induced hot plasmas, has given rise to a renewed interest in soft x-ray and x-uv reflectivity measurements. Such studies were, for a long time, hindered because of mainly two reasons. First, it was really difficult to generate soft x-rays in the laboratories and second, there was no possibility for practical implementation and design of optical systems, such as focusing elements, mirrors, etc. associated with that particular wavelength region. Soft x-rays, with wavelength range usually from 10 to about 200 angstroms, can produce images of higher resolution than visible light due to their shorter wavelength. For years, physicists have wanted to construct an x-ray microscope that would exploit the ability of soft x-rays to detect small structures. The need for such an instrument is clear. The resolution of light microscopes is limited by the comparatively long wavelength of visible light. Although transmission electron microscopes have much higher resolution, they are weak in penetrating power and are, therefore, limited to very thin specimens. Therefore, transmission electron microscopy involves extreme care in sample preparation. Such preparations which might alter the very structure of a biological sample, would not be required in x-ray microscopy. The difficulties in constructing an x-ray microscope, however, have proved to be irresistible, because of the fact that soft x-rays cannot be brought together to form an image. In other words, soft x-rays cannot be made reflected from any known single surface at normal or near-normal incidence. The only possibility that existed until now employs grazing incidence, the only form of focusing x-ray optics. But their quality (resolution) has been limited because they must be machined in the form of a paraboloid or hyperboloid. Lenses of the kind used in ordinary optical microscopes cannot be made for use at wavelengths less than about 1000 angstroms. There are two reasons for this. First, there is only a tiny difference in the refractive indices among the different materials at soft x-ray wavelengths. Second, soft x-rays are strongly absorbed by all materials and cannot penetrate any conceivable lens, used in ordinary optical microscopes or telescopes. A major advance in x-ray optics holds a great promise both in the fields of high resolution scanning x-ray microscopy, lithography and substantial improvements in the quality of x-ray telescopes. Recent improvements in the techniques for quality control of evaporated and sputtered films have led to the interest in the controlled fabrication of multilayered structures known as 'Layered Synthetic Microstructures', to be used as mirrors for the extreme ultraviolet and soft x-ray regions. These can be produced with virtually any layer spacing greater than approximately 10 angstroms and they have a considerably high diffraction efficiency at normal or near-normal incidence. This remarkable enhancement in normal-incidence reflectivities at x-uv domain of the electromagnetic spectrum leads to another innovative application of these microstructures, i.e. the production of x-ray lasers with high gain-length products, where the enhanced normal-incidence reflectivity of the multilayers has been applied for multiple pass gain of the laser media by increasing the effective path lengths of the plasma columns. The present article covers the theoretical considerations, development and different techniques of controlled fabrication of layered synthetic microstructures along with their potential applications in the fields of x-ray spectroscopy, microscopy, x-ray laser production and lithography.

Essai technique d'automatisation des groupages HLADR

Two types of anti-class II coated magnetic beads were compared for the use in isolation of B lymphocytes from whole blood. The HLA DR typing was performed in parallel after separation of the cells by the rosette technique. The depletion was monitored by Flow cytometry using CD2 and CD20 antibodies. Typing was performed using classic lymphocytotoxicity test of Propidium-Iodide-Carboxyfluorescein diacetate fluorescence. The results were scored with an ordinary microscope or by photometric evaluation. These techniques lead to simplification in cell isolation from small amounts of blood with an important gain of time and a less subjective interpretation of DR phenotyping was until now long and difficult.

Structural change of salted jellyfish during cooking.

Salted jellyfish are eaten after desalting in water, boiling, and dipping in water for several days. In this work, the structural change of salted jellyfish during the process described above has been studied by observation with a scanning electron microscope and with ordinary microscopes. The samples became shortened in the direction along the surface of the skin by boiling at 80°C for 30min, so that the thickness which was perpendicular to the surface increased. The change is due to the shrinkage of collagen in a part of the umbrella of the jellyfish by heating, which is ascertained by the stain method and differential scanning calorimetrical measurement. A network structure was observed on the surface of the heated sample. The network structure of the heated sample was destroyed after dipping in water for 6 days. Also, the surface of the both the unheated and heated sample was peeled by dipping in water, and the change was larger in the heated one. Not only the thermal denaturation of collagen but these structural changes may contribute to the texture of cooked jellyfish.

