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.

Tomographic 3D-reconstructions from tilt series of projections differ in two major aspects when moving from biochemistry (e.g.) to materials science applications: (I) Crystalline inorganic materials fail the weak phase image formation approximation and suffer from dynamic and nonlinear contrast contributions (which depend for both CTEM and HREM on atomic lattice plane orientation). (II) exposure tolerance increases by two orders of magnitude. We therefore propose energy filtered tomography with inelastic electrons (electron spectroscopic imaging, ESI) as a new method for 3D-reconstruction of free or buried subsurface nanostructures or nanoparticles. Energy filters are not used here to improve SNR by removing inelastic background , but to provide the necessary projection relationship between a 3D concentration distribution and the (processed) image intensity. Ideally, a 4-dimensional data space is recorded for a single tilt axis tomography experiment, D=D(δE, Θ,x,y), (with 5E as energy loss, Θ as tilt, and (x,y) as image coordinates). The material has to withstand exposures of T = t × Ntilt × Nloss (t = 5...20sec per image, Ntilt >12 directions, Nloss = 5....20 energy loss values).

Chromosome alignment during mitosis and meiosis is mediated through the interaction between kinetochores and spindle microtubules (Mts). Kinetochores are fibrous mat-like structures, located at the primary constriction of chromosomes, that bind spindle Mts, generate poleward chromosome motion, and delay progression through mitosis until chromosome alignment is complete. Mts are polar polymers that function as tracks for the movement of a variety of cellular organelles and vesicles. Chromosome alignment requires a unique form of Mt-based motion because kinetochores capture and bind the plus ends of a bundle of Mts, rather than moving along the lateral surface of a single Mt . As a result, the direction of chromosome motion must be coordinated with the growth and shrinkage of Mts. Furthermore, the kinetochore maintains continuous attachment to growing and shrinking Mts and simultaneously permits the addition and dissociation of tubulin subunits within its confines

During the last decade, computation of a three-dimensional image from a tilt series (3D reconstruction) has become a well established method, of which a variety of implementations are available. The term “electron tomography” is now generally used for this type of data acquisition and 3D reconstruction. An overview over the techniques involved is given in.

With the introduction of micro-processor-controlled TEMs and cooled slow-scan CCD cameras and with the progress in performance of high-speed computers, automation of complex imaging procedures became mainly a task of developing appropriate software, using the control facilities of the microscope. in this way, automated electron tomography was realized in 1990 at the Max- Planck-Institute for Biochemistry in Martinsried, and at about the same time at the University of California in San Francisco (UCSF). New techniques for automatic focusing and alignment, developed somewhat earlier , have been integrated in these automated tomography procedures. in the following we discuss the requirements of automatic data acquisition and the present implementation for several TEMs.

We have developed methods for generating tomographic reconstructions of large cellular volumes efficiently and at relatively high resolution. One key component of these methods is the ability to capture images from our high voltage electron microscope (HVEM) with a sensitive lenscoupled CCD camera. to overcome the limited resolution and field of view of this 1k × 1K camera, we have used the image shift capability of the HVEM to develop automatic montaging of images from an array of positions, with about 10% overlap between adjacent positions. We have also developed automatic repositioning and autofocusing after each tilt of the specimen, so that it is now possible to acquire single-frame or montaged images at a series of tilt angles, with no operator intervention. with these capabilities, up to 2 GB of image data can be acquired in a single session.

Tilt series are processed with the IMOD software package developed in our laboratory, which now runs on a PC under Linux, as well as on SGI computers. Several features are notable for working with large data sets.

The application of electron tomography to cell biology has led to important insights into the 3D fine structure of subcellular processes and organelles. Tomography has been particularly useful for studying relatively large, multi-component structures such as the Golgi apparatus, mitochondria and synaptic complexes. When combined with very powerful high voltage electron microscopes, tomography has also provided high resolution quantitative views of extended structures such as neuronal dendrites in very thick sections (4 μm) at electron microscopic resolution. The utility of tomography is twofold: first, it provides 3D examination of subcellular structure without the need for serial section analysis; second, because the computed slices through the tomographic volumes can be much thinner than is possible to produce by physical sectioning, it reveals structural detail in the range of 5-30 nm that tends to be obscured in conventional thin sections. Tomographic analysis has forced re-assessment of long-standing views of organelles such as mitochondria and the Golgi apparatus and as the technique advances, additional insights are likely forthcoming.

Electron tomography is an expensive technique, both in terms of the instruments used and the computational resources required. The three major high voltage electron microscope resources in the United States, San Diego, Boulder and Albany, all are actively engaged in tomographic research and offer this important technology to the scientific community at large.

In skeletal muscle, depolarization of the plasma membrane, which is initiated at the neuromuscular junction, is transduced to a rise in cytoplasmic calcium at specialized structures known as triad junctions (TJs). TJs occur in the myofiber’s interior at regions near the z-lines, where transversely oriented tubular invaginations of the plasma membrane (T-tubules) form junctions with two elements of the sarcoplasmic reticulum (SR). Isolation of membrane fractions that are enriched in junctional complexes and which retain function has been reported.

Figure 1 shows a region of an electron micrograph containing an isolated TJ in the frozen-hydrated state. in the orientation shown, two SR-derived vesicles sandwich a flattened vesicle derived from the T-tubule. The junctional regions contain a complex distribution of density, presumably due to proteins that are known to be present in TJs. Electron tomography offers the means to determine the three-dimensional mass density from such micrographs, which would greatly aid in their interpretation. Only recently has the automated data collection technology for determining tomograms of non-stained, frozen-hydrated specimens become available. Here we describe the first tomographic reconstruction of a frozen-hydrated triad junction by automated electron tomography.

The pinceau is a cerebellar structure formed by GABA-ergic descending basket cell axonal terminals converging on the initial axonal segment of Purkinje cell. Although it exerts a powerful inhibitory influence on the output of the cerebellar cortex, the exact ultrastructural organization and function of this structure are not understood. The unique morphology of the pinceau lies in several elements: lack of synaptic contacts between basket cell terminals and the initial axonal segment, the absence of postsynaptic densities, and heavy ensheathment of the initial segment with glial processes, making astrocytes the only intervening cellular element between the two types of axonal structures. Several plasticity related proteins in this area have been previously characterized by immunocytochemistry. Those include nitric oxide synthase, PSD95, GABA transporters, and several voltage gated potassium channel subunits. We are using a combination of confocal and electron microscopy, correlated photooxidation, EM and tomography to reveal the ultrastructural distribution of novel proteins and their relationships to morphological compartments in the pinceau.

We describe here Kv3.2, a recently discovered member of the Shaw-like subfamily of potassium channels, and the correlated distribution of other proteins described below. Pre-embedding immunolabeling, electron microscopy and tomography showed that Kv3.2 was selectively localized to basket cell axons.

We have used electron tomography of rigorized fibers of insect flight muscle swollen by immersion in dilute buffer (†1mM MOPS, pH 6.8), to reveal the 3-D arrangement of the myosin S2 fragment for the first time. S2 is an α-helical coiled-coil structure that connects the myosin head to the thick filament backbone. It is usually invisible in electron micrographs because of its close proximity to the backbone. The increase in inter-thick filament spacing from 45 nm to 55 nm pulls the S2 away from the filament backbone making it easier to identify. A total of 4 tomograms have been calculated by our standard method which involves collecting images using non-uniformly spaced tilt angles, cross correlation alignment, area matching and Whittaker-Shannon interpolation of the 3-D transform. A projection of one of these tomograms is shown in Figure 1. in raw tomograms S2 appears as a rod-like structure that originates at the thick filament surface to connect to both lead and rear crossbridges of rigor. S2 segments are particularly well resolved at the ends of the thick filament (Fig. 2), where in some cases it appears that the myosin subfilaments are separated from the backbone. Both the azimuthal direction and axial angle of S2 are variable.

Recent development in confocal and multi-photon microscopy allows 3D imaging of plant tissue in high resolution. However, other than physical sectioning, macroscopical study of plant organs in 3D remains a difficult task. Among various available technologies for macroscopical imaging (e.g., Xray macro-tomography, optical coherent tomography and MRI), MRI is an ideal choice for its contrasting modality in volumetric imaging of soft tissues. A 3T Biospect MRI system (Brucker, Germany)(FIG 1) equipped with a 6cm inner diameter micro-quadrature coil (FIG 2) for RF transmission and reception of MRI signals was used in this study. Spin echo based RARE sequence was used to obtain T2 weighted images with TR/TE = 3160.5/58.5ms and field-of-view of 1.67cm × 1.67cm (256 × 256 pixels) at a slice thickness of 0.8mm. This corresponds to a voxel size of 65 × 65 × 800μm. Data was obtained within 1/2 hour with number-of-excitations (nex) set at 16. Figure 4 (a-x) shows a series of MRI sections through a stem node (the node below the main ear insertion) from field-grown maize (Zea mays, van Odyssey sweet corn). The stem was fixed in 1:3 EtOH/acetic acid, washed thoroughly in water prior to imaging. Air bubbles trapped in the tissue were removed by vacuuming, to avoid imaging artifact due to low magnetic susceptibility of air. Figure 5 (a-g) shows reconstructed longitudinal sections. Three-dimensional reconstruction (FIG. 3) was performed by using Vaytek VoxBlast™ and AutoQuant’s AutoVisulize 3D™ software. in combination with image segmentation and tracing tools, the MRI technology will greatly enhance our capability in the understanding of vascular architecture and its development in plants.

One of the most promising and exciting developments in electron tomography is its application to ice-embedded cells and cellular organelles. The primary goal of cellular electron tomography is not to obtain a high-resolution structure of a particular protein complex, but to identify a macromolecule by virtue of its structural signature and to locate it in the context of the native cellular environment . We present a computationally feasible approach, which demonstrates that an objective identification of large macromolecular complexes in cells is possible, given that a highresolution reconstruction and the structure of the molecular assembly under scrutiny are available. Our aim is to quantify the identification results using a realistic test specimen, open for the modification of experimental parameters like thickness, protein density or protein composition. in this study, the reliability of the algorithm applied to electron tomograms of ‘phantom cells’ - mimicking a real cell - is shown.

We prepared phospholipid vesicles that were filled with different macromolecular complexes. First, a phospholipid solution (SOPC) was sonicated and mixed with His-tagged macromolecular complexes, such as thermosomes, 20S proteasomes or a mixture of both. The lipid-protein mixture was subjected to several freeze-thaw cycles and extruded to produce uni-laminar ‘phantom cells’ of various sizes.

The multi-layered architecture of alphaviruses is built up with the registration of nucleocapsid (NC) proteins and envelope spike proteins across a lipid bilayer. At the surface of the NC, capsid proteins are organized as 42 capsomers such that the three tails from each spike complex bind three capsomers. Alphaviruses have been extensively studied using cryo-EM and X-ray crystallography at near-atomic resolution (see review by Sedzik et al.). This work involves averaging images of many isolated virions, and makes use of the symmetry of the particle. The study of assembly and budding of virus particles at the cell surface, however, requires structural determination of individual virions in situ. Electron tomography offers a means for such studies.

Small features are obscured in typical thin-section (ca. 50nm) TEM micrographs due to the projection in 2D of all the structures throughout the section thickness. However, slices thinner than lnm can easily be obtained from tomographic reconstructions. Thus, small structures such as capsomers can be seen, even in thick (>100nm) specimens (Fig. 1b).