STUDIES ON THE BIOCHEMISTRY AND FINE STRUCTURE OF SILICA SHELL FORMATION IN DIATOMS : I. The Structure of the Cell Wall of Cylindrotheca fusiformis Reimann and Lewin

An electron microscope study on the cell wall of the diatom Cylindrotheca fusiformis was carried out using stereoscopic and sectioning techniques. Material prepared by an enzyme treatment or by a mechanical method showed that the wall consists of two major components: a silica shell and organic material. Vapor of hydrofluoric acid was employed to remove the silica and thereby reveal the arrangement of the organic material. An attempt was made to increase the contrast of the organic component by "staining." Uranylacetate not only increased the electron opacity of the organic material but also apparently decreased the electron opacity of the silica shell. In ultrathin sections of complete cells, the structure as revealed by stereoscopy could be confirmed and extended. Every part of the silica shell is tightly enclosed by organic material. In the valve region the silica enclosed in this way is located between other layers of organic material. The whole cell wall is surrounded by a mucilaginous substance which stains with ruthenium red.     

Fixation and imaging of biological elements: heavy metals, diffusible substances, ions, peptides, and lipids

We tested various fixation and analysis methods to demonstrate by electron microscopy elemental imaging in tissues and cells, i.e., soluble substances such as many kinds of ionic elements, water soluble low molecular peptides, and even organic solvent soluble substances such as lipids. For the ionic elements, we tested frozen dried or freeze-substituted methods and organic or inorganic special chemical precipitation methods combined with microwaved fixation methods. The data were analyzed with electron beam X-ray microanalysis, electron energy filtered imaging analysis, and electron microscope autoradiography. The data were demonstrated as elemental distribution images and were calculated quantitatively. For the soluble low molecular peptides, we developed a tannic acid and aldehyde method combined with microwaved fixation. We discuss the theoretical background of the tannic acid fixation and microwaved fixation methods. For the organic solvent soluble substances, i.e., lipids including steroids, we successfully tested the use of a mixed fixative of aldehyde and osmium, digitonization, and osmification with the use of p-phenylendiamine or imidazole. We also proposed some new ideal biotracers for electron beam X-ray microanalysis and electron energy filtered imaging analysis.     

The Wettability and Mechanism of Geometric Non-Smooth Structure of Dragonfly Wing Surface

Scanning electron microscope and optical contact angle measuring instruments were used to investigate the microstructure and wettability of geometric non-smooth structure of dragonfly wing surface. Results show that the geometric non-smooth structure of dragonfly wing surface is one part of epicuticle, some organic solvents can effectively dissolve the main ingredient of non-smooth structure. The hydrophobicity of dragonfly wing surface is induced by the co-coupling of the non-smooth structure and the waxy layer covering.     

Nanobiology of the Ocean

Concepts of nanobiotic forms and their relationships with oceanic organic matter and feeding, as well as of their role in oceanic communities, are proposed on the basis of long-term electron microscope and microbiological studies initiated in 1969 in the Barents Sea, the Atlantic and Pacific oceans, and the marginal seas of the Arctic Ocean.     

Early post-mortem calcified Devonian acritarchs as a source of calcispheric structures

Uniquely preserved Late Devonian calcispheres were found in a core of the deep borehole Sosnowiec IG-1 (Upper Silesia, southern Poland). These enigmatic calcareous microfossils are interpreted here as acritarchs that underwent an early post-mortem calcification. Remnants of organic walls preserved in the calcispheres suggest that they represent various acanthomorphic acritarchs, characteristic members of the Palaeozoic marine phytoplankton. Taphonomic analysis combined with the light microscope and scanning electron microscope (SEM) observations of mineral and organic components of the investigated calcispheres suggest that a complex multi-stage process led to calcification of their in vivo non-mineralized acritarch forerunners. The ubiquity of acanthomorphic calcispheres in many Devonian shallow-water limestones is a testimony to little, thus far, documented acritarch crops that must have existed over extensive areas of carbonate-producing epicontinental seas. The scarcity of acritarchs described from Devonian shallow-water limestones may thus represent a taphonomic bias rather than real rarity or absence.     

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。     

THE USE OF SPECIFIC ANTIBODY IN ELECTRON MICROSCOPY : I. Preparation of Mercury-Labeled Antibody

The preparation of antimyosin conjugated with mercury and fluorescein is described. The mercury was introduced to permit visualization of the antibody in the electron microscope. An organic mercurial, tetraacetoxymercuriarsanilic acid, was prepared and coupled to the antibody through the diazonium salt. The fluorescein was coupled through the isocyanate by a modification of the procedure described by Coons and Kaplan. The antibody conjugate retained its specificity of reaction with the tissue antigen. This was demonstrated by the staining pattern obtained in fluorescence microscopy.     