Fast bevel cross sectioning of quantum-well semiconductor layers with high resolution

An improved bevel cross-sectioning method is presented which makes it possible to measure the thickness of relatively thin semiconductor surface layers. A staining procedure to enhance the contrast of the layers for two material systems, GaAs/GaAlAs and InP/InGaAsP, has been developed. Applying a newly constructed ball-lapping-apparatus, an ordinary optical microscope and a surface-profiler, relatively thin MBE-grown quantum-well layers (6.5 nm) can rapidly be measured with an accuracy of better than 10%, which is improved for thicker, normal layers (1000 nm) to better than 5%.

コンフォーカル光学系における分光法の一提案

Confocal microscope system, which has higher resolution than an ordinary microscope, has spectrometric capability with no special optical elements. It is found by calculation that the confocal microscope system with SFS1 lens which has 20.9 aberration number has 0.6 nm spectral resolution. This report describes about some applications of the system.

The ordinary light microscope: an appropriate tool for provisional detection and identification of crystals in synovial fluid.

To determine if the ordinary light microscope is a useful tool in the detection and identification of crystals in synovial fluid 63 fluids (13 with monosodium urate (MSU), 14 with calcium pyrophosphate dihydrate (CPPD), one with both types of crystal, and 35 without crystals) were examined blindly by two observers, first with an ordinary light microscope, then with a polarising light microscope. Tentative identification of the crystals by their shape was attempted. The sensitivity of the ordinary light microscope for the detection of crystals was 96.2% and 100%, with specificity of 100% and 97.1% for observers 1 and 2, both with respect to the polarising light microscope. The concordance between observers for the ordinary light microscope was chi = 0.90 (0.897, 0.902) and for the polarising light microscope chi = 0.96 (0.958, 0.961). The specificity for identification of MSU and CPPD crystals by their shape with respect to the polarising light microscope was 92.3% and 85.7% for observers 1 and 2. When a polarising light microscope is not available examination of synovial fluid by an ordinary light microscope may allow a provisional diagnosis; definitive identification of the crystals requires a polarising light microscope.

Modified TEM-STEM for magnetic imaging

An electron microscope for magnetic imaging (magnetic domain observation) is described. The microscope includes a magnetic-field-free objective lens and a large bulk specimen stage 30 × 50 mm 2 in size. Its resolution as an ordinary transmission electron microscope is 0.7 nm and it has three Lorentz microscopy modes for magnetic imaging: Lorentz TEM (transmission electron microscopy), DPC (differential phase contrast)-STEM (scanning transmission electron microscopy) and Type-2 magnetic contrast SEM (scanning electron microscopy). Resolution of the domain image is 20 nm in the DPC-STEM mode. The minimum magnification for Type-2 SEM is ×5 on a 15×20 mm 2 specimen area. Quantitative measurement of magnetization can be made in DOC and Type-2 imaging because the magnetic contrast is proportional to the magnetic induction.

Application of an ultrahigh-resolution scanning electron microscope (UHS-T1) to biological specimens.

In 1985 we developed an ultrahigh-resolution scanning electron microscope with a resolution of 0.5 nm. It is equipped with a field emission gun and an objective lens with a very short focal length. In this study we report a survey of some different preparation techniques and biological specimens using the new scanning electron microscope. Intracellular structures such as cell organelles were observed surprisingly sharper than those observed by ordinary scanning electron microscopes. However, at magnifications over 250,000 x, platinum particles could be discerned as scattered pebbles on the surface of all structures in coated materials. Using an uncoated but conductively stained specimen, we successfully observed ribosomes on a rough endoplasmic reticulum at a direct magnification of 1 million. In these images some protrusions were recognized on the ribosomes. Ferritin and immunoglobulin G were used as samples of biological macromolecules. These samples were observed without metal coating and conductive staining. The ferritin particles appeared as rounded bodies without any substructure on the surface and immunoglobulin G as complexes of three-unit bodies. In the latter the central body might correspond to the Fc fragment and two side ones to Fab fragments. We assume that ultrahigh-resolution scanning electron microscopy is an effective means for observation of the cell fine structures and biological macromolecules. It will open a new research field in biomedicine.

A confocal laser microscope scanner for digital recording of optical serial sections.

