bluuebunny/crossref_metadata_2025 huggingface dataset:
Organization of microtubules in cochlear hair cells
Authors: Furness David N. , Hackney Carole M. , Steyger Peter S.
Published: February 2005

The organization of microtubules in hair cells of the guinea‐pig cochlea has been investigated using transmission electron microscopy and correlated with the location of tubulin‐associated immunofluorescence in surface preparations of the organ of Corti. Results from both techniques reveal consistent distributions of microtubules in inner and outer hair cells.

In the inner hair cells, microtubules are most concentrated in the apex. Reconstruction from serial sections shows three main groups: firstly, in channels through the cuticular plate and in a discontinuous belt around its upper perimeter; secondly, forming a ring inside a rim extending down from the lower perimeter of the plate; and thirdly, in a meshwork underlying the main body of the plate. In the cell body, microtubules line the inner face of the subsurface cistern and extend longitudinally through a tubulo‐vesicular track between the apex and base.

In outer hair cells, the pattern of microtubules associated with the cuticular plate is similar, although there are fewer present than in inner hair cells. In outer hair cells from the apex of the cochlea, microtubules occur around an infracuticular protrusion of cuticular plate material. In the cell body, many more microtubules occur in the region below the nucleus compared with inner hair cells.

The possible functions of microtubules in hair cells are discussed by comparison with those found in other systems. These include morphogenesis and maintenance of cell shape; intracellular transport, e.g., of neurotransmitter vesicles; providing a possible substrate for motility; mechanical support of structures associated with sensory transduction.
Ultrastructure of Paneth cells in the intestine of various mammals
Authors: Satoh Yohichi , Yamano Miki , Matsuda Mitsuyoshi , Ono Kazuyuki
Published: February 2005

Paneth cells in the following species were observed under an electron microscope: human, rhesus monkey, hare, guinea pig, rat, nude rat, mouse, golden hamster, and insect feeder bat. Secretory granules containing homogeneous electron‐dense materials were observed in the Paneth cells of humans, monkeys, hares, guinea pigs, and bats; mouse Paneth‐cell granules were bipartite (central core and peripheral halo), and the Paneth cells in rats and golden hamsters had secretory granules showing various electron densities. In humans, monkeys, and bats, immature granules near the Golgi apparatus sometimes showed bipartite substructure. The number and size of secretory granules were also diverse among various animal species. Some lysosome‐like bodies were commonly observed in peri‐ or supranuclear regions, though the size and shape of the bodies differed from cell to cell. In apical cytoplasm, small clear vesicles (100–200 nm diameter) were more‐or‐less observed in all species examined, and it was especially note that rat Paneth cells contained many clear vesicles. Small dense‐cored vesicles (150–200 nm diameter) were rare. It is unlikely that the various ultrastructural features of Paneth cells correlate with the phylogenetical classification.
A new method for phase identification for electron diffractionists
Authors: Mighell Alan D. , Himes Vicky L.
Published: February 2005

An accurate analytical procedure for phase identification for electron diffractionists has been developed. The method opens new frontiers in the identification of solid‐state materials, as crystalline samples in the size range 10 μm to 10 Å can be accurately characterized. Research with NIST CRYSTAL DATA (a large database with chemical, physical, and crystallographic data on solid‐state materials) has proved that a material can be uniquely characterized on the basis of its lattice and chemical composition. To characterize a material, it is sufficient to determine any primitive cell of the lattice and the element types present. Using a modern analytical electron microscope (AEM), the experimentalist can collect the required data on an unknown sample. The lattice information is obtained by rotation of the sample to obtain two or more planes of data. From these planes, a unit cell defining the lattice can be deduced. The chemical data are determined by energy‐dispersive spectroscopy (EDS). Once the experimental data are measured, the unknown is identified against the database of knowns using lattice/element‐type matching techniques. The basic strategy consists of three conceptual steps. First, the unknown lattice is searched against the database to find all lattices that are the same or related; the results are kept in set 1. Second, the unknown is searched against the database to find all materials with the same or similar element types; the results are kept in set 2. Finally, the results in sets 1 and 2 are combined to obtain the answer set. Experience has proved that the procedure is highly selective and reliable.
Transformation of the amphibian oocyte into the egg: Structural and biochemical events
Authors: Bement William M. , Capco David G.
Published: February 2005

Amphibian oocytes, arrested in prophase I, are stimulated to progress to metaphase II by progesterone. This process is referred to as meiotic maturation and transforms the oocyte, which cannot support the early events of embryogenesis, into the egg, which can. Meiotic maturation entails global reorganization of cell ultrastructure: In the cell cortex, the plasma membrane flattens and the cortical granules undergo redistribution. In the cell periphery, the annulate lamellae disassemble and the mitochondria become dispersed. In the cell interior, the germinal vesicle becomes disassembled and the meiotic spindles form. Marked changes in the cytoskeleton and mRNA distribution also occur throughout the cell. All of these events are temporally correlated with intracellular signalling events: Fluctuations in cAMP levels, changes in pH, phosphorylation and dephosphorylation, and ion flux changes. Evidence suggests that specific intracellular signals are responsible for specific reorganizations of ultrastructure and mRNA distribution.
Modification of the Polaron sputter‐coater unit for glow‐discharge activation of carbon support films
Authors: Benada O. , Pokorný V.
Published: February 2005

