Atomic force microscopy study of the collagen fibre structure

Observations of intact reconstituted and native collagen fibres were performed with the atomic force microscope. The results are compared between the two types of fibres and with those obtained previously with the electron microscope on freeze-etched or negative stained samples. Some of the findings presented here indicate that the specimens observed in air with the atomic force microscope were still in a hydrated state.     

Assembly of microspheres from acidic proteinoids and histones or histone-like proteinoids

Basic polyamino acids, whether organismic or synthetic in origin, alter the morphology of proteinoid microspheres in which they are included. The particles obtained with histone are essentially indistirguishable under the light microscope from those obtained with proteinoids of histone-like composition (histonoids). In each instance, structures of morphological complexity similar to those of some microfossils are obtained.     

Confocal scanning light microscopy of the Escherichia coli nucleoid: comparison with phase-contrast and electron microscope images.

The nucleoid of living and OsO4- or glutaraldehyde-fixed cells of Escherichia coli strains was studied with a phase-contrast microscope, a confocal scanning light microscope, and an electron microscope. The trustworthiness of the images obtained with the confocal scanning light microscope was investigated by comparison with phase-contrast micrographs and reconstructions based on serially sectioned material of DNA-containing and DNA-less cells. This comparison showed higher resolution of the confocal scanning light microscope as compared with the phase-contrast microscope, and agreement with results obtained with the electron microscope. The effects of fixation on the structure of the nucleoid were studied in E. coli B/r H266. Confocal scanning light micrographs and electron microscopic reconstructions showed that the shape of the nucleoid remained similar after OsO4 or glutaraldehyde fixation; however, the OsO4 nucleoid appeared to be somewhat smaller and more centralized within the cell.     

Measuring elastic properties of cells by evaluation of scanning acoustic microscopy V(Z) values using simplex algorithm

In this paper a new technique is proposed to determine the acoustic properties as well as the thickness (and volume) of biological cells. Variations of thickness, density, acoustic wave velocity, stiffness, and attenuation coefficient of a living or dead cell are obtained by scanning the cell by an acoustic microscope. The distance between the cell and the microscope lens is varied and several voltage curves are thus obtained. These curves are then inverted by simplex optimization technique to obtain the cell parameters. The spatial resolution of the method is limited to the resolution of the scanning acoustic microscope. It allows to take advantage of the full range of frequencies and amplification of the microscope. Characteristic distributions of stiffness are exemplified with an endothelial cell in culture. The main part of the thin, lamellar cytoplasm has high stiffness, which drops close to the lamella/cell body transition region and only slightly increases again through the central part of the cell. Acoustic attenuation seems to be related to two factors, cytoplasm accumulation (in the lamellar parts) and scattering in the central part rich in organelles.     

Mapping of intracellular halogenous molecules by low and high resolution SIMS microscopy.

The subcellular distribution of halogenous molecules has been studied by SIMS microscopy in cultured cells of a human breast carcinoma (MCF-7 cell line). Two instruments of microanalysis were used. A low lateral resolution ion microscope (SMI 300 CAMECA) and a prototype scanning ion microscope equipped with a cesium gun that gives high lateral resolution images. This apparatus has been developed by G Slodzian, in Onera Laboratories (Office National d'Etudes et de Recherches Aérospatiales). Molecules studied by low lateral resolution ion microscope were halogenous steroids: fluorometholone, triamcinolone, bromocriptine and bromoandrosterone. Analytical images show that the first two compounds are mainly localized in the nuclear structure of MCF-7 cells whereas the last two molecules are localized in cytoplasm of these cells. Images were obtained with a resolution of 1 micron. With the scanning ion microscope, it is now possible to obtain images at the ultrastructural level. Four analytical images can be simultaneously obtained by a single scan of the imaged area, corresponding to a depth of erosion of the section of ten nm. The intranuclear distributions of three pyrimidine analogs, 5-bromo-2'-deoxyuridine, 5-iodo-2'-deoxyuridine and 5-fluorouracil have been studied in phase S and M of MCF-7 cells and these images have been compared to the distribution of sulfur, nitrogen and phosphorus. All these images have been obtained with a lateral resolution better than 100 nm.     

Scanning electron microscope studies of the endometrium of the cyclic mare.

Endometrial biopsies obtained from mares at different stages of the oestrous cycle, during anoestrus and in various abnormal conditions were examined with the scanning electron microscope. Preliminary observations suggest that the patterns of secretory and ciliary activity in the uterine epithelium are similar to those observed by electron microscopical techniques in laboratory and other large domestic animals. The response of the epithelial cells to hormonal variations and infections is compared with that of the endometrium as seen with the light microscope.     

AOTF microscope for imaging with increased speed and spectral versatility.