A confocal laser microscope scanner developed at our institute is described. Since an ordinary microscope is used, it is easy to view the specimen prior to scanning. Confocal imaging is obtained by laser spot illuminatin, and by focusing the reflected or fluorescent light from the specimen onto a pinhole aperture in front of the detector (a photomultiplier tube). Two rotating mirrors are used to scan the laser beam in a raster pattern. The scanner is controlled by a microprocessor which coordinated scanning, data display, and data transfer to a host computer equipped with an array processor. Digital images with up to 1024x1024 pixels and 256 grey levels can be recorded. The optical sectioning property of confocal scanning is used to record thin (about 1 microm) sections of a specimen without the need for mechanical sectioning. By using computer-control to adjust the focus of the microscope, a stack of consecutive sections can be automatically recorded. A computer is then used to display the 3-D structure of the specimen. It is also possible to obtain quantitative information, both geometric and photometric. In addition to confocal laser scanning, it is easy to perform non-confocal laser scanning, or to use conventional microscopic illumination techniques for (non-confocal) scanning. The design has proved reliable and stable, requiring very few adjustments and realignments. Results obtained with this scanner are reported, and some limitations of the technique are discussed.

A modification of testicular biopsy reporting.

A modified method has been devised which enables the pathologist to evaluate more efficiently the seminiferous tubules and to present a more detailed histological report on testicular biopsies in patients investigated for infertility. This basically involves the production of a diagrammatic representation of the actual biopsy onto a specifically designed reporting form: a detachable prismatic arm attached to an ordinary light microscope enables the pathologist to transfer an actual outline of each of the seminiferous tubules contained in the biopsy onto this form. The seminiferous epithelium and Leydig cells are scored using the method of Johnsen. This method was evaluated using 20 consecutive biopsies. Skill in its usage was quickly acquired and it has been found to be a more clinically acceptable way of histopathological reporting, further facilitating standardization and inter-laboratory comparison.

Wet-Mount Examination for the Diagnosis of Trichomoniasis

To the Editor. —Krieger et al 1 have done a beautiful study on the diagnosis of Trichomonas vaginalis infection. The only criticism I have concerns the preparation of the wet mount. A Popsicle stick (or Papanicolaou smear scraper), not a cotton-tip swab, should be dipped in the vaginal puddle and immediately transferred to a drop of saline already placed on a clean glass slide. This preparation should then promptly (preferably less than a minute and certainly no more than minutes) be examined under the ordinary light microscope at low power. Trichomonads will lose motility rapidly with delay or under the hot light of the microscope. A coverslip should not be applied until after initial perusal under low power. The two fast legs system is necessary to have a high percentage of positive wet preparations. 2

Direct determination of the aliphatic carbon content of individual macerals in petroleum source rocks by near-infrared microspectroscopy

In order to determine the aliphatic carbon content in sedimentary organic particles near-infrared (NIR) microscope spectroscopy has been employed for the first time. An ordinary lens microscope photometer common for vitrinite reflectance measurements has been modified to detect hydrocarbon absorbance differences with a spatial resolution of about 30 μm. Microscopic studies of natural and artificial samples under reflected light condition were shown to be unsuitable for quantitative aliphatic C determination. Transmitted light measurements ofliptinites in polished thin sections, however, could be calibrated against synthetic polymer blends without exact knowledge of the sample thickness. In contrast to the established microscopic techniques for characterizing coal and petroleum source rocks NIR microspectroscopy is able to follow the chemical alterations of liptinites directly with comparatively low cost.

Growth of NdP 5O 14 single crystals by the cold-finger technique

Single crystals of neodymium pentaphosphate, NdP 5O 14, were grown from a solution of pyrophosphoric acid containing neodymium oxide by the cold-finger technique. The growth patterns on the crystal surface, the shapes of chemical etch pits, and the internal defects due to flux inclusions and voids were observed using an ordinary reflection microscope and an interference contrast microscope. The optimum growth condition of the crystals and the growth mechanism are discussed briefly.

Microcirculatory methods for evaluating the effect of vasoactive drugs in clinical practice.

Microcirculatory methods have to be used in order to be able to evaluate the effect of vasoactive drugs in ischemic skin areas. During the past two decades several new such techniques for studying the microcirculation in man have been developed. These techniques have shown to be of great value for evaluating drug effects in patients with disturbed peripheral circulation. One such method is the non-invasive Laser Doppler technique. It measures both the nutritional and the non-nutritional thermoregulatory vascular bed of the skin. The data achieved are only semiquantitative, but the method has been shown to be clinically very useful for studying the dynamics of total skin microcirculation in a specific area. In order to be able to study directly what happens in the nutritional capillaries microscopical methods have to be used. Two such techniques are available in clinical practice. By an ordinary light microscope the capillaries can be directly visualized. A specific classification system can be used to evaluate the degree of ischemic damage to an area. The effect of different kinds of therapeutic procedures for improving the microcirculation in an ischemic area can be easily evaluated. By the combination of a light microscope and a sensitive TV-camera the blood flow in single skin capillaries can be measured at a magnification of 250-1 000 times. This method is well suited for testing the immediate effects of vasoactive substances on the nutritional skin circulation both in healthy subjects and in patients with different kinds of cardiovascular diseases.

