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).

Electron microscopy is a technology fully capable of bridging the light microscopic world with the world of protein structure at atomic resolution. There are numerous examples of cases where cryoelectron microscopy has been used to fit an atomic structure into a structure that has been difficult to crystallize for high resolution structure analysis. More difficult is the fitting of atomic structures into more conventionally prepared tissue like that used in freeze substitution.

We have been using electron tomography to obtain 3-D images of insect flight muscle in various states, including those produced in an active isometric contraction as well as in rigor. These tomograms were obtained using non-uniform tilt angles, cross correlation alignment, area matching and Whittaker-Shannon interpolation of the 3-D transform. The fitting of atomic structures into raw tomograms is a difficult proposition because of the relatively highly noisy feature of the images. Better results are obtained when averages are produced. We have used two method for producing averages. One of these, the so-called column average is used when the muscle filaments are relatively straight with good axial order, or can be straightened in 3-D but their lateral ordering is poor. Column averaging improved considerably reduced the noise in tomograms of contracting muscle enabling the four major crossbridge conformation to be observed (Fig. 1).

In order to extend the resolution available in electron tomograms, we have been experimenting with various deconvolution procedures that will ultimately enable correction of the focus gradient thereby allowing the resolution to be extended to beyond the first zero in the contrast transfer function (CTF). This effort requires first a determination of the amount of amplitude and phase contrast in the typical specimen used by us.

For the determination of the percentage of amplitude contrast a focus series of 19 images was taken at a magnification of 13500 on a CM300. The micrographs were digitized on a PDS scanner, the pixel size was 0.888 nm. After correction of translational shifts, rotations, and defocus dependent magnification differences the image restoration was carried out according to [1,2]. From the resulting complex wave function an averaged amplitude contrast amount of 18% was obtained for the stained specimen. The phase contrast part and amplitude contrast part are shown in Figs, 1b, and 1c, respectively, and can be compared with the micrograph taken at 960 nm underfocus (Fig. 1a).

The ability of cornea to transmit light whilst being mechanically resilient is directly attributable to the formation of an extracellular matrix containing orthogonally arranged sheets of densely packed uniformly-narrow collagen fibrils, in which each sheet contains parallel arrays of fibrils. An understanding of the molecular basis of tissue organisation requires knowledge of the detailed 3-dimensional structure of individual collagen fibrils and how they interact to generate higher order structures. in this study adult bovine corneal collagen fibrils were examined using transmission electron microscopy and automated electron tomography to generate three-dimensional volumes of individual fibrils which had been negatively stained. Tilt series images were collected with an angular increment of 2° over a range of ± 70° using a Philips CM200FEG coupled to a TVIPS automated electron tomography system. A microfibrillar substructure was observed throughout the thickness of the fibril (Figure 1). The microfibrils were ∼4 nm in diameter and were oriented in a right-handed helical twist along the length of fibrils. The microfibrils were observed at an angle of 15° to the long axis of the fibril. As the technique of electron tomography generates a threedimensional volume from the data it was possible, for the first time, to visualise the lateral arrangement of molecules within individual fibrils. There were regions where there was a distinct lateral order and other regions which showed a more fluid-like arrangement. The axial positions of most crystallinity corresponded with the location of the C-telopeptides and N-telopeptides as well as a region close to the d-band in the gap structure of the fibrils. The study also demonstrated that the microfibrils are organised in a quasi-hexagonal packing arrangement

The smallest apatite mineralites found in nature reside in vertebrate bone tissue. These nanoapatites participate in the formation of a protein/mineral composite with unique physical and biological properties. The composite has distinct structural features at the 10 nm, 100 nm, 1 um, 100 um, and 1 cm length scales. We analyzed the natural material on the 1-1000 nm length scale with the purpose of setting the design criteria for synthetically duplicating the material. Using a biomimetic motif, we synthesized nanomineralites with size and shape approximately equal to the natural material.

We show that our synthetic mineralites compare with the natural apatites. Recently the average size of naturally occurring mineralites in mature bovine bone was shown to be 12 nm X 10 nm X 0.61 nm. Similar experiments on 1-3 week old bovine bone mineralites showed an average size of9nm X 6nm X 2.0 nm. Within a standard deviation, the length and width of the two populations are equal.

The need to optimize adhesion between the femoral stem and surrounding tissue has led to the development of plasma-sprayed HA (PSHA) coatings as attachment facilitators for hip prostheses. The mechanisms underlying this facilitation are incompletely understood, and the long-term efficacy of HA coatings in maintaining bonding to bone remains largely unexplored. in this study, two in vivo models—canine and human—were employed for study by scanning electron microscopy (SEM) and transmission electron microscopy (TEM) to elucidate the sequence of early bone mineralization and the longer term fate of HA coatings. High-resolution SEM and TEM of whole bone and anorganic bone were employed to establish the structure of mature bone for comparison.

Whole and Anorganic Bone.Anorganic bone derived from bovine trabecular bone was examined in low-voltage field-emission SEM(LV-FESEM) and high-resolution TEM to establish the morphology of the mineral component of bone [1,2]. Trabeculae were seen to comprise oriented fiber bundles (Fig. 1), each fiber comprising an array of oriented apatite platelets (Fig. 2) arranged like a sheared stack of dominoes with approximately the 64-nm stagger of collagen hole zones.

many naturally occurring structures are, in fact, composite materials with functionally graded mechanical properties. in manufacturing, achieving such graded properties is critical when joining two materials of significantly different elastic moduli, thermal or electronic characteristics. One assumes that similar imperatives are at play in the profusion of biological examples of gradients. Two gradient biosystems, in particular, have been the focus of our attention: These are mussel byssal threads and polychaete jaws. Mussel byssal threads are composites consisting of anisotropically packed discontinuous fibers in an amorphous matrix all coated by a protective cuticle. The threads are produced by process resembling reaction injection molding and imparted with a longitudinal gradient of mechanical properties ranging from stiff and tough distally to extensible and rubbery proximally. Collagen is the principal tensile polymer as determined by fiber x-ray diffraction, however, the mechanical properties of typical tendon are poorly matched with those of byssal threads.

Structural and dynamic studies of the adsorption behavior of small molecules within and on the surface of porous systems are of great interest for designing biocompatible and sustainable biodevices. Porous vycor glass is an ideal system for such studies because of its well characterized hydrophilic network of cylindrical pores. Moreover, the pore sizes in vycor are directly comparable to pore sizes of systems like macromolecules and biological membranes. Molecular dynamics simulations of vycor-water interactions as well as some structural data from neutron scattering and diffraction experiments are available. Close to the pore surface the number of water-water hydrogen bonds is reduced, and compensated by vycor-water hydrogen bonds. Insight into the behavior of water molecules in those confined geometries could provide necessary and relevant information for designing biodevices for tissue generation, as well as about contamination- and inflammation affinities of implant materials.

Here we present an Atomic Force Microscopy (AFM) study of vycor at different levels of surface hydration.

Dental adhesives are bonded to a wet surface of live tissue, dentin. There are two important stages of surface preparation for adhesive application: 1) Etching the tooth structure creating a layer of collagen network free from mineral, and 2) Applying a primer, in this case a water solution of hydrophilic monomer of 2-hydroxyethylmethacrylate (ScotchBond Multipurpose®, 3M). During the priming stage, the resin monomer should penetrate the fibril network and coat collagen fibrils. Subsequent adhesive resin penetration into the collagen network (hybrid layer) is not uniform, influenced by the presence of residual water and degree of resin polymerization. Dependence of bond strength of dental adhesives on time or intensity of light activation is well known, but no direct microscopic observations of the dynamics of polymerization were performed. This was due to the fact that during specimen dehydration for conventional SEM the polymerization process is still proceeding and residual monomer could be washed out.

Tissues are characterized by highly distinctive microarchitecture and overall form that are critical to tissue specific function. with increasing interest in tissue engineering to create surrogates for human tissues and organs, innovative techniques have been developed to generate scaffolds that display form, and microarchitecture mimicking physiological structures. While this artifice may generate pre-programmed shapes, it fails to take advantage of the inherent ability of cells to organize themselves and their surroundings. The generation of mechanical forces by the cellular cytoskeleton plays a critical role in the organization of matrix and of cellular colonies. The anchorage of the cytoskeleton to a substrate is essential for cellular tractional processes. in tissues, cells are anchored in a stationary, 3-D network of collagen in a highly characteristic spatial arrangement. This spatial order arises from vectorial deposition of matrix and its subsequent tractional organization by cells. The junction of the cell binding domain of collagen with specific integrin receptors, and the cells’ cytoskeleton comprises the apparatus for the interconversion of mechanical and biochemical energy.