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).

When an electron beam interacts with a solid target a number of interactions occur which produce electrons, x-rays and light. Typically in an electron microprobe analyser (EPMA) both the electron and x-ray signals are collected for analysis and imaging. However, if the EPMA is equipped with an optical spectrometer then all three signals can be collected. Commonly, the optical or CathodoLuminescence (CL) spectrometer is a monochromator type and can only collect a single frequency or small range of frequencies at a time. Simultaneous collection of the complete visible spectrum is not possible. The collection optics associated with the spectrometer often must be moved into place to start collection, this then obscures the other detectors and prevents simultaneous collection. At CSIRO Minerals an optical spectrometer has been integrated into a JEOL 8900R EPMA and allows simultaneous collect of all light, x-rays and electron signals. This form of mapping, termed Holistic Mapping, has significant advantages over traditional mapping in that it removes the need to have a priori knowledge about what the important frequencies are that will provide the solution to the problem at hand.

Oil immersion lenses have been used in optical microscopy for a long time. The light’s wavelength is decreased by the oil’s index of refraction n and this reduces the minimum spot size. Additionally, the oil medium allows a larger collection angle, thereby increasing the numerical aperture. The SIL is based on the same principle, but offers more flexibility because the higher index material is solid. in particular, SILs can be deployed in cryogenic environments. Using a hemispherical glass the spatial resolution is improved by a factor n with respect to the resolution obtained with the microscope’s objective lens alone. The improvement factor is equal to n2 for truncated spheres.

As shown in Fig. 1, the hemisphere SIL is in contact with the sample and does not affect the position of the focal plane. The focused rays from the objective strike the lens at normal incidence, so that no refraction takes place.

The production of semiconductor chips and packaging materials involves the use of a wide array of materials, from solvents and polymers, to photoresists, to metal and dielectric layers, to conductive and thermal pastes. Characterization of these materials, both in raw form and as formulated for in-process use, is integral to successful use of them in microelectronic manufacturing. Physical and chemical analytical techniques are employed to determine parameters such as composition, cure state, and interface chemistry. More often than not, it is the successful combination of complementary analyses which provide the complete understanding of material parameters needed.

This paper reports the use of Raman microprobe spectroscopy as a characterization technique for microelectronic materials. Several examples will be given, illustrating the type of information which can be obtained and the complementary use of Raman spectroscopy in conjunction with other analytical techniques such as Fourier transform infrared spectroscopy (FTIR) and x-ray photoelectron spectroscopy (XPS) to characterize microelectronic packaging materials and organic materials used in semiconductor manufacture.

FTIR and Raman microanalysis may be used as a powerful combination to determine the identity, and hence infer the source, of contaminant particles which diminish yields of semiconductor components and devices. The complimentarity of these techniques arises from the underlying spectroscopic selection rules.

Vibrational spectroscopic techniques are commonly used to characterize the molecular structure of bulk organic materials. These bulk materials typically represent purified fractions of components which may be further investigated with various classical instrumental techniques such as Differential Scanning Calorimetry (DSC), Nuclear Magnetic Resonance (NMR) spectroscopy, UV-Vis spectroscopy. However, these classical technique may have limited value for the interrogation of small impure particles or materials of limited quantity(ng.).

Elemental techniques such as Scanning Electron Microscopy coupled to Energy Dispersive Spectroscopy are enhanced by the specificity of FTIR Microprobe Spectroscopy and Raman Microprobe Spectroscopy which are now used in process laboratories to characterize and identify particulate and thin film residues with the intent of device yield enhancement.

The analytical needs in semiconductor technology have developed according to the increasing demands imposed by size and complexity. Early optical visualization, localization and counting of ever-smaller particles extended almost immediately to their morphological and chemical characterization. This evolution was due to the shifting balance from quality control to root-cause analysis. Root-cause analysis requires a deeper analytical approach to the characterization of the observed defects. Atomic composition may suffice at times as an answer. However, this information may be ambiguous and inadequate. Therefore, deeper chemical information regarding molecular structure and crystallinity of the particle is now required.