We have developed a new fluorescence microscope that addresses the spectral and speed limitations of current light microscopy instrumentation. In the present device, interference and neutral density filters normally used for fluorescence excitation and detection are replaced by acousto-optic tunable filters (AOTFs). Improvements are described, including the use of a dispersing prism in conjunction with the imaging AOTF and an oblique-illumination excitation scheme, which together enable the AOTF microscope to produce images comparable to those obtained with conventional fluorescence instruments. The superior speed and spectral versatility of the AOTF microscope are demonstrated by a ratio image pair acquired in 3.5 ms and a micro-spectral absorbance measurement of hemoglobin through a cranial window in a living mouse.     

The optical near-field and cell biology

A new form of scanning light microscopy is described in which the lens is replaced by a point of light that is smaller than the wavelength. Resolution is obtained that is defined not by the wavelength but by the size of the spot of light. This is the case so long as the point of light is within the dimension of a wavelength from the surface that is to imaged or within the optical near-field. This new form of light microscopy is called near-field scanning optical microscopy (NSOM). Resolutions are being obtained with NSOM that are similar to scanning electron microscopy but without the destructive effects of a vacuum or of an electron beam. In addition such a microscope is readily interfaced with fluorescent and non-fluorescent contrast enhancing stains that are commonly used in cell biology. The possibility of a near-field/far-field microscope is discussed with overlapping resolutions from a few hundred of a conventional microscope to the tens of thousand that can be obtained with NSOM.     

Determination of absolute coccolith abundances in deep-sea sediments by spiking with microbeads and spraying (SMS-method)

A quick new method is described for the quantification of absolute nannofossil proportions in deep-sea sediments. This method (SMS) is the combination of Spiking a sample with Microbeads and Spraying it on a cover slide. It is suitable for scanning electron microscope (SEM) analyses and for light microscope (LM) analyses. Repeated preparation and counting of the same sample (30 times) revealed a standard deviation of ±10.5%. The application of tracer microbeads with different diameters and densities revealed no statistically significant differences between counts. The SMS-method yielded coccolith numbers that are statistically not significantly different from values obtained from the filtration-method. However, coccolith counts obtained by the random settling method are three times higher than the values obtained by the SMS- and the filtration-method.     

Field observations on the use of anti-sporozoite monoclonal antibodies for determination of infection rates in malaria vectors

Samples of indoor-resting Anopheles gambiae s.1. from Mali and Burkina Faso (West Africa) were processed in order to compare Plasmodium falciparum sporozoite rates obtained by immunoradiometric assay (IRMA) with circumsporozoite (CS) monoclonal antibody and by microscope examination of salivary glands. The immunological method provided sporozoite rates always higher than those obtained by microscope examination. This result does not appear to be related to cross-reactions involving non-sporozoite antigens. A small fraction of IRMA-positive mosquitoes is necessarily negative by microscope, since these mosquitoes actually contain the CS antigen only in the abdomen, presumably in connection with the presence of fully mature oocysts. However, the frequency of these mosquitoes cannot explain in itself an average ratio of 1:2 between microscope and IRMA sporozoite rates. A more important source of difference appears to depend on the detection of positive mosquitoes with low sporozoite numbers which remain more frequently undetected by microscope examination. Failure of salivary gland penetration by sporozoites is also considered as a possible source of discrepancy between the two methods.     

Demonstration of epithelium-associated microbes in the oesophagus of pigs, cattle, rats and deer

Scanning electron microscope or light microscope examination of oesphageal specimens demonstrated the association of microbial cells with the epithelial surface. Rod-shaped bacteria and cocci were seen in specimens obtained from pigs and rats, cocci in specimens from cattle, and rods     

Pigmentary system of the adult alpine salamander Salamandra atra atra (Laur., 1768).

The pigmentary system of the skin from adult specimens of the black alpine salamander Salamandra atra atra was investigated by light microscope, electron microscope, and biochemical studies. Results were compared with those obtained in previous study of the subspecies Salamandra atra aurorae. Unlike Salamandra atra aurorae, which presents epidermal xanthophores and iridophores, Salamandra atra atra is completely melanized, presenting only epidermal and dermal melanophores. The melanosomes in both the epidermis and the dermis appear to derive from a multivesicular premelanosome similar to that in the goldfish, and the epidermal melanosomes are smaller than those in the dermis. Premelanosomes with an internal lamellar matrix were not observed. The biochemical results have shown that in the ethanol extracts obtained from the skin in toto and from the melanosomes, pteridines and flavins are always present and are the same as those extracted from the black skin areas of Salamandra atra aurorae.     

Practical aspects of diagnostic electron microscopy.

The electron microscope technique of negative staining was first used to obtain fundamental information about virus morphology, but in recent years it has developed into a practical method for virus diagnosis. The methods employed are both simple and rapid. The following review discusses in detail the steps that must be taken to obtain good electron microscope preparations and illustrates some of the results that can be obtained.     