We describe a modification of the Polaron sputter‐coater unit series 11 HD enabling activation of carbon support films for electron microscopy of macromolecules and macromolecular assemblies. The modification is simple and the device can be used in two modes, for sputter‐coating of SEM samples and for glow‐discharge activation of carbon support films. Examples of protein‐free spreading of DNA and negative staining of bacteriophage particles on activated carbon support films are presented.
Ultrastructural correlates of meiotic maturation in mammalian oocytes
Authors: McGaughey Robert W. , Racowsky Catherine , Rider Virginia , Baldwin Kendall , DeMarais Alyce A. , Webster Scott D.
Published: February 2005

Immature mammalian oocytes reside in ovarian follicles with junctionally coupled granulosa cells. When released from a currently undefined meiotic arresting influence, these oocytes resume meiosis to progress from late diplotene (germinal vesicle stage) through the first meiotic division to metaphase II. Oocytes remain at metaphase II until fertilization activates them to complete meiosis. This review summarizes ultrastructural events that occur during meiotic maturation in mammals. Developmental correlates that promise a clearer understanding of regulatory mechanisms operating to control maturation are emphasized. By use of TEM of thin sections, freeze‐fracture analysis, and replicated oocyte cortical patches, we demonstrate stage‐specific changes in the oocyte nucleus, reorganization of cytoplasmic organelles, correlations between oocyte maturational commitment and the junctional integrity of associated granulosa cells, and definition of the components comprising the oocyte cortical cytoplasm.
Morphology of mammalian sperm membranes during differentiation, maturation, and capacitation
Authors: Bearer Elaine L. , Friend Daniel S.
Published: February 2005

The mammalian spermatozoon is a highly polarized cell whose surface membrane can be divided into five functionally, structurally, and biochemically distinct domains. These domains are formed during spermatogenesis, continue to be modified during passage through the epididymis, and are further refined in the female reproductive tract. The integrity of these domains appears to be necessary for the sperm to perform its function—fusion with the egg and subsequent fertilization. The domains can be identified morphologically by their surface contours and texture, the content, distribution, and organization of intramembranous particles after freeze‐fracture, and by the density of surface and cytoplasmic electron‐dense coatings in thin sections. By using a variety of labels that stain carbohydrates (lectins), lipids (filipin and polymyxin B), and monoclonal antibodies to specific membrane constituents, the biochemical composition of these contiguous membrane regions has also been partly elucidated. We review here what is known about the structure, composition, and behavior of each membrane domain in the mature sperm and include some information regarding domain formation during spermatogenesis. The sperm is an excellent model system to study the creation and maintenance of cell polarity, granule exocytosis, and fertilization. Hopefully this review will provide impetus for future studies aimed more directly at addressing the relationship of its morphology to its functions.
Three‐dimensional organization of the golgi complex observed by scanning electron microscopy
Authors: Tanaka Keiichi , Fukudome Hatsuko
Published: February 2005

With the development of new specimen preparation techniques and improvements in instrumental resolution, scanning electron microscopy (SEM) became an effective means of studying the three‐dimensional organization of the Golgi complex. When specimens prepared by the osmium‐DMSO‐osmium method are observed with high‐resolution SEMs, cis‐most cisternae of Golgi stacks appear as sieve‐like plates with many small perforations. In some cell types, larger fenestrations are also present. The trans‐most cisternae showed a fenestrated or retucular pattern. At the trans‐side of Golgi stacks, distinctive structures, such as a well‐developed tubular plexus or a single widely extended cisterna, are observed in some types of cells. In lacrimal gland cells, Golgi stacks are linked by an irregular network of anastomosing branches extending throughout the cytoplasm. In these cells, the piled cisternae seemed to be connected to each other either directly within the stack or via cisternae of other stacks. Connections between Golgi stack and rough endoplasmic reticulum (ER) were often found in our SEM observations. In nerve cells, interconnecting tubules arise from rough ER and most often fuse with the rim of the cis‐cisternae of Golgi stacks.
Trans‐Golgi reticulum
Authors: Geuze Hans J. , Morré D. James
Published: February 2005

The trans‐Golgi apparatus reticulum is that portion of the Golgi apparatus located in the trans‐most aspect of the stack exhibiting certain characteristic morphological and functional characteristics. The membranes of the trans‐Golgi reticulum are reticular in form, thickened with plasma membrane‐like characteristics and with a considerable portion of their surface covered by clathrin coats. The enzymes thiamine pyrophosphatase and sialyl‐ and galactosyl transferases are functional markers. Correlative studies show the trans‐Golgi apparatus reticulum to be involved in glycoprotein, enzyme and receptor processing and sorting along multiple pathways. Sorting and transfer of constituents to lysosomes, to secretory granules, or to the plasma membrane emerge as dominant functions.
Analysis of protein transport through the golgi in a reconstituted cell‐free system
Authors: Wattenberg Binks W.
Published: February 2005

The processes which transport membrane proteins between compartments of the Golgi apparatus have been reconstituted in vitro using isolated Golgi fractions. This cell‐free system allows a detailed analysis of protein transport not possible in intact cells. Transport of the membrane glycoprotein (G protein) of vesicular stomatitis virus (VSV) is measured from a “donor” to an “acceptor” Golgi fraction. The donor Golgi fraction is prepared from VSV‐infected Chinese hamster ovary (CHO) mutant cells deficient in the glycosylation enzyme N‐acetylglucosamine transferase I. “Acceptor” is prepared from uninfected wild‐type CHO cells. Transport is measured by the addition of N‐acetylglucosamine to G protein, which can occur only upon movement of G protein from donor to acceptor. Transport requires physiological pH and osmolarity, is dependent on nucleotide triphosphates, and is mediated by proteins both from cytosol and on the Golgi membranes. Protein movement is inhibited by the non‐hydrolyzable GTP analogue, GTPγS. The process of transport proceeds through the budding, pinching off, targeting, and fusion of transport vesicles. In this system these vesicles are initially coated with a non‐clathrin coat and are targeted with this coat intact. Several of the proteins which mediate transport have been characterized, and isolated to homogeneity. The successful development of this assay has led to the formulation of cell free assays for protein transport between other compartments. Comparison of these systems indicates that some common mechanisms of vesicular movement are used in transport between a variety of membrane compartments.
High‐Angle annular dark‐field imaging on a tem/stem system
Authors: Otten Max T.
Published: February 2005

The technique of high‐angle annular dark‐field (HAADF) imaging, which is highly sensitive to atomic‐number contrast, can be performed on TEM/STEM systems using the standard annular dark‐field detector. For optimum HAADF imaging, the TEM/STEM must have a high maximum diffraction angle, small minimum camera length, and a descanning facility. The sensitivity of the technique is demonstrated to be about 105 to 106 times higher than energy‐dispersive X‐ray spectroscopy. Examples are shown from semiconductor, catalysis, ceramics, and particle analysis applications.
Gamete interactions and the fate of sperm organelles in fertilized echinoderm eggs
Authors: Longo Frank J.
Published: February 2005

Investigations of gamete fusion, sperm entry and the fate of the sperm nucleus, plasma membrane, mitochondrion, and axonemal complex in fertilized echinoderm eggs are reviewed. The timing of gamete fusion with respect to the onset of electrical activity characteristic of the activated egg and the affects of fixation conditions on the stability of fusing membranes are discussed. Observations from investigations using cationized ferritin labeled gametes and immunogold cytochemistry to demonstrate the mixing of sperm plasma membrane components within the egg plasma membrane, in particular along the surface of the fertilization cone, are compared with results from studies in somatic cells. Transformations of the sperm nucleus into a male pronucleus, consisting of sperm nuclear envelope breakdown, chromatin dispersion, and formation of a pronuclear envelope, are correlated with recent biochemical observation of similar processes in other cellular systems. Fates of the sperm mitochondrion and axonemal complex are examined.
Multiple intracellular signals coordinate structural dynamics in the sea urchin egg cortex at fertilization
Authors: Chandler Douglas E.
Published: February 2005

Fertilization of the sea urchin egg is accompanied by a sequence of structural changes in the egg cortex that include exocytosis, endocytosis, and microvillar growth. This architectural reorganization is coordinated by two intracellular signals: a rapid, transient rise in cyto‐solic free calcium and a slower, longer lasting increase in cytoplasmic pH. In this report we provide ultrastructural views of these events in quick‐frozen eggs and discuss their relationship to the calcium and pH signals.
Fertilization of the mouse oocyte
Authors: Calarco Patricia G.
Published: February 2005

This paper presents morphological data on mouse oocyte maturation and fertilization, reviews evidence supporting the existence of a sperm receptor, and suggests future directions for this line of research. We used scanning electron microscopy to examine oocytes under a variety of conditions. The surfaces of mature mouse oocytes are seen to be similar whether maturation occurs in vivo or in vitro. Capacitated sperm (both acrosome‐intact and acrosome‐reacted) are observed to interact with the microvilli of the oocyte surface.

Little is known about oocyte surface proteins that mediate fertilization in mammals. Data of ours and others show that enzyme treatment of live unfertilized eggs interferes with sperm binding. Enzyme treatment (trypsin, chymotrypsin treatment, or pronase) reduces the number of bound sperm, suggesting removal of a surface protein involved in fertilization. Trypsin treatment also causes some lengthening of surface microvilli in a belt surrounding the metaphase II region. After metabolic labeling, proteins of zona‐free unfertilized eggs can be identified by SDS‐PAGE and autoradiography. Comparison of 1‐D gels from untreated and enzyme‐treated eggs show the nearly complete disappearance of proteins of 263, 170, 137, 97, and 87 kD after digestion; an increase in a 66 kD protein after trypsin or chymotrypsin; and a major new band of 20 kD after chymotrypsin treatment. Fertilized eggs show the loss of a 255–265 kD band among other changes. Proteins of 97 kD and 87 kD were seen previously by surface labeling (Johnson and Calarco, 1980b), and our 97 kD and 66 kD bands are similar in molecular weight to those identified by Boldt et al. (1989). Taken together, these data identify a few candidate proteins for the role of sperm receptor on the egg surface.

Future work should focus on identification of the surface protein(s) which functions physiologically in fertilization by developing fertilization‐blocking antibodies. Relatedness to other mammalian sperm receptors and identification of the genes involved would provide valuable information to our understanding of fertilization and to the problems of infertility and contraception.
The three‐dimensional architecture of the mitotic spindle, analyzed by confocal fluorescence and electron microscopy
Authors: Merdes Andreas , Stelzer Ernst H. K. , De Mey Jan
Published: February 2005

Fluorescence microscopy techniques have become important tools in mitosis research. The well‐known disadvantages of fluorescence microscopy, rapid bleaching, phototoxicity and out‐of‐focus contributions blurring the in‐focus image are obstacles which still need to be overcome. Confocal fluorescence microscopy has the potential to improve our capabilities of analyzing cells, because of its excellent depth‐discrimination and image processing power. We have been using a confocal fluorescence microscope for the study of the mechanism of poleward chromosome movement, and report here (1) a cell preparation technique, which allows labeling of fixation sensitive spindle antigens with acceptable microtubule preservation; (2) the use of image processing methods to represent the spatial distribution of various labeled elements in pseudocolour; (3) a novel immunoelectron microscopic labeling method for microtubules, which allows the visualization of their distribution in semithin sections at low magnification; and (4) a first attempt to study microtubule dynamics with a confocal fluorescence microscope in living cells, microinjected with rhodamine labeled tubulin.

Our experience indicates that confocal fluorescence microscopy provides real advantages for the study of spatial colocalization of antigens in the mitotic spindle. It does not, however, overcome the basic limits of resolution of the light microscope. Therefore, it has been necessary to use an electron microscopic method. Our preliminary results with living cells show that it is possible to visualize the entire microtubule network in stereo, but that the sensitivity of the instrument is still too low to perform dynamic time studies. It will be worthwhile to further develop this new type of optical instrumentation and explore its usefulness on both fixed and living cells.
Ultrastructural cryoimmunocytochemistry is a convenient tool for the study of DNA replication in cultured cells
Authors: Raska Ivan , Michel Laurée Salamin , Jarnik Michal , Dundr Miroslav , Fakan Stanislav , Gasser Susan , Gassmann Max , Hübscher Ulrich , Izaurralde Eliza , Martinez Ernest , Richter Arndt , Dubochet Jacques
Published: February 2005

In the present study, we have optimized an immunocytochemical ultrastructural approach for in situ localization of newly synthesized DNA in unsynchronized as well as in synchronized human HeLa cells and in exponentially growing mouse P815 cells, which had incorporated bromodeoxyuridine (BrdU) during short pulses varying from 1 to 20 minues. The incorporated BrdU was detected in hydrolyzed ultrathin cryosections or Lowicryl sections by means of a monoclonal antibody, revealed by secondary colloidal gold‐labeled probes. The results demonstrate our ability to study, with high resolution and reproducibility, DNA replication during consecutive periods of the S‐phase, which is monitored by the incorporation of tritiated thymidine. In addition, this approach allows one to perform a concomitant mapping of replicated DNA and various enzymes of the replisome.
Effects of substrate texture and curvature on the morphology of cultured cells
Authors: Hogan Margaret E. , Degaetano Douglas H. , Klomparens Karen L.
Published: February 2005

The ultrastructural characteristics of several growth matrices were examined using two cell types chosen for their distinct growth habits. Chinese hamster ovary cells and Balb‐c 3T3 mouse fibroblasts were grown on flat substrates (glass, tissue culture plastic, Millipore filters) as well as spherical (glass, tissue culture plastic, cross‐linked dextran) substrates. Cells were plated maintaining equal densities and growth surface area. Once the majority of the cells reached confluency, the cell's morphology on each matrix was examined using scanning electron microscopy. Digital analysis was performed on cell attachment area to compare the effect of each matrix on cell spreading.

Variation in cell shape was dramatic between matrices, being most noticeable between a textured surface (filter, dextran bead) and that of a smooth (glass) surface. Even within smooth surfaces, some variation was observed. There was also an effect of matrix curvature on cell attachment area, the greatest being in the 3T3‐c Balb cells, causing an overall decrease in the area of attachment between cell and matrix. The changes seen could also be related to the particular cell type used. Hamster ovary cells tended to be cylindrical and showed little effect between matrices, whereas the mouse fibroblasts, which are more flattened, showed the matrix effect to a greater degree.

This study demonstrates the necessity of being aware of substrate‐induced cell changes in tissue culture, where some variation in cell shape may be due to the surface on which the cells are grown as opposed to the experimental procedure.
Electron crystallography at atomic resolution: The structure of the odd‐chain paraffin n‐tritriacontane
Authors: Dorset Douglas L. , Zhang Weiping
Published: February 2005

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 00ℓ reciprocal row. The phase set is sufficient to calculate an electrostatic potential map which is directly interpretable as a structure image at atomic resolution.
The effect of aluminium coating on elemental standards in X‐ray microanalysis
Authors: Hopkins Diane M. , Jackson Alan D. , Oates Kenneth
Published: February 2005

The standardisation of frozen hydrated bulk biological specimens using gelatin standards is described. The relationship between corrected elemental X‐ray counts and ionic concentration was found to be linear, and minimum detectable limits for each element are stated. Variations in uncorrected standard curves were found to be due to changes in aluminium coating thickness. There was an inverse relationship between coating thickness and elemental X‐ray counts. The factors causing this are discussed. To avoid errors arising from inconsistent aluminium thickness, experimental material should only be compared with standards of similar aluminium net counts. This can be achieved most easily by mounting and analysing specimen and standard together.
Determination of the physical structure of biological materials at biosensor interfaces by techniques of increasing magnification from microscopic to molecular scale
Authors: Krull U. J. , Brown R. S. , Vandenberg E. T. , Heckl W. M.
Published: February 2005

Chemical selectivity of biosensors is derived from biological materials interfaced to the surface of transducing devices. Molecular recognition events lead to macroscopic function suitable for analytical measurements. The structure‐function relationships of biochemical species at interfaces must be established to characterize and optimize biosensor operation. The techniques of ellipsometry, fluorescence microscopy, electron microscopy, and scanning tunneling microscopy are used to investigate the structure of monolayers and multilayers of proteins and lipids at interfaces that are prepared by Langmuir‐Blodgett techniques and by self‐assembly from bulk solution. The relative merits and limitations of the measurement techniques in the determination of aspects of interfacial structure are considered.
Tridimensional ultrastructure of perfusion fixed gastrointestinal epithelial cells by high resolution scanning electron microscopy
Authors: Kendall Cyril W. , Venketeshwer Rao A. , Janezic Susan A. , Temkin Robert J. , Hollenberg Martin J. , Lea Peter J.
Published: February 2005

Improvements in the design of modern scanning electron microscopes (SEM) and new methods of specimen preparation incorporating chemical removal of the cytosol and cytoskeleton, now make it possible to view cells and their organelles in three dimensions (3D) at high magnification. In this experiment, high resolution SEM (HRSEM) utilizing new methods of tissue preparation was used to study the intracellular structures of the mouse ileum. In addition, in vivo intestinal perfusion was used to further enhance cellular preservation. Using these modifications it was possible to visualize, in 3D, the fine structure of intestinal epithelial cells and intracellular organelles such as the nucleus, mitochondria, endoplasmic reticulum, and Golgi complex, as well as microvilli and cell membrane. Whole mitochondria appeared as irregularly shaped organelles which contained tubular cristae. Plate‐like cristae were not observed. The brush border was found to be a closely packed array of cylindrical projections. The extensive folding and structural intricacy of lateral cell membranes between absorptive cells could only be appreciated by viewing this tissue with 3D HRSEM. The use of HRSEM to study 3D ultrastructure of cells and their organelles will improve our understanding of the structure‐function relationships in both the healthy and diseased gastrointestinal tract.
Resolution limit of serial sections for 3D reconstruction of tubular cristae in rat liver mitochondria
Authors: Winslow J. L. , Hollenberg M. J. , Lea P. J.
Published: February 2005

Following stereo, high resolution scanning electron microscopy (HRSEM) of various rat tissue mitochondria prepared by mild glutaraldehyde fixation followed by freeze cleavage and extraction of the cytosol (Lea and Hollenberg, 1989a), a new model for the ultrastructure of mitochondrial cristae has been developed (Lea and Hollenberg, 1989b). The cristae mitochondriales, previously considered in most cells to have a shelf like structure, were found in many cell types including hepatocytes by HRSEM to be tubes which spanned the mitochondrial matrix and were continuous with the inner mitochondrial membrane at both ends. Despite computer aided, serial, reconstruction of serial thin sections examined in the transmission electron microscope (TEM), tubular cristae in mitochondria have not been resolved entirely, even though circular cross sectional profiles of cristae have been observed in the TEM. The current study was undertaken to probe this deficiency and to understand its cause.

In this study, the problems of inherent contrast and resolution loss in the digitized image have been reduced by using a computer based, background, extraction process analogous in the computer to the chemical cytosol extraction process used for HRSEM (Lea and Hollenberg, 1988; Hollenberg et al., 1989). Despite the use of this membrane enhancement algorithm, it was still not possible to reconstruct entire tubular cristae, as observed by HRSEM, even when the micrograph negatives were digitized and reprocessed (Lea and Hollenberg, 1989b). A comparison of serial section thickness deduced from the diffraction colour of the respective section (60 nm) to the measured diameter of tubular cristae profiles (30 nm), with membrane thickness of 10 nm, suggested that this size disparity is responsible for the apparent loss of resolution. The sampling theorem from signal analysis indicates that the signals of interest (X, Y vs. Z coordinate values of mitochondrial internal structure) must be sampled at least twice the frequency of their highest frequency. Because the structures are small and embedded in image noise, even thinner serial sections are required in order to obtain enough information for adequate reconstruction. Consequently in practice, the thickness of the serial sections would have to be reduced by four times to less than 8 nm in order to be able to reconstruct tubular mitochondrial cristae 30 nm in diameter and less than 2 nm for membrane shape. Ultra‐thin sections, some 15 nm in thickness, have been reported (Buchanan et al., 1988). However, these sections would be only thin enough to resolve structures greater than 30 nm in diameter. Observation of cristae by HRSEM therefore is essential in order to verify ultrastructure derived by computer based three dimensional reconstruction.
Image analysis of mineralized and non‐mineralized type I collagen fibrils
Authors: Larry Arsenault A.
Published: February 2005

Turkey leg tendons at an early stage of mineralization have been thin sectioned and imaged by electron microscopy. At this stage collagen‐associated mineral apatite was found to be present within both the gap and overlap zones. The earliest apatite occurs in a microcrystalline form which gives a rather generalized and characteristic density to both the gap and overlap zones; with subsequent development larger defined apatite crystals arise which span gap/overlap zones. Fourier transformation of such images revealed the major 67 nm axial repeat of the gap/overlap zone plus four other maxima corresponding to repeat spacings of 22, 16, 13, and 11 nm respectively. Computer imaging techniques were used to reconstruct images by using selected spatial frequencies from such transforms. In this manner the subperiodic distributions of mineral were visually enhanced. These subperiodicities are positioned in an asymmetric fashion over the entire D unit repeat aligning with the molecular orientation of the fibril. Analyses of both negatively stained collagen and computer‐generated maps of collagen hydrophobicity were compared to the mineral distribution of collagen. Densitometric comparisons showed a positional correlation between the axial banding patterns of mineralized fibrils and those of negatively stained non‐mineralized fibrils. Comparable spatial frequencies were also present in transforms between hydrophobic maps and mineral distribution of collagen. These results suggest that the lateral clusterings of hydrophobic residues which span the fibril at specific sites in both the gap and overlap zones serve to prohibit early mineral deposition. This observed hydrophobic influence in combination with the gap space appear as contributing factors in the observed axial distribution of mineral within collagen.
Structural organization of the thylakoid membrane: Freeze‐fracture and immunocytochemical analysis
Authors: Olive J. , Vallon O.
Published: February 2005

This article summarizes our ultrastructural studies on the organization of the thylakoid membrane of green algae and higher plants. We have used freeze‐fracture and immunogold labeling to investigate the lateral distribution of the components in the membrane, their interactions, and the folding of their polypeptide chains in the membrane.
Quantitative electron microscopic analysis of DNA‐protein interactions
Authors: Cam Eric Le , Théveny Bernard , Mignotte Bernard , Révet Bernard , Delain Etienne
Published: February 2005

Electron microscopy offers a unique potentiality to visualize individual molecules. For the last 30 years it has been used to study the structure and the interactions of various biological macromolecules. The contribution of electron microscopy is important because of its capacity to demonstrate the existence of conformational structures such as kinks, bents, loops, etc., either on naked DNA, or on DNA associated with various proteins or ligands. Increasing interest was given to such observations when it was found that they provide a direct visualization of interacting molecules involved in DNA metabolism and gene regulation. Technical advances in the preparation of the specimens, their observation in the electron microscope, and the image processing by computers have allowed the shifting from qualitative to quantitative analysis, as illustrated by a few examples from our laboratory.
Structural analysis of two‐dimensional arrays of cholera toxin b‐subunit
Authors: Mosser Gervaise , Brisson Alain
Published: February 2005

Two‐dimensional arrays

The term array is used to describe a regular arrangement of motifs without implying the existence of translational symmetry elements between motifs, by distinction with a crystal.

of cholera toxin B‐subunit (CTB) have been obtained by specific interaction with lipid films, as described by Ludwig et al. (1986). The relationship between two types of array, of either rectangular or hexagonal geometry, was analyzed using crystallographic methods of electron image analysis. Our results showed that the type of array obtained was highly dependent on the negative stain used and that both arrays presented related lattice parameters, indicating that they originated from a common unstained structure. Image analysis of hexagonal arrays at 17 Å resolution revealed variable CTB projected structures, ranging from annularly symmetric particles to highly asymmetric particles, very distinct from the pentameric structure resolved from rectangular crystals. The present data suggest that hexagonal arrays result from an imperfect staining of CTB rectangular crystals. The staining distortion is such that the stain layer does not match faithfully the pentameric protein distribution whereas the regular organization of the specimen is maintained.
Advantages of fast‐freeze fixation followed by freeze‐substitution for the preservation of cell integrity
Authors: Nicolas Gisèle
Published: February 2005

The use of fast‐freeze fixation (FFF) followed by freeze‐substitution (FS) has supplied new data in a variety of domains due to the rapidity of this fixation compared to the slow process of the conventional chemical fixation. FFF completely arrests all the cell's biological reactions, and physically immobilizes most molecules within a few milliseconds. This review deals with the main results we obtained concerning the preservation of organelles particularly sensitive to osmotic shock, the maintenance in situ of soluble or depolymerizable cell components, the visualization of brief transient events, and the opening up of new prospects in the field of immunocytochemical labeling.
Preparation of ultra‐thin oxide windows on titanium for tem analysis
Authors: Rådegran G. , Lausmaa J. , Mattsson L. , Rolander U. , Kasemo B.
Published: February 2005

Using submerged jet electropolishing, extremely thin (<10 nm), continuous, thermal oxide “windows” have been prepared on poly crystalline titanium (Ti). The preparation technique is described in detail. It has allowed a systematic investigation of the structure of thermal surface oxide layers on Ti in the thickness range 6–40 nm, corresponding to oxidation temperatures 100–450°C. Auger electron spectroscopy was used for oxide characterization and for depth profiling to determine oxide thickness.

The thinnest oxides, < 10 nm, are amorphous, morphologically homogeneous, and with essentially no contrast in the transmission electron microscopy (TEM) pictures. As the oxide thickness is increased up to 40 nm, a texture corresponding to the grain structure of the oxidized metal becomes gradually more visible. At the same time the oxide becomes increasingly more crystalline. The results are compared with previously published corresponding results for thicker anodic oxides on Ti.
Routine cryofixation of plant tissue by propane jet freezing for freeze substitution
Authors: Ding Biao , Turgeon Robert , Parthasarathy M. V.
Published: February 2005

Cryofixation and freeze substitution methods were developed for ultrastructural studies of cells in complex plant tissues. Leaf tissues and root tips of tobacco (Nicotiana tabacum L. var. Maryland Mammoth) were frozen with a RMC MF7200 propane jet freezer and freeze substituted sequentially with tannic acid and osmium tetroxide/uranyl acetate in acetone. High quality preservation was consistently obtained for epidermal and phloem cells of the leaf, and epidermal, cortical, meristematic, and cap cells of the root tip. Leaf mesophyll cells were also often well frozen. Organelles, including nuclei, endoplasmic reticulum, mitochondria, Golgi bodies, and plastids, showed excellent structural integrity and contrast. Most notable is the superior preservation of the cytoskeleton. Our results demonstrate that the propane jet freezer can be used routinely for high quality cryofixation of higher plant cells in certain complex tissues. This could have important implications for the use of cryofixation approach in a wide range of research in plant biology.
Fine structural characteristics of testicular cord formation in the developing rabbit gonad
Authors: Wartenberg Hubert , Kinsky Inge , Viebahn Christoph , Schmolke Cordula
Published: February 2005

This paper presents morphological (light‐ and electron‐microscopical) evidence for the role of the mesonephros in contributing cells to the differentiating indifferent gonad and, after sexual differentiation, to the testis. A continuous process is revealed during which segregation of cells occurs from the developing and regressing mesonephros. Additionally, the complementary role of the coelomic epithelium in gonadal ridge and testis formation is demonstrated. The differentiation of testicular cords, their remodelling from a primary reticulum, and the composition and further change of the cellular content during the period after sexual differentiation is described using a computer‐aided three‐dimensional reconstruction system. Apart from these morphogenetic events, cytodifferentiation in the somatic cells of the indifferent gonad and of the early differentiated testis is demonstrated using indirect immunof luorescence in combination with monoclonal antibodies to the intermediate filament proteins keratin 8 and 18 and vimentin. The immunohistochemical results show that different forms of cytodifferentiation coexist among the somatic cells present in the indifferent gonad and in the testis early after sexual differentiation.
Capillary pericytes: Perspectives and future trends
Authors: Tilton Ronald G.
Published: February 2005

A complete understanding of the microcirculation requires full knowledge of the structure and function of each of the constituent cells, including pericytes. Vascular endothelium and smooth muscle cells have been investigated intensively during the last two decades, but much less is known about the metabolism and function of capillary pericytes. However, the development of new electron microscopy techniques and the application of new cell culture and molecular biology techniques should allow for the rapid elucidation of the cellular biochemistry and the microvascular function and pathology of this ubiquitous capillary cell.
Vasculature of the fish gill: Anatomical correlates of physiological functions
Authors: Olson Kenneth R.
Published: February 2005

The fish gill is a multifunctional organ responsible for respiration, osmoregulation, acid base balance, nitrogen excretion, and metabolism of circulating hormones. Two or more microcirculatory systems subserve these activities and form one of the most complex vascular networks found in any vertebrate. In this article the vascular anatomy of the teleost gill and the role of gill vessels in mediating physiological function are examined. Vascular corrosion replication techniques have been instrumental in resolving the spatial organization of gill microcirculation. Variations in the replication procedures provide information on the interrelationships between the vascular pathways, factors that govern flow distribution, and the physical characteristics of the vessels themselves. Anatomically, gill vessels are as diverse as their physiological functions. Pillar cells, unique to the fish gill, form the lining of the respiratory vasculature and may have substantial metabolic effects on circulating hormones. The non‐respiratory pathways appear to be lined with both typical and unusual endothelial cells, although the fine structure and function(s) of these vessels are largely unknown. To date most of the information on gill vessels has been derived from descriptive morphological studies. Further evaluation of the anatomical and physiological correlations of these tissues is predicated upon the application of histocytochemical, morphometric, and other quantitative methodologies as well as an examination of gills from fish with various evolutionary and environmental backgrounds.
Interrelationships of ultrastructure and function in the microvasculature of normal and ischaemic myocardium
Authors: Gavin John Bevan , Maxwell Linda , Sage Martin David
Published: February 2005

This paper reviews various methods involving electron microscopy that have been used to investigate the ultrastructure of the vasculature of the normal and diseased heart. Whereas scanning electron microscopy is more commonly employed to record surface topography, it can be used to examine freeze‐fracture planes within the myocardium and, using heavy‐metal staining and back‐scattered electron imaging, to examine large 2‐μ‐thick resin‐embedded sections through the heart. The latter technique allows the comparison of structural alterations across the wall of the heart and thus accurate definition of the transmural progression of pathological processes. Transmission electron microscopy can then be used to provide more detailed information from precisely localised regions. Human myocardium can be usefully studied up to 12 hours post‐mortem provided that suitable control material is included. Intravascular tracers including low‐viscosity resin and nuclear track emulsion can be used to determine whether or not particular vessels allow flow at the time of fixation, and thus changes in the pattern of flow through the microvasculature due to ischaemia and reperfusion can be quantified and defined. Particular care is required in the fixation of ischaemic tissues because oxygen dissolved in the fixative can lead to the rapid formation of oxygen‐free radicals on contact with the tissue. This produces artefactual reoxygenation damage characterised by membrane disruption and cell and organelle swelling, which has previously been attributed to ischaemic injury per se. Bubbling glutaraldehyde with nitrogen substantially reduces this artefact.
The anatomy, physiology and comparative aspects of the repugnatorial glands of Orthocricus arboreus (Diplopoda:Spirobolida)
Authors: Woodring J. P. , Blum M. S.
Published: February 2005

The quinoidal secretion, secreted by holocrine action from most gland cells (no cytoplasmic inclusions), of Orthocricus arboreus may ooze out or be sprayed over 30 cm. A thick gland intima and basement membrane protects internal organs. The gland grades into a neck leading to an external orifice. An ejaculatory duct, its lumen closed by cuticular spring action, is formed by invagination of the neck. Part of the duct forms a tongue, which can independently unplug the orifice. Retractor muscles open the ejaculatory duct, secretion flows in from the gland, and the part of the duct nearest the gland closes. Sequential relaxation of the rest of the retractor muscles build up fluid pressure, which results in a spray when the orifice is unplugged. A ligament opposes the pull of the retractor muscles. Opening both duct and orifice results in secretion ooze. No intrinsic gland muscles were found. Intersegmental muscles may produce hemolymph fluid pressure on the gland, thereby contributing to secretion ooze. Illustrations are presented. A table comparing the two basic types of millipede repugnatorial glands (spirobolid and polydesmid), on the levels of structure, histology, secretion, chemistry and function is presented. The polydesmid type (2‐chambered) probably evolved from the spirobolid type (1‐chambered). The secretion of one type would not work in the other type of apparatus. A second table corelates the secretion and ejection mechanism of 23 species of millipeds with their phylogenetic position in the class Diplopoda.
A morphological and histochemical study of the reproductive system of the slug, Philomycus carolinianus (Bosc)
Authors: Kugler Otto E.
Published: February 2005

Glycogen, determined by Best's carmine and/or PAS Tripas staining, and alkaline phosphatase, determined by the Gomori technic, occur together throughout the epithelium of the genital tract. Sites of high alkaline‐phosphatase activity are the acinar epithelium, nurse cells, and follicular cells of the ovotestis. Sperm heads are also strongly positive for the enzyme. The nidamental gland cells and the subepithelial mucous cells associated with the loop of the hermaphroditic duct and with the spermatic groove are thionine metachromatic, alcianophilic, PAS‐positive and amylase‐fast. The mucous cells of the genital atrium are PAS‐positive and amylase‐fast but lack both thionine metachromasia and affinity for Alcian blue.