The analytical capabilities and versatilities associated with optical spectroscopy inevitably have led to their increasing applications in the semiconductor industry. in particular, the complementary Infrared and Raman spectroscopies on one hand provide huge insight into the molecular structure and crystallinity of the particles, on the other hand Raman and Photoluminescence (PL) spectroscopies are easily coupled to systems providing particle visualization and localization.

With the advent of easy to use analytical and research instrumentation the Raman technique has finally become accepted and is now widely used by pharmaceutical and biomedical companies. Applications range from simple polymorphic crystal form and protein studies to Raman mapping studies of active/excipient distribution and association that give a better understanding of drug dissolution and effectiveness. Examples of such work will be discussed.

More recently with the growing confidence in the power of Raman spectroscopy pharmaceutical and biomedical companies have teamed with instrument companies to develop specific Raman tools. One example of this is the development of a Raman mutiwell/microarray analyzer. High Throughput screening (HTS) is the latest approach in analytical technology. Used particularly in the pharmaceutical industry and in the life science laboratories, this method enables systematic screening of drug design and high throughput analysis of batch production. Combinatorial chemistry is a statistical approach in synthetic chemistry.

Many drugs are known to crystallize in different polymorphic forms or as solvates. Solubility, melting point, density, hardness, optical properties, vapor pressure, and a host of other physical properties may all vary with polymorphic form. Not only do the various crystal structures of a given pharmaceutical compound affect the efficacy of the drug, but they may also carry enormous legal implications. Much product revenue can depend upon the identification and patent protection of certain polymorphic forms. Thus, the control of crystallization is a very important process parameter, and techniques such as X-ray crystallography, infrared spectroscopy, Raman spectroscopy, and polarized light microscopy are routinely used in the characterization of crystalline drugs.

This presentation will involve the investigation of a variety of pharmaceutical polycrystalline films using infrared (IR) spectroscopic imaging. Preliminary data was collected using a conventional FT-IR microscope with visible polarized light capabilities. Correlating data was then collected using a commercially available IR imaging microscope.

In work reported previously we, and the McCreery Group at Ohio State University, have proposed the use of glass luminescence standards for the calibration of the Raman spectral intensity. We now have a program underway that is directed at the development of such secondary standards for the calibration of the Raman instrument response function. The first Raman intensity standard to be certified and issued by NIST this year will be for the relative intensity calibration of Raman spectra excited at 785 nm. in general, for any laser excitation wavelength, the calibration procedure is based on the use of luminescent glasses, doped with either transition metal or rare earth ions, that will furnish structureless, broad-band emission spectra upon laser excitation, over any chosen Raman range. For any selected glass standard, the intensity of the luminescence spectrum is calibrated versus the radiant output of a NIST-calibrated white light source, thereby transferring the white light calibration onto the output emission of the luminescent glass.

Single point near-infrared (NIR) spectroscopy is used extensively for characterizing raw materials and finished products in a wide variety of industries: polymers, paper, film, pharmaceuticals, paintings and coatings, food and beverages, agricultural products. As advanced industrial materials become more complex, their functionality is often determined by the spatial distribution of their discrete sample constituents. However, conventional single point NIR spectroscopy cannot adequately probe the interrelationship between the spatial distribution of sample components with the physical properties of the sample. to fully characterize these samples, it is necessary to probe simultaneously spatial and chemical heterogeneity and correlate these properties with sample characteristics.

Recently, we have developed a novel NIR imaging spectrometer that can deliver spatially resolved chemical information very rapidly. in contrast to conventional, single point NIR spectrometers, the imaging system uses an infrared focal-plane array (FPA) to collect up to 76,800 complete spectra, one for each pixel on the array, in approximately one minute.