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.

A widely accepted tenet of biomaterials research is that the initial step following contact of a synthetic material with blood is the rapid adsorption of plasma proteins. The composition of this adsorbed protein layer is dependent on a variety of factors, including the surface properties of the implant material and the nature of the adsorbing proteins, and the composition and function of this protein layer is important in the subsequent biological response and ultimately the success or failure of the implanted material. While a great amount of effort has gone into developing structure/function responses for implanted biomaterials, there is still much about the molecular level interactions to be determined. We utilized atomic force microscopy (AFM) to investigate the molecular-level interactions of proteins with model biomaterial substrates. The AFM is unique in that it offers the opportunity to characterize interfacial environments, determine material properties, measure protein/surface interaction forces, and visualize the tertiary structure of adsorbed proteins.

The primary function of lung surfactant monolayers (LS) is to control and reduce surface tension in the lung permitting easy breathing. to do this, the monolayer must maintain coverage of the entire lung surface during the rapid surface area changes which accompany breathing. We use a magnetic needle viscometer and optical microscopy to determine the relationship between monolayer shear viscosity, which controls its two-dimensional flow properties, and synthetic lung surfactant composition. We show that, within a specific range, palmitic acid interacts with DPPC/POPG to make lung surfactant rigid at low surface tension (or high surface pressure) and fluid at high surface tension. This enables the monolayer to attain near zero surface tensions during compression yet respread easily at high surface tension More palmitic acid makes the monolayer more rigid and eventually increases the collapse surface tension while reducing the readsorption rate at high surface tension.

Micro-patterned surfaces are of interest in biology and chemistry due to the ability to confine functional sample materials to specific areas. If patterns are visible, either through optical microscopy, or topographically, with scanning probe microscopy, investigating samples in a manner that is cheap and accessible to most laboratories is possible. Examples include micropatterning cells in tissue engineering, and protein micro-array analysis. We have created a micro-patterned surface with tailored optical and topographic properties. These were investigated using the microscopy techniques, confocal microscopy (CM), atomic force microscopy (AFM) and near field scanning optical microscopy (NSOM). AFM and CM were used to investigate different aspects of the micro-pattern. to confirm the properties of the micro-pattern, we show the advantages of NSOM in investigating surfaces with both optical and topographic properties simultaneously. Micro-patterns were fabricated using a soft lithography technique, micro-contact printing, where reagents are ‘stamped’ upon substrates using an elastomeric moulding of a micro-template.

Although scanning probe microscopy (SPM) can generate images of surface topography, this class of techniques is exceptionally valuable in its ability to provide quantitative and chemically specific information about biomaterial surfaces with high spatial definition. Since engineered biomaterials are designed to deliver chemically defined information, often arrayed in specific geometries, tools that can characterize such materials are needed.

A few years ago, we demonstrated how the atomic force microscope (AFM) could precisely distinguish between each of the four nucleotide bases that comprise DNA, measure the nucleotide-nucleotide force of interaction and spatially localize that information on a surface (1). in particular, we found that the nucleotide bases could self-assemble on gold. The assembly process was imaged using scanning tunneling microscopy (STM) and this led to an understanding of the structure of the assembled film. The assembled film structure was further characterized using electron spectroscopy for chemical analysis (ESCA) and secondary ion mass spectrometry (SIMS).

The ability of ESEM to image insulating and/or moist specimens without the need for the removal of volatile components or the application of a conductive coating has significantly increased the potential range of experiments and observations that can be performed at the high resolution of electron microscopy. Such a technological advance has particularly important implications for the study of soft matter, complex fluids and biological specimens.

An important area of study to which ESEM can be readily applied is that of materials for biomedical applications. Hydroxyapatite (HA) ceramics and HA/polymer composites (e.g. HAPEX™) are being developed for use as synthetic scaffolds in bone tissue engineering. The bioactivity of these materials is dependent upon such factors as phase composition, chemical composition, surface activity, crystallinity and microstructure. Using ESEM it is possible to obtain surface-sensitive, specimen-dependent secondary electron images (in the absence of specimen coating), yielding potentially new perspectives on microstructure to complement information derived from other techniques.

The geochemistry and toxicity of chromium is controlled by its valence state. Chromium is a redox active 3d transition metal with a wide range (− to +6) of possible oxidation states; however only Cr(III) and Cr(VI) are stable. Hexavalent Cr(VI) can be readily reduced to the trivalent state by Fe2+, S2−, organic compounds, wetland plants, and several species of microorganisms. The associated mechanisms of Cr(VI) reduction are technologically and biologically important because they convert a toxic, mobile element into a less toxic, immobile form.

Reduction of Cr(VI) by the bacterium, Shewanella oneidensis (previously classified Shewanella putrefaciens strain MR-1), was studied by absorption spectrophotometry and in-situ, environmental cell (EC) - transmission electron microscopy (TEM) coupled with electron energy loss spectroscopy (EELS). Shewanella oneidensis (S. oneidensis), a gram-negative, facultative bacterium is capable of respiring aerobically and anaerobically using a variety of compounds, including O2, Fe(III), Mn(IV), NO2−, NO3−, SO2, SO32−, thiosulfate (S2O32−), trimethyamine oxide, fumarate, U(VI), and Cr(VI) as terminal electron acceptors.