Silica in the Mesophyll Cell Walls of Italian Rye Grass (Lolium multiflorum Lam. cv. RvP)

Isolated cell envelopes from the leaf mesophyll of Italian ryegrass were examined in a transmission electron microscope equippedwith an energy-dispersive X-ray spectrometer. Silicon was detectedin these walls at an estimated concentration of 1–2 percent. Samples were also subjected to a range of techniques,for the removal of organic matter, which confirmed the presenceof silica throughout the cell walls. Lolium multiflorum Lam, Italian rye grass, cell walls, silica, electron microscopy, X-ray microanalysis     

Microarchitecture of Body Wall of Extant Cyclostome Ectoprocts

Scanning electron microscope and light microscope studies ofthe body wall in cyclostome stenolaemate ectoprocts show thatthis wall has a distinctive microarchitecture. The microstructuresconsist of a linear series of longitudinal canals in the centralregion. Adjoining regions on either side have laminae of overlappingcalcareous tablets. In some cyclostomes at certain stages ofgrowth, laminae may show a less orderly meshwork pattern nearthe canals. The laminate layers of calcareous tablets are enclosedin an organic matrix and are penetrated by tubuli which passfrom the inner part of the body wall to the canals and alsolink the canals. The canals with organic and mineralized matricesopen into pores in the body wall. This elaborate meshwork andcanal and pore system provide the framework for growth and resorptionof the body wall and facilitate transport of cellular materialsfrom one zooid to another in a colony. Calcification at thedistal part of a colony seems to proceed in a series of stagesaround sites of calcification. The pattern of the body wallof the cyclostome ectoproct does not parallel a molluscan patternof skeletal growth as previously proposed. The hypothesis ofa hypostegal coelom in some cyclostome ectoprocts is rejected.     

Fine structure of the chitin-protein system in the crab cuticle

The fine structure of the organic matrix of the shore crab cuticle (Carcinus maenas L.), observed in transmission electron microscopy, reveals three different levels of organization of the chitin—protein complex. The highest level corresponds to the ‘twisted plywood’ organization described by Bouligand (1972). Horizontal microfibrils, parallel to the cuticle plane, rotate progressively from one level to another. When viewed in oblique section this structure gives superimposed series of nested arcs, visible in light microscopy or at the lowest magnifications of the electron microscope, in all the chitin-protein layers. At the highest magnifications of the electron microscope and with the best resolution, when the ultrathin sections are exactly transverse to the microfibril, a constant pattern can be observed which consists of rods transparent to electrons, which are embedded in an electron-opaque matrix. In cross-section, these rods often form more or less hexagonal arrays. We call a microfibril one rod and the adjacent opaque material, and question the usual interpretation of the microfibril molecular structure. Between these two levels of organization, there is an intermediate level, which corresponds to the grouping of microfibrils. Microfibrils form a dense structure, with few free spaces in the membranous layer, the deepest and non-calcified layer of the cuticle. In other parts of the cuticle, microfibrils are grouped into fibrils of various diameters or form a reticulate structure, the free spaces of the organic matrix being occupied by the mineral.     

Scanning and transmission electron microscopy of human ejaculate spermatozoa with special reference to their abnormal forms

Summary Spermatozoa from fertile and infertile human ejaculates were observed under the scanning electron microscope. A parallel study of sections was performed by transmission electron microscope.The normal head shows under the scanning electron microscope vesicular elevations in the region of the acrosome and a smooth and rigid appearance corresponding to the postnuclear cap whose occurrence is confirmed under the transmission electron microscope. Immediately anterior to this cap a shallow furrow transverses the head. Duplicated, unusually large or small and deformed heads are found under the scanning electron microscope. Most of these abnormal heads show no surface structure suggesting an acrosome.The neck and middle piece are occasionally, though frequently in abnormal spermatozoa, covered by a cytoplasmic droplet. Otherwise, the mitochondrial sheath is recognized under the scanning electron microscope as a beaded thickening in the middle piece. The lack of mitochondria is manifested by a smooth middle piece thinner than the principal portion. Transmission electron microscopy of sections reveals various types of anomalies in the number of cores, core filaments and mitochondria embedded in the cytoplasmic droplets.Abnormalities in the principal portion of the tail such as duplication, unusual thickness and length are shown under the scanning electron microscope.The investigation indicates that scanning electron microscopy is suited for the clinical as well as cytological examination of human ejaculate spermatozoa.     

Improvement of biodesulfurization rate by assembling nanosorbents on the surfaces of microbial cells

To improve biodesulfurization rate is a key to industrialize biodesulfurization technology. The biodesulfurization rate is partially affected by transfer rate of substrates from organic phase to microbial cell. In this study, gamma-Al2O3 nanosorbents, which had the ability to selectively adsorb dibenzothiophene (DBT) from organic phase, were assembled on the surfaces of Pseudomonas delafieldii R-8 cell, a desulfurization strain. gamma-Al2O3 nanosorbents have the ability to adsorb DBT from oil phase, and the rate of adsorption was far higher than that of biodesulfurization. Thus, DBT can be quickly transferred to the biocatalyst surface where nanosorbents were located, which quickened DBT transfer from organic phase to biocatalyst surface and resulted in the increase of biodesulfurization rate. The desulfurization rate of the cells assembled with nanosorbents was approximately twofold higher than that of original cells. The cells assembled with nanosorbents were observed by a transmission electron microscope.     

Three-dimensional diffractive imaging for crystalline monolayers with one-dimensional compact support

The use of a compact support constraint along the beam direction is considered as a solution to the phase problem for diffraction by two-dimensional protein crystals. Specifically we apply the iterative Gerchberg-Saxton-Fienup algorithm to simulated three-dimensional transmission electron diffraction data from monolayer organic crystals. We find that oversampling along the reciprocal-lattice rods (relrods) normal to the monolayer alone does not solve the phase problem in this geometry in general. However, based on simulations for a crystalline protein monolayer (lysozyme), we find that convergence is obtained in three dimensions if phases are supplied from a few high resolution electron microscope images recorded at small tilts to the beam direction. In the absence of noise, amplitude-weighted phase residuals of around 5 degrees, and a cross-correlation coefficient of 0.96 between the true and estimated potential are obtained if phases are included from images at tilts of up to 15 degrees. The performance is almost as good in the presence of noise at a level that is comparable to that commonly observed in electron crystallography of proteins. The method should greatly reduce the time and labor needed for data acquisition and analysis in cryo-electron microscopy of organic thin crystals by avoiding the need to record images at high tilt angles.     

Studies on the biochemistry and fine structure of silica shell formation in diatoms

Summary The fine structure and pigmentation of an apochlorotic diatom isolated from decaying Macrocystis pyrifera is described. The morphology of acid cleaned shells suggests that the isolate is Nitzschia alba Lewin and Lewin. Light microscope observations indicated a centrally located nucleus and numerous highly refractile bodies which stained differentially with Nile blue and Sudan black B. The stained globules could be correlated in thin-sectioned profiles with either electron dense or lucent areas depending on the fixation technique. In the electron microscope the nucleus, Golgi complex, and mitochondria were similar in appearance to those described for other diatoms. Proplastid-like organelles, delimited by a double membrane, and containing small vesicles were also observed. Neither carotenoids nor chlorophylls could be detected by spectroscopic or spectrofluorometric analysis in vivo or in organic solvent extracts. Deposition of new walls was initiated by formation of silicon deposition-vesicles in the central region of dividing cells. The acentric raphes were deposited last. The genesis and interrelationship of the old plasmalemma, silicalemma, and newly formed plasmalemma are discussed.     

Scanning electron microscope observations of the organic matrix in the otolith of the teleost fish Fundulus heteroclitus (Linnaeus) and Tilapia nilotica (Linnaeus)

Scanning electron microscope observations of sagitta otoliths of Fundulus heteroclitus (Linnaeus) and Tilapia nilotica (Linnaeus) have revealed that the “discontinuous zone” is a narrow band of organic matrix consisting of fibers ≈900 Å thick, that in turn are composed of thin fibers ≈200 Å thick. The “incremental zone” is the crystalline layer with crystals elongated perpendicular to the otolith periphery that are usually terminated at the discontinuous zones. The crystals are embedded in organic matrix fibers that appear similar to and continuous with the fibers of the discontinuous zones. Frequently, these fibers aggregate into matrix sheets. Based on these findings, a possible process of otolith formation is proposed.     

Visualizing "greengold" clusters in the STEM.

"Greengold" is a large metallic cluster thought to contain 75 gold atoms in a compact 1.4-nm-diameter core surrounded by an organic shell. Scanning transmission electron microscope imaging shows uniform mass and size distributions with an apparent mass of 24 kDa, unaffected by radiation damage. The signal-to-noise ratio is adequate for visualization at low dose and in the presence of a relatively thick biological matrix. Under some conditions these clusters have a slight tendency to form linear chains and 2-D hexagonal arrays with a spacing of 2.6 nm. The parameters presented permit estimation of the feasibility of proposed labeling experiments.     

Improvement of the morphometry method and its use in electron microscopic studies

The paper describes morphometric device for electron microscope. Using this gauge one can get morphometric data from the screen of the electron microscope. Perspectives of morphometric analysis of ultra-structures in the electron microscope are discussed.     

Cinnabar mineralization in fossil small mammal remains as a consequence of diagenetic processes

The peculiar red colour of fossil small mammal remains from a late Miocene section in southern Spain suggests an unusual diagenetic alteration. These remains have been studied by means of environmental scanning electron microscope equipped with different detectors, energy dispersive X‐ray spectroscopy and an inductively coupled plasma optical emission spectrometer. The red colour is caused by the presence of cinnabar (HgS) in the pores of the fossil bones and teeth, filling the dentinal and bone tubules and other cavities that at life were filled by organic matter. A nearby cinnabar outcrop in the metamorphic materials of Sierra Nevada is interpreted as the source of mercury. This element was mobilized by meteoric diagenesis and incorporated as cinnabar in a palustrine environment, occupying pores and cavities of mammal remains shortly after the degradation of the organic matter (post‐mortem enrichment), during the early diagenesis.