Analysis of organic acids in fruit-bodies of Tricholoma giganteum by high performance liquid chromatography

A rapid method for determining the sensitivity of Staphylococcus aureus to beta-lactams is described. The method involves the culture of microcolonies of the bacterium on the surface of antibiotic-containing agar medium on microscope slides. The areas of the microcolonies formed are measured by means of a microscope and image analyzer. The sensitivity of the bacterium can be estimated in 4 h by this technique and the results are comparable to those obtained by overnight incubation.     

网脊石耳及网脊平盘石耳分类中微形态学的新证据

虽然网脊石耳与网脊平盘石耳因各自具不同类型子囊盘而彼此易于区别,但是,由于它们的子囊盘极为少见,而且二者的叶状体上表面都具有明显的网状褶皱,因而在实际分类工作中往往难以区分。本文作者用解剖镜和扫描电镜进行对比研究时,为该两种地衣的分类从微形态学方面提供了新的证据。     

A Novel Phase-contrast Transmission Electron Microscopy Producing High-contrast Topographic Images of Weak objects

We report a novel class of transmission electron microscope (TEM), the difference-contrast electron microscope (DTEM), which displays nanostructures of thin specimen objects in a topographical manner. Topography obtained by the difference-contrast develops shadowgraphs in pseudo three-dimension, namely volume-like representation of projected objects as if things are illuminated by light from one direction. The specific optical device tomanipulate electron waves for DTEM is the hemicircular phase-plate, which appears to be quite distinguishable from the Zernike phase plate utilized in Zernike phase-contrast TEM, while both have to be placed onto the back-focal plane of the objective lens. The topographic images obtained with DTEM for ultrathin sections of kidney cells were compared with those obtained with conventional TEM. DTEM confirmed the experimental advantage of high contrast topography by visualizing ultrastructural details inside the cells.     

An electron microscope study of lampbrush chromosomes

Lampbrush chromosomes were isolated from germinal vesicles of oocytes from Necturus maculatus, Triturus viridescens, Pseudotriton montanus and Rana pipiens. After treatment of isolated nuclei with 10 per cent sucrose, chromosomes free of nuclear sap are obtained for examination in either the light microscope or in the electron microscope. For electron microscopy the chromosomes were prepared either by Anderson's critical-point procedure or were embedded in methacrylate and sectioned. The evidence presented in favor of the view that the loops, axis, and the chromomeres of lampbrush chromosomes are formed by two chromonemata is based on the following observations: 1. Treatment of isolated chromosomes with 0.002 M KCN loosens the structure of the loops, and a more or less coiled organization is then observed in most of them with the light microscope. At the electron microscope level, each loop consists of a bundle of microfibrils. The latter are 500 A in diameter, and their complex arrangement within the loops is best studied in stereoscopic preparations. 2. Treatment of chromosomes with 0.002 M KCN also unravels the "chromomeric" regions of the axis. A fibrillar organization then becomes visible in the light microscope. In the electron microscope, wide strands are seen within some chromomeres; their diameter corresponds closely to that of the chromonemata forming the loops associated with the same chromomeres. In thin transverse sections of isolated chromosomes, no special structure is visible in the axial region except random profiles of fibrils similar to those seen in the loops of the same preparations. 3. Two strands sometimes connect adjacent chromomeres. Where gaps exist along the axis, after stretching of the chromosomes, a loop occasionally straddles the break and returns to a chromomere on each side.     

Histological Preparation of the Undecalcified Rat Otic Bulla for Mesoscopic Observations

Otic bullas of the rat, obtained by excision and formalin fixed, are successfully embedded in methylmethacrylate by dehydration and subsequent infiltration with plastic under vacuum. Sections 10 μm thick are obtained by cutting the trimmed and sandpapered acrylic blocks on an LKB multirange microtome. The sections are collected on adhesive tape and stained with a Trichrome stain (modified Weigert-van Gieson). Finally, the sections attached to the tape are mounted on microscope slides with glycerin-gelatin and sealed in the same medium. Serial sections are used for three-dimensional graphic reconstruction.     

Direct observation of the transition from calcite to aragonite growth as induced by abalone shell proteins

The mixture of EDTA-soluble proteins found in abalone nacre are known to cause the nucleation and growth of aragonite on calcite seed crystals in supersaturated solutions of calcium carbonate. Past atomic force microscope studies of the interaction of these proteins with calcite crystals did not observe this transition because no information about the crystal polymorph on the surface was obtained. Here we have used the atomic force microscope to directly observe changes in the atomic lattice on a calcite seed crystal after the introduction of abalone shell proteins. The observed changes are consistent with a transition to (001) aragonite growth on a (1014) calcite surface.     

Simple Densitometer for Microbiology Laboratories

A densitometer composed of a microscope and a colorimeter is described. Results obtained with the microscope densitometer and with a standard densitometer compare favorably.