Manipulation of gold nanoparticles: influence of surface chemistry, temperature, and environment (vacuum versus ambient atmosphere).

We have manipulated raw and functionalized gold nanoparticles (with a mean diameter of 25 nm) on silicon substrates with dynamic atomic force microscopy (AFM). Under ambient conditions, the particles stick to silicon until a critical amplitude is reached by the oscillations of the probing tip. Beyond that threshold, the particles start to follow different directions, depending on their geometry and adhesion to the substrate. Higher and lower mobility were observed when the gold particles were coated with methyl- and hydroxyl-terminated thiol groups, respectively, which suggests that the adhesion of the particles to the substrate is strongly reduced by the presence of hydrophobic interfaces. Under ultrahigh vacuum conditions, where the water layer is absent, the particles did not move, even when operating the atomic force microscope in contact mode. We have also investigated the influence of the temperature (up to 150 degrees C) and of the geometrical arrangement of the particles on the manipulation process. Whereas thermal activation has an important effect in enhancing the mobility of the particles, we did not find differences when manipulating ordered versus random distributions of particles.

Combinatorial electrochemical synthesis and characterization of tungsten-based mixed-metal oxides.

Automated systems for electrochemical synthesis and high-throughput screening of photoelectrochemical materials were developed and used to prepare tungsten-based mixed-metal oxides, W(n)O(m)M(x) [M = Ni, Co, Cu, Zn, Pt, Ru, Rh, Pd, and Ag], specifically for hydrogen production by photoelectrolysis of water. Two-dimensional arrays (libraries) of diverse metal oxides were synthesized by automated cathodic electrodeposition of the oxides on Ti foil substrates. Electrolytes for the mixed oxides were prepared from various metal salts added to a solution containing tungsten stabilized as a peroxo complex. Electrodeposition of the peroxo-stabilized cations gave rise to three distinguishable oxide groups: (1) mixed-metal oxides [Ni], (2) metal-doped tungsten oxides [Pt, Ru, Rh, Pd, Ag], and (3) metal-metal oxide composites [Co, Cu, Zn]. The oxides typically showed n-type semiconducting behavior. Automated measurement of photocurrent using a scanning photoelectrochemical cell showed the W-Ni mixed oxide had the largest relative zero bias photocurrent, particularly at a low Ni concentration (5-10 atomic percent Ni). Pt and Ru were also found to increase the photoactivity of bulk tungsten oxide at relatively low concentrations; however, at concentrations above 5 atomic percent, crystallization of WO(3) was inhibited and photoactivity was diminished. Addition of Co, Cu, and Zn to WO(3) was not found to improve the photoelectrochemical activity.

Chemically induced electronic excitations at metal surfaces.

The energy released in low-energy chemisorption or physisorption of molecules on metal surfaces is usually expected to be dissipated by surface vibrations (phonons). Theoretical descriptions of competing electronic excitations are incomplete, and experimental observation of excited charge carriers has been difficult except at energies high enough to eject electrons from the surface. We observed reaction-induced electron excitations during gas interactions with polycrystalline silver for a variety of species with adsorption energies between 0.2 and 3.5 electron volts. The probability of exciting a detectable electron increases with increasing adsorption energy, and the measured time dependence of the electron current can be understood in terms of the strength and mechanism of adsorption.

31P NMR spectroscopy of developing cartilage produced from chick chondrocytes in a hollow-fiber bioreactor.

31P NMR was used to measure the concentrations and spin-lattice relaxation times of phosphorus-containing metabolites in neocartilage developing in an NMR-compatible hollow-fiber bioreactor over four weeks. Separate studies were performed for tissue developing from chondrocytes taken from the proximal and the distal sternum of the chick embryo. The metabolite ratio beta-ATP/Pi did not change significantly with development (proximal: beta-ATP/Pi = 0.38+/- 0.12 at one week, beta-ATP/Pi = 0.44+/-0.07 at four weeks, P< 0.63; distal: beta-ATP/Pi = 0.39+/-0.05 at one week, beta-ATP/Pi = 0.66+/- 0.26 at four weeks, P<0.28). ATP spin-lattice relaxation times were found to be comparable to those in muscle and brain tissue (proximal: T(1)(beta-ATP) = 0.5+/-0.06 sec at one week, T(1)(beta-ATP) = 0.4+/- 0.01 sec at four weeks; distal: T(1)(beta-ATP) = 0.3+/-0.12 sec at one week, T(1)(beta-ATP) = 0.4+/-0.04 sec at four weeks). A large increase in the spin-lattice relaxation time of inorganic phosphate, from 1.2+/-0.13 sec to 3.8+/-0.04 sec (P<0.0001) over four weeks of growth, was observed in tissue developing from chondrocytes harvested from the proximal sternum. No comparable increase in T(1)(Pi) was found in tissue developing from chondrocytes harvested from the distal portion of the sternum, which ossifies later in vivo.

Cartilage formation in a hollow fiber bioreactor studied by proton magnetic resonance microscopy.

The ideal in vitro system for investigating the regulation of cartilage formation and maintenance would allow for three-dimensional tissue growth, a wide range of biochemical interventions, and non-destructive evaluation. We have developed a hollow fiber bioreactor (HFBR) system which meets these criteria. After injection with embryonic chick sternal chondrocytes, neocartilage is elaborated around the hollow fibers, reaching a thickness of up to a millimeter after four weeks of growth. This process was monitored over time with nuclear magnetic resonance (NMR) microimaging and correlative biochemical and histologic analyses. Tissue volume and cellularity increased greatly during development. This was accompanied by changes in magnetic resonance properties consistent with increased macromolecular content. Further, tissue heterogeneity, observed as regional variations in cell size in histologic sections, was also observed in quantitative NMR images.

A radiobiological probe for simultaneous NMR spectroscopy and 192Ir gamma irradiation of Saccharomyces cerevisiae.

A novel nuclear magnetic resonance (NMR) spectroscopy probe was designed and constructed for the study of transient metabolic changes in cellular systems during exposure to ionizing radiation. The probe incorporated a bioreactor, a radiation source, and a radiofrequency detection circuit tunable between 100 and 300 MHz for in vivo NMR spectroscopy of 23Na, 13C, and 31P at 11.7 Tesla. The bioreactor system allowed perfusion, oxygenation, and temperature control of cultured cells during irradiation and while performing simultaneous spectroscopic experiments. The concentric design of the bioreactor allowed for the insertion of a 192Ir gamma ray source (E(gamma) = 370 keV) to allow irradiation of the bioreactor system during the acquisition of NMR spectra. Initial results of 31P spectra obtained during simultaneous gamma irradiation of Saccharomyces cerevisiae at approximately 8 Gy/hr show rapid decreases in adenosine triphosphate (ATP) and polyphosphate at the onset of irradiation followed by a slow recovery of polyphosphate.

Magnetic resonance microscopy studies of cation diffusion in cartilage.

The diffusion of copper ions in bovine nasal cartilage (BNC), a dense connective tissue, was investigated to further the understanding of ion transport in charged biopolymer systems. Using an inversion-recovery null-point imaging technique, it was found that the diffusion rate of divalent copper ions into cