The long bones of vertebrates, such as the tibiae and femurs of humans, extend in length by means of genotypic and phenotypic changes orchestrated by the chondrocytes comprising growth plate cartilage. Among the constituents synthesized by these cartilage cells, osteopontin (OPN), a phosphorylated glycoprotein, is thought to play a critical role in events leading to normal growth plate function and ultimate mineralization of the deeper zones of this cartilage region. The precise role of OPN, however, is uncertain with regard to mineralization, and present evidence supports the alternative possibilities that the protein may be either facilitative or inhibitory to mineral deposition. in order to investigate OPN function in a model growth plate, cartilage from normal 1-11 day old postnatal mice was examined by the novel techique of laser capture microdissection (LCM) followed by RT-PCR to obtain a measure of OPN gene expression by chondrocytes of known age and specific location in the plate. LCM permits identification of individual or clusters of cells within a tissue section and subsequent unique isolation (“capture”) of such cells for a variety of molecular analyses.

In this study, mouse tibiae were dissected, placed in RNAlater (Ambion, Austin, TX) to preserve message, and stored at −20°C. Sections (5 μm thick) of fresh frozen developing epiphyseal growth plates were obtained in a cryostat maintained at −20°C, fixed briefly in 70% ethanol, and stained with eosin. Sections were examined in a Pixcell LCM system (Arcturus Engineering, Mountain View, CA) where chondrocytes were attached to the surface of polymer film substrates and lifted free of sections. in separate experiments, ∼200-1200 cells were captured and analyzed. Substrates were transferred to Eppendorf tubes containing RNA extraction buffer. RNA was extracted from cells by microisolation (Stratagene, La Jolla, CA), DNAse-treated, reverse-transcribed, and then subjected to PCR (40 cycles) with AmpliTaq DNA polymerase (PE Applied Biosystems, Foster City, CA). Ethidium bromide agarose gels revealed OPN mRNA from groups of chondrocytes isolated from whole cartilage and from resting, proliferating, and hypertrophic growth plate zones from the mouse tibiae. Brain cells captured by LCM from the same mouse sections served as positive controls and reactions containing no reverse transcriptase were negative controls. 18S rRNA was used as a marker for semiquantitation and standardization of expressed message from captured cells.

For industrial-scale culture of anchorage-dependent mammalian cell, such as vero cell, growth on the microcarriers offers certain advantages. As previous research reported, growth process can be illustrated by a growth curve - cell density versus time, which shows the change of cell density through whole growth process including: first adhesion stage; spreading and division stage; and last confluent stage. The growth curves are controlled by the culture condition (culture medium, temperature, pH value, inoculation concentration, etc.) and space condition (kind of microcarrier, microcarrier size, and surface property, etc.). Therefore, the cell growth curve is not only the important basis for studying growth kinetics, but also for designing biological reactors.

Traditionally, total cell concentration for the microcarrier cultures were determined using the crystal violet method, and the cell nuclei were counted with a hemacytometer. in this method, the cells attached on each microcarrier were trypsinized and dyed by crystal violet dye, so that the number of nuclei counted was statistically representative. in this work, the cells and microcarriers were examined by using an environmental SEM - KYKY1500. As a result, the moist cells and microcarriers in SEM images show their natural characteristics, because this environmental SEM permits biological sample to be observed directly in an environmental specimen chamber without normal preparation for biological sample. As for the normal preparation, it needs a long duration, e.g. 2 days normally, to do fixing, desiccating and gold-coating. in contrast, the operation for observation of the biological samples in this SEM is much simpler. in view of this, a statistical counting method based on the SEM images for determining cell concentration during microcarrier culture was developed.