The elastin-mimetic tri-block copolymer, C5, with the polypeptide repeat [((VPAVG)(IPAVG)3(VPAVG))m((VPGVG)2(VPGEG)(VPGVG)3)n((VPAVG)(IPAVG)3(VPAVG))m] was produced via protein hyper-expression utilizing a pET plasmid and Escherichia coli (BL21- Gold(DE3)) as the bacterial host. The gene coding for the polypeptide was produced by a multimerization protocol that has undergone recent modifications. A sample of the elastinmimetic block copolymer, C5, was prepared as a 20 to 25 percent protein solutions in sterile ddH20. Approximately 10 μL of the solution was pipetted into 3 mm gold planchets (Balzers BU 012 130T), which had been pre-equilibrated to 4°C in an isothermal environmental cooler (Torrey Pines Scientific). The temperature of the cooler was raised to 20°C and allowed to stabilize for 10 minutes. The solidified samples, were plunge-frozen in liquid ethane at its melting point (−183°C); the samples were stored in liquid nitrogen (LN2). A sample was transferred to and mounted on the precooled (∽−170°C) Oxford CT-3500 cryo-stage held in the cryo-preparation chamber.

Death and disability due to cardiovascular disease and congenital anomalies remains a significant health problem in the United States. Despite improvements in detection, patient management, surgery and preventative medicine, the quality of life for people who suffer from cardiovascular dysfunction has a major impact on our society. The intact heart is an elaborate three-dimensional structure that insures the orderly propagation of electrical signals coordinating the contraction and relaxation of the ventricular wall. Localized loss of muscular tissue as a result of congenital defect or disease process alters this structural arrangement and impairs overall cardiac function. Conventional surgical techniques cannot begin to adequately restore the subtle structural and functional relationships in the heart. The ability to construct a tissue-engineered prosthesis composed of cardiac muscle cells in a collagen-based scaffold may potentially offer a superior alternative to currently available surgical techniques.

A tissue engineered myocardium must have the following components: First, it must develop and maintain the correct cellular phenotype as well as a functioning contractile apparatus of parallel myofibrils, Second it must be able to form gap junctions within itself and with the native tissue and these gap junctions must be competent at conducting electrical pacing as well as other biochemical signals, and Third, it must be capable of participating in and contributing to the rhythmic contraction of normal myocardium as well as accommodate the changes in contraction frequency ubiquitous in the cardiac environment

Bone is known to alter its architecture, mass, composition, metabolic state, development and function in response to the effects of external forces applied to it, whether those forces be loading or unloading in nature (1). These adaptive changes by bone and the vertebrate skeletal system in general may be imparted by mechanical forces, gravity, buoyancy, or other such influences and manifest themselves in a number of ways. The skeleton of an animal, for example, may increase in mass through exercise and heightened activity, it may lose mass as a result of extended immobilization or weightlessness, or it may remodel during fracture repair processes. A mechanism explaining the adaptation by bone and the skeleton to the presence or absence of applied forces is not completely understood, but the changes are thought to occur ultimately at the cellular level of structure. Studies presented here have examined the relationship between forces and bone cell response in this context: Spaceflight and weightlessness have been utilized to investigate the influence of gravitational unloading on a model of cultured osteoblasts derived from normal embryonic chicken calvaria.

The development of a seamless, collagen-based vascular prosthetic scaffolding (< 4 mm I.D.) has been accomplished using an electrospinning fiber production technique (Patents Pending). Electrospinning is the deliberate application of the phenomenon of electrostatic spraying which occurs when electrical forces at the surface of the polymer solution overcome the surface tension, creating a splay. The splay produces fibers with diameters in the nano-scale range (<500 nm). The collagen-based scaffold production method (flexible, quick, and simple) utilizes the splaying of the collagen-based solution from a nozzle to a grounded rotating mandrel (4 mm O.D. cylindrical mandrel for the preliminary vascular tissue engineering). in splaying, the solvent evaporates creating nano-scale fibers of polymerized collagen (and elastin in some variations). The collagen fibers are collected on the mandrel forming a cylindrical tube (media equivalent scaffolding). to date, mixtures of collagen containing Type I and 80:20 Type l/Elastin have been utilized to produce vascular scaffolds of various thickness (50 - 2,000 microns).

The objective of this study is to develop a skeletal muscle prosthetic composed entirely of natural or otherwise bioresorbable materials that recapitulates the structure and function of intact muscle. in our fabrication strategy we begin by electrospinning (see Mathews et al., this volume) a hollow, cylindrical fascial sheath composed of poly-glycolic acid (PGA), poly-lactic acid (PLA), Type I collagen microfibers or a blend of these materials (figure 1). The resulting fascial sheath is a highly porous fabric composed of interwoven fibers that range from less than 1 micron to 2-5 microns in diameter (patents pending). Next, we isolate neonatal rat skeletal myoblasts by enzymatic dissociation. The dissociated tissue is subjected to an interval of differential adhesion to enrich the preparation in skeletal muscle myoblasts. The final cell pellet of myoblasts is then suspended at high density in Type I collagen. This suspension is polymerized at 37°C for 10-15 minutes and used to fill a fascial sheaths (patents pending).