A simple method to detect bacterial associations in bivalve gills

Recent discoveries have shown that endosymbiotic bacteria may be widespread among bivalve species in different habitats. A simple method is described which allows the detection of bacterial‐gill association with an ordinary light microscope.

Indirect alkaline phosphatase immunoenzymatic staining for the detection of antibodies to Epstein-Barr virus-induced virus capsid antigens and early antigens

An indirect alkaline phosphatase immunoenzymatic staining technique was developed for the detection of antibodies against Epstein-Barr virus-induced virus capsid antigens and early antigens in cell smears. The presence of antibodies against Epstein-Barr virus-induced virus capsid antigens and early antigens was revealed by a dark blue staining of cells expressing the antigens. The alkaline phosphatase assay gave a permanent record of the reaction that could be visualized under an ordinary light microscope. The titers obtained with this assay on 91 serum samples were significantly correlated with the titers obtained with an immunofluorescence technique.

Birefringent objects visualized by circular polarization microscopy.

In the standard polarizing microscope, birefringent material appears bright only if its optic axis is oblique to the axes of the polarizer and analyzer filters; consequently, an object may be visualized as several disconnected bright regions. This confusing appearance is avoided if the crossed plane polarizers of the conventional microscope are replaced by circular polarizers of opposite handedness. All orientations of the optic axis in the focal plane then become equivalent; objects generally appear uniformly bright. Ordinary microscopes are easily modified to use this technic with readily available components.

Alkaline phosphatase immunoenzymatic staining for detection of antigens induced by cytomegalovirus.

An indirect alkaline phosphatase immunoenzymatic staining method was developed to localise antigens in human embryo fibroblasts that have been induced by cytomegalovirus. The enzyme label was developed with a naphthol salt and fast blue to obtain a bright blue staining of the antigens that could be clearly visualised under an ordinary light microscope. The procedure is rapid, sensitive, and specific and can be used in diagnostic laboratories to detect active infection caused by cytomegalovirus.

Muscle and sarcomere lengths in the hind limb of the rabbit (Oryctolagus cuniculus) during a galloping stride

Rabbits were filmed galloping, and the length changes of the principal hind limb muscles were determined. Sarcomere lengths were measured in carcasses set by rigor mortis in four of the positions adopted during a stride. These sarcomere lengths were measured by means of a diffraction technique, devised for the purpose, using an ordinary microscope. Expected sarcomere lengths for three of the positions were predicted from that observed in the fourth, together with muscle length changes. A theoretical length‐tension curve for rabbit muscle was constructed, using A and I filament lengths, it was shown that when the muscles were active, their sarcomere lengths corresponded to the plateau of the length‐tension curve.

Golgi apparatus is one continuous organelle in pancreatic exocrine cell of mouse.

The three-dimensional structure of the Golgi apparatus (GA) in mouse pancreatic exocrine cells was studied under an ordinary transmission electron microscope and also a high voltage electron microscope. From observations of thick sections of ZIO (Zinc-Iodide-Osmium solution)-reacted pancreatic tissues, GA was found to be a complex three-dimensional structure with its basic configuration summarized as follows: 1. Golgi cisterna is a long elongated ribbon-like structure and Golgi stack is composed of several ribbon-like cisternae organized from cis to trans. 2. The ribbon-like Golgi stack has wide expanded areas in some portions where it branches into 2 or 3 ribbon-like stacks. 3. The Golgi stack often shows a huge circular arrangement, about 10μm in diameter, maintaining the polarity from cis to trans. 4. In some portions the direction of polarity of the stack changes: the Golgi stack in some parts rotates in the long expanding direction (X axis) maintaining each cisternal continuity. 5. According to the observations of 1μm thick serial sections, the continuity and polarity of the Golgi stack are maintained three-dimensionally (X, Y and Z axes) and GA, as a whole, was found to be one huge continuous reticular organelle in the cell.