In the embryologic development of the nephron, the glomerular tuft (renal corpuscle) and the excretory tubules develop from two distinct embryologic origins or anlagen. Nephrotomes constitute the earliest formation of the excretory tubules and appear first as segmentally arranged cell clusters, which grow and become canalized, forming nephric tubules. Formation of the glomerulus begins when the dorsal aorta gives rise to small branches, consisting of tufts of capillaries. Bowman’s capsule forms as a blind terminal expansion at the proximal end of the nephron. in the final stage of development, Bowman’s capsule becomes deeply indented by the glomerular tuft. Which leads to a differentiation of it into 2 layers: 1) an inner, visceral layer and 2) an outer, or external, parietal layer. The two layers are separated by a crescent shaped space, known as Bowman’s or the urinary space.

The inner, visceral layer encloses, and, comes into close apposition with the glomerular capillaries. At the vascular pole of the glomerulus, the visceral layer is reflected off these vessels and becomes continuous with the external, or parietal layer of Bowman’s capsule, which is lined by a very flat polygonal epithelium. Traditionally, it has been depicted that there is a direct opening of the urinary space into the tubule and that the flat cells of the parietal layer of Bowman’s capsule become continuous with the cuboidal epithelium in the neck of the proximal convoluted tubule.

This investigation shows that the junction of the renal corpuscle and the proximal convoluted tubule is a more complicated one. Scanning electron microscopic studies of microdissected nephrons enabled the visualization of the glomerulus, with the two vessels at the vascular pole and the proximal convoluted tubule at the urinary pole. An unexpected finding, was the presence of a gourd-like out-pouching at the urinary pole. (Fig. 1, “A”) Higher magnification showed a saucer shaped depression (50 μm × 25 μm) on its free border. (Fig. 2). This was a consistent finding in the material that was studied.

The Chlamydomonas zygospore is formed after gametic cell fusion by a progression of ultrastructural and physiological changes, including the synthesis of a protective and elaborately sculptured secondary wall (Figure 1). Assembly of the secondary wall occurs within the confines of a transient primary zygote wall, synthesized immediately after gamete fusion, and assembled beneath the residual gamete walls. Upon completion of secondary wall assembly, the primary zygote wall is shed from the maturing zygospore (Figure 2). This primary zygote wall, composed primarily of a β-1,3 glucan (callose), may be important in the regulation of secondary zygospore wall formation.

A temperature-sensitive mutant strain of C. monoica, zym30, has been isolated that shows marked abnormalities in both primary and secondary wall ultrastructure. The degree of wall formation in the mutant strain is inversely proportional to temperature. At 25° C most zygotes fail to synthesize either the primary or the secondary wall. At 15° C both primary and secondary wall assembly occurs to some extent and as many as 50% of the zygotes develop a “pseudo-wildtype” phenotype (Figure 3). Although release of the primary wall rarely occurs in the zym30 strain (even at permissive temperatures), those walls that are released appear thinner than the walls shed from wildtype zygospores, as viewed by phase contrast microscopy. Fluorescence images incorporating aniline blue, a fluorochrome specific for β-1,3 glucans, show decreased fluorescence suggesting reduced callose synthesis (Figure 4).

Several permanent, ultrastructurally distinct layers comprise the secondary wall, the assembly of which may be controlled, in part, by the primary wall. One or more of these secondary wall layers contain the highly durable, autofluorescent biopolymer, sporopollenin. Autofluorescence of zym30 zygospores produced at the permissive temperature is enhanced relative to wildtype zygospores. This correlates with ultrastructural changes in the primary wall and overproduction and/or misassembly of the surface secondary wall layer (Figure 5). Work is in progress to determine whether the environmental resistance, and/or viability of the zym30 “pseudowildtype” zygospores are reduced relative to wildtype zygospores.

The concept of prion (infectious protein) originated in studies of transmissible spongioform encephalopathies (TSE) in mammals, but more recently, two nonchromosomal genes of yeast- [PSI] and [URE3] - were identified as prions. While the agents of TSEs kill infected cells and [URE3] or [PSI+] only slow growth at most, these infections are believed to have similar mechanisms, i.e. self-propagating amyloids. TSEs are often associated with amyloid deposition in infected tissues, and both Sup35p and Ure2p have been shown to form amyloid in vitro.

[URE3] is a prion of the Ure2 protein, that normally regulates nitrogen catabolism. Its ‘prion’ domain (residues 1-65) is necessary and sufficient for propagation of the prion, whereas the C-terminal portion (residues 81-354) is sufficient to carry out the nitrogen regulation function. The prion domain peptide spontaneously forms amyloid filaments in vitro. Full-length native Ure2p is a stable soluble dimer, but forms co-filaments when the prion domain peptide is added. This in vitro amyloid formation is highly specific and self-propagating, thus providing a possible explanation for the [URE3] prion. We have sought to clarify this hypothesis by examining the state of Ure2p in [URE3] cells by thin-section electron microscopy.

Yeast cells with the [URE3] prion and control [ure-o] cells were thin-sectioned after fixation and embedding in an epoxy resin. We found distinctive filamentous aggregates in the cytoplasm of [URE3] cells of a strain that overexpresses Ure2p (Fig. 1). These aggregates were seen in some cell profiles represented in 50-70 nm sections, and were never seen in control sections of [ure-o] cells. in cell sections showing these structures, there was typically one such aggregate, which could be quite large - up to several μm across - and approximately globular in outline. They contain irregularly associated filaments about 25 nm in diameter.

Shells of agglutinated foraminiferan protists are composed of mineral grains bound by secreted adhesives. As such, they are useful models for examining the evolution of “primitive” exoskeletons. Previous studies revealed the ultrastructure of shells in the giant Antarctic foraminiferan Astrammina rara and demonstrated that shucked specimens would reconstruct shells using glass beads. Here we further investigate shell architecture in this model species.

For micromechanical testing, an intact A. rara shell was placed between a fixed plate and a facing plate in series with a calibrated load cell. Displacement was effected by a high-precision drive, and 2-3 loading cycles were used to determine shell material properties. to assay tensile properties of the adhesive matrix, a network of pseudopodia and extracellular matrix fibers (i.e., the shell adhesive component) was obtained by incubating shucked cell bodies on 200-mesh gold grids. Pseudopodia were subsequently removed by detergent washes. Fibers in the resultant isolated matrix were severed with a Nd: YAG laser using an inverted DIC light microscope equipped with a 60× objective lens. Preliminary loading experiments using glass needles showed that Sepharose 2B beads were suitable strain gauges to assess compression within reconstructed shells (Fig. 1). in this assay, shucked cell bodies were incubated with a mixture of glass and Sepharose beads, and the reconstructed shells were examined by SEM.

Repeated loading and unloading demonstrated the elastic behavior of intact shells (Fig. 2). Adhesive matrix fibers snapped towards their attachment sites within 2 sec after cutting with a laser (Fig. 3), demonstrating that they are deployed under tension. SEM images of shells reconstructed with Sepharose show compressed particle profiles (Fig. 4).

In this study a combination of scanning (SEM) and transmission electron microscopy (TEM) was used to examine leaf cells of the common weed Indian strawberry (Duchesnea indica) infected by the biotrophic rust fungus Frommeëla mexicana var. indicae. For SEM, free-hand sections of infected leaves cut with a razor blade were placed in a 5% (wt/vol) aqueous solutions of Ariel (Proctor and Gamble, Cincinnati, OH), a washing powder containing a bacterial protease. This treatment removed exposed host cell cytoplasm so as to reveal details of fungal haustoria and host cell wall responses to infection (Figs. 1, 2). Samples then were processed for study according to procedures described previously. For TEM, samples were prepared for study using high pressure freezing followed by freeze substitution. This fixation procedure has been used with excellent results to study hostpathogen interactions in rust-infected plants.

Frommeëla mexicana var. indicae produced specialized hyphal branches known as haustoria that extended through the walls of infected cells. Each haustorium possessed a long, slender neck region (Fig. 3) and an expanded body (Figs. 1, 4) that was separated from the host cell cytoplasm by the invaginated host cell plasma membrane. At maturity the neck region was surrounded by a extensive labyrinth cell wall in-growth or elaboration (Figs. 1-3) that appeared to be identical to wall elaborations that characterize plant transfer cells. Such cells are thought to be involved in intensive solute transfer over short distances. While transfer cells have been reported within cells of the haustoria of various plant parasitic higher plants and in plant giant cells induced by certain plant parasitic nematodes, this appears to be the first report of their formation in response to infection by a plant pathogenic fungus. Not only did these elaborations form in the immediate vicinity of haustoria, they also arose apart from haustoria in infected cells. However, such elaborations were not found in cells of uninfected leaves.

Type 1 pili are outer membrane organelles formed by 80% of all E. coli strains. They are up to 2 μm long, rod-like appendages covering the bacterial body like spines on a hedgehog (Fig. 1). Type 1 pili expressed by uropathogenic E. coli strains mediate adhesion to specific receptors of the bladder epithelium. They allow colonization of the cell surface as a prelude to infection. Type 1 pili are composed of a rigid rod and a fibrillar tip (Fig. 2a). The minor subunit FimH mediates adhesion and is located at the fibrillar tip as demonstrated by immunolabeling (Fig. 2b). The pilus rod is built from a structural protein, the major subunit FimA. Brinton analyzed parameters describing the helix of the type 1 pilus rod based on paracrystalline arrays. Accordingly, the data predicted one helical repeat to be composed of 25 subunits in 8 turns with a helical pitch of either 2.32 or 2.4 nm.

Here, the 3-D helical structure of the pilus rod has been determined from isolated negatively stained type 1 pili imaged in the annular dark-field mode by scanning transmission electron microscopy (STEM) (Fig. 3a). The power spectra of unbent rods (Fig. 3b) exhibit the strongest layerline at a spacing of (2.42 ± 0.04 nm)−1 representing the pitch of the putative one-start genetic helix (Fig. 3c). The axial distance to its nearest layerline corresponds to the helical repeat (Fig. 3c). Averaging by a linear superposition, as employed for structural analysis of F18 fimbriae, another type of adhering E. coli appendages, confirmed the helical repeat to be 19.31 ± 0.34 nm. Mass-per-length (MPL) determination of unstained type 1 pili by STEM revealed one helical repeat to be composed of 26 ±1 15.8 kDA FimA molecules. All possible helical selection rules were tested by D(Z,k) filtering. Thereby, any data inconsistent with the employed helical selection rule are excluded (Fig. 3d). Hence, the helical selection rule yielding the most power recovery represents the closest selection rule describing the helix. We found l = 8n + 21m to be the optimum helical selection rule. Fig. 3e represents the filtered image. Accordingly, one helical repeat is made of 8 turns containing 27 FimA monomers occurring with an axial rise of 0.72 ± 0.01 nm. D(Z,k) filtering with the helical selection rule assumed by Brinton resulted in significant loss of structure. Hence, we conclude to have refined the previous indexation. The resulting 3-D structure of the type 1 pilus unveils a 6.8 nm wide right handed helical tube harboring an axial cavity (Fig. 4). The tube wall is formed by a coiled fiber comparable to the metal thread of a spring.

Napsin is an aspartic proteinase which has two essential asparagine residues in the active peptide (Tatnell et al., 1997). Napsin may participate in the processing of pulmonary surfactant protein B (Weaver et al., 1992; Chuman et al., 1999). in addition, changes in napsin expression have been linked to primary adenocarcinoma as well as kidney dysfunction (Hirano et al., 2000; Schauer-Vukasinovic et al., 2000). Localization studies by light microscope immunocytochemistry and in situ hybridization detected napsin in the lungs as well as in the kidney (Tatnell et al., 1997; Chuman et al, 1999; Schauer- Vukasinovic et al., 1999). However, these studies did not resolve the ultrastructure localization of napsin. in this study, EM cryoimmunogold labeling was used to characterize the subcellular distribution of napsin in lung and kidney cells.

A truncated murine napsin propeptide construct (1212 b.p.) without the signal peptide sequence (47 b.p.) was cloned into pProEX™HTb plasmid vector for the generation of recombinant protein. Polyclonal antibodies were generated against purified recombinant protein. The specificity of polyclonal antibodies was determined by immunoprecipitation and Western blotting using isolated mouse alveolar type 2 epithelial cells and kidney homogenates. The napsin antibodies detected a single protein with Mr= 43 kD.

Localization of napsin by EM cryoimmunogold labeling was performed using protein A gold probes as previously described (Na et al, 2000). in the lungs, napsin was localized in both alveolar type 2 epithelial cells and in alveolar macrophages. in alveolar type 2 epithelial cells, napsin was detected in multivesicular bodies and in lamellar bodies; internalized BSA gold colocalized with napsin in the lysosomes of alveolar macrophages (Figure 1A and 1B), consistent with the observation that napsin is active at low pH (Schauer-Vukasinovic et al., 1999). Although Schauer-Vukasinovic et al. (1999) reported that napsin was expressed by some bronchiolar cells, we did not observe any significant napsin labeling in Clara cells or in ciliated epithelial cells.

Zanichellia palustris is a submerged halophyte which occurs in waters of low salinity (5%o to 10%o). It is not capable of surviving at high salinity. Reasons for its inability to tolerate hypersaline conditions is important to our understanding of its biology. in the present study, leaf ultrastructure of plants growing at normal salinity was compared with plants growing at high salinity so as to assess the effects of hypersalinity. Attention was focused on ultrstructural changes occurring in leaf blade epidermal cells.

Plants were grown in seawater at two salinities : 10%o (control) and 20%o (high salinity). Pieces of mature leaf blades from both treatments were harvested and prepared for Transmission Electron Microscopy (TEM) following standard procedures. in addition, the overall distribution and concentration of chlorine (Cl) in the leaves was ascertained since it is the most abundant anion in seawater and is important in considerations of salt tolerance in submerged halophytes. Cl was determined by means of a nuclear microprobe. Pieces of sectioned leaves were rapidly frozen, freeze dried and carbon coated. Elemental distribution maps of Cl were obtained using the true elemental imaging system (Dynamic Analysis). Two complementary techniques, Proton Induced X-Ray Emission (PLXE) and Proton Back Scattering (BS) were performed simultaneously using the GeoPIXE suite of programmes. Sample thickness and composition of major elements were obtained from the BS spectra.

The ultrastructural morphology of leaf blade epidermal cells, which possess transfer cell characteristics differed at each salinity. While the cytoplasm was well preserved in epidermal cells of plants at the control salinity : 10%o (Fig 1), considerable cytoplasmic damage occurred in epidermal cells of plants at high salinity, in the form of breakdown of membranes such as the plasmamembrane and tonoplast, and senescence of various organelles including chloroplasts and mitochondria (Fig. 2).

Growth is “anisotropic” when growth rates in different directions are not equal. Anisotropic elongation is controlled by cortical microtubules and cellulose microfibrils of the cell wall. Distorted anisotropic growth results when there are aberrations in either the cellulose network or microtubule cytoskeleton. in this ultrastructural study, the roots of wild type (control) and mutants of Arabidopsis thaliana (L.) Heynh, ecotype Columbia, were compared to determine the role of microtubule organisation, cellulose synthesis and cytokinesis on root expansion.

Three mutations, obtained by treating seeds of A. thaliana with ethane-methylsulfonate and backcrossing once to wild type after four selfed generations, were isolated and designated as rsw (radially swollen). These phenotypes are all temperature sensitive, growing and appearing as wild type at 19°C but expressing the mutant phenotype at or above 30°C. Segments of intact root tips from the three mutants and wild type were prepared and examined for TEM with a Jeol 1200EX. The overall appearance of rsw seedlings at 19°C is the same as wild type. At 30°C these phenotypes lead to extensive swelling of the root apex. Despite considerable alteration in the morphology of the root apex, the appearance of the root cap and root hairs seems essentially normal. When mutant roots expressing these phenotypes at 30°C are returned to 19°C, a wild type appearance of the root apex and elongation gradually returns.

The primary root of the wild type has a remarkably uniform cellular organisation with regularly arranged dense cytoplasmic cells (Fig. 1). At 19°C, rsw4, rsw6 and rsw7 were indistinguishable from wild type; however, at 30°C, the well-defined anatomy was distorted in all tissues by additional cell walls in unusual positions (Fig. 2).

Chloroplasts, present in leaves of higher plants have an extensive, folded network of photosynthetic membranes. These membranes have closely appressed (grana) and non-appressed (stroma) regions and they are responsible for the conversion of solar energy into biochemically useful forms. Light plays an important role for the development of mature chloroplasts from proplastids. However, when seeds germinate in the dark, the proplastids do not form mature chloroplasts and instead they form etioplasts. The etioplasts contain a compact, highly regular lattice of inner membranes called prolamellar body. Exposure of dark grown seedlings to light induces formation of fully mature chloroplasts from etioplasts. Plant growth and development is dependent on various environmental factors and drought has profound effect on plant metabolism. Leaves depend on adequate water for both turgor pressure and photosynthesis. Turgor pressure enables nutrients in the form of carbon and minerals to be transported from soil to leaves where they are needed for synthesis of organic compounds. in this study we evaluated the influence of drought on the ultrastructure of developing chloroplasts in pepper plants.

Jalapeno pepper plants were grown from seeds in the dark and drought was induced to experimental group of plants after they developed the first pair of primary leaves. The dark grown control plants received water throughout the drought induction period. At the end of tenth day of drought, plants in both control and experimental groups were exposed to light for up to 48 hours and leaf samples from both groups were obtained at various time intervals after exposure to light. The samples were fixed, dehydrated and embedded for transmission electron microscopy. Ultrathin sections were made, observed under a Hitachi H7000 TEM and data were collected for ultrastructural study. The mature chloroplast and etioplast structures were compared between experimental and control group of plants.

Vero cells (African green monkey kidney epithelium) contain very dense cytoplasm with numerous ribosomes and mitochondria. They also contain an impressive network of cytoskeletal proteins, especially vimentin intermediate filament. Vimentin is present in all cells of mesenchymal origin, but their localization in the cell is usually restricted to a perinuclear position. Vero cells exhibit large bundles of vimentin filaments in a network, coursing throughout the cell. Additionally, many vimentin filaments connect to each other in an intermediate filament focus, or vertex. We investigated these vimentin vertices using immunofluorescence, scanning and transmission electron microscopy.

The processing of cultured cells for transmission electron microscopy is a difficult task, especially when one desires to preserve in situ relationships. When sectioning epithelial cell lines, a single cell layer (approximately three micrometers) of tissue is available, so a few thick sections will remove all the cells in the block. A traditional method for TEM of cultured cells is to disrupt these cells from their substrate and then centrifuge these cells into a pellet which can be sectioned. While it may be easier to obtain thin sections of cultured cells employing this method, many characteristics of the cells cannot be adequately investigated, including cell-to-cell junctions, cell shape on a substrate, cell morphology associated with a substrate, position of organelles in a cell growing on a substrate, and morphology and relationship of mitotic cells.

Growing cells on Thermanox cover slips and embedding in LR White polymerized in gelatin capsules at 55°C affords an effective method of viewing and labeling cells in culture. When the resin has been polymerized, the Thermanox coverslip is carefully removed from the block by using a razor blade to pry it off. The cells remain in the LR White, exposing them to the surface of the block. in fact, the very first sections of the block contain cells. Although this prevents one from facing the block, the exposed surface is usually flat enough to obtain good thin sections.

Glutathione (GSH) is a tripeptide thiol found in virtually all mammalian cells. GSH is a cofactor in several enzymatic reactions and is important in intracellular cysteine storage. Probably the most interesting of its many functions is its role in protecting the cell against the detrimental effects of ionizing radiation, reactive oxygen compounds, free radicals, and toxic xenobiotics. GSH is the reduced form which can be oxidized to a disulfide form (GSSG) or to form mixed disulfides with proteins. The GSH oxidation-reduction cycle maintains a high GSH to GSSG ratio which provides a reservoir of reducing equivalents that can nullify reactive oxygen species and detoxify xenobiotics. The study of glutathione in the fields of cancer research and immunology has attracted much attention, especially with regard to changes with age.

In this study, we have determined the cellular distribution of glutathione using immunoelectron microscopy. A mouse monoclonal antibody (8.1-GSH) against the glutathione adduct with Nefhylmaleimide (NEM) was used.

The reactive oxygen species (ROS) such as superoxide radical (O2), hydrogen peroxide (H2O2), and hydroxyl radical (OH), plays a critical role in the pathogenesis of hypertension, atherosclerosis, ischemia-reperfusion injury, stroke, myocardial infarction, and congestive heart failure. A recent study demonstrated that the frequency of mutation in a reporter gene increased in cortical DNA after forebrain ischemia-reperfusion. Eight DNA lesions that are characteristic of DNA damage mediated by free radicals were detected. Among the different oxidative-damage DNA products, 8-oXo-7,8-dihydrodeoxyguanine (8-oxoG) is the most stable and deleterious adduct. Recent studies demonstrated that human MutY homologue (hMYH) protein plays important role in repairing oxidative damage. to date, there is no information regarding the role of hMYH expression in human myocardium with congestive heart failure (CHF). Therefore, the current study designed to investigate the levels of hMYH expression and 8-oxoG in myocardium in the absence or presence of end-stage congestive heart failure. The hypothesis of this study is that increasing DNA damage such as 8-oxoG generation is associated with a reduction of hMYH expression in human myocardium with congestive heart failure.

We developed a new type 3-Dimensional Internal Structure Microscope (3D-ISM) for the observation of internal structures of samples. The internal structure of a sample is obtained by cutting it into thin slices and observing the cutting side continuously while cutting. The position of the camera, as well as the sample position are fixed. Therefore there is no shift between the sections, and this system can obtain a true color image of the sample, which is a high resolution and a high-quality three-dimensional image compared with X-ray CT and MRI. After repeatedly slicing a sample, the digital image data from the sectional views is transferred to a computer, where 3-dimensional images of the internal structure of the sample are reconstructed. Using this system we analyzed many biological organisms. Just now, we are developing the system which builds in a fluorescent microscope. in this paper, We therefore observed fluorescent-stained samples by a fluorescence microscope installed in the 3D-ISM instrument are shown.

This article presents the outline of the device and the principle of the observation. This system, consisting of a slicing device, reflecting optical microscope, a CCD camera, a laser video recorder, a PC-based control system and an image processing workstation. FIG. 1 shows a schematic view of the cross secsional machine. Before slicing, the sample is embedded into either paraffin or OCT compound. Subsequently, the sample is positioned into a cross-sectioning machine for the slicing operation, which is performed using a cutting blade. The maximum size of the sample which can be observed with this device is ϕ 8x10mm. The thickness of the slices can be adjusted mechanically between 0.5-10 μ m. The cutting speed can range from 0.5 to 4 slices/ s. Each cross-section is viewed by employing a epi-fluoresence microscope and a CCD camera. The object lens used for the observation can use the x2-80. The resulting image is recorded on a laser video disc. After the slicing operation, the 3D sample image is reconstructed on the basis of volume rendering by using a Workstation. These observations can be completely done by full automation.

Semaphorins are primarily known for the important role they play in the guidance of growth cones during neuronal development. There is evidence, however, that semaphorins are expressed outside the nervous system as well, suggesting a wider scope for semaphorin function. The overall objective of our study is to identify the functions of semaphorins outside central nervous system especially in T cell development. Some of the 20 semaphorins have been shown to have extra-neural functions that include (for different semaphorins) bone differentiation, promotion of B-cell survival and aggregation, and activation of T-cells. Apart from central nervous system statement of most semaphorins, one semaphorin (CD 100) has transcripts in T cells, B cells, neutrophils, monocytes and granulocytes. EST analysis suggests that other semaphorins are expressed in lymphoid tissues such as thymus, spleen, tonsil, and the interfollicular areas and germinal centers of lymph nodes.

Semaphorins have been related to several cell survival mechanisms, immunosuppression and promotion of cell death resistance. in preliminary studies our lab found that viral semaphorins inhibit the migration of human T cells and human SEMA3A can inhibit migration of human neutrophils.

Electron tomography is a three-dimensional (3D) imaging method with transmission electron microscopy (TEM) that provides high-resolution 3D images of structural arrangements. with electron tomography a series of images is acquired of a sample that is tilted over a large angular range (±70°) with small angular tilt increments. For the 3D-reconstruction, the images of the tilt series are aligned relative to each other and the 3D-reconstruction is computed. Electron tomography is the only technique that can provide 3D information with nm-scale resolution of individual and unique samples. Routine application of electron tomography will comprise a major step forward in the characterization of complex materials and cellular arrangements. When collecting tilt series for electron tomography image shifts and defocus changes have to be corrected for by the human operator. The repetitive correction of these changes is highly time consuming, error prone and very hard to carry out under low-dose imaging conditions.

Many practical problems are overcome when electron tomography data collection is performed in an automated fashion. Automation includes the (a) image acquisition on a (digital) CCD camera, which implies that (b) changes in image position and defocus can be detected by on-line image processing and (c) immediately be corrected for by computer control of the microscope, (d) Finally, tilt series are directly available in digital format for subsequent processing. Typically, carrying out such an experiment would take a day, and the actual data collection 2-4 hours. in spite of the enormous progress made in terms of data collection speed during the last few years, the current status of automated tomography still does not meet the requirements that would make it a routinely applicable tool. For a great number of biological assays and research projects, results obtained under different experimental conditions have to be compared, and series of experiments have to be carried out. Therefore, we propose a novel approach for recording a tilt series that significantly increases data collection speed, and widens the applicability of the technique.

Gap junction channels serve important functions for the homeostasis of organ systems. These membrane channels serve as low-resistance pathways for the diffusion of small molecular species such as ions, metabolites and second messengers between two neighboring cells (1). Recent intense investigations have been pursuing the effects that defects in gap junction proteins, the connexins, have in hereditary diseases. Mutations in the primary sequence of several connexins have been shown to be responsible for several hereditary human diseases including X-linked Charcot-Marie- Tooth demyelinating neuropathies, nonsyndromic sensorineural deafness, erythrokeratodermia and congenital cataractogenesis (c.f. (2, 3) for recent reviews). Many of the published mutations are being investigated thoroughly to determine at what stage and why the protein fails to make functional channels. Steps involved in connexin trafficking and assembly are: translation of the protein; transport from the endoplasmic reticulum to the Golgi apparatus; oligomerization of the subunits into a hexamer (called a connexon); transport of the connexon to non-junctional areas of the plasma membrane; correct docking with a partner connexon from an apposing cell to form a functional intercellular channel; and incorporation into aggregates (gap junction plaques).

Currently, the morphology of carbon black is described by distributive morphological properties that are measured using two dimensional TEM projections of the carbon black aggregates. The area, perimeter and several Feret diameters of 2000 such aggregate projections are measured with an automated system and used to calculate a number of parameters including the mean primary particle diameter and the surface area of the carbon black in m2/g. Several assumptions must be made in order to carry out these calculations, the first being that the aggregates are either isotropic or randomly oriented. True random orientation is impossible due to the flat surface on which the carbon black is deposited. For relatively compact spherical, ellipsoidal and linear aggregates this assumption works fairly well. However, the more highly structured a carbon black aggregate is, the worse this assumption becomes. Studies of these types of aggregates have shown that the two dimensional projected area and perimeter can decrease up to 25% as the viewing angle is increased from 0° to 45°. The use of electron tomography to determine surface area will eliminate this problem. in addition, the use of tomographically reconstructed carbon black aggregates for the determination of surface area is a more direct measurement technique, although limitations to its accuracy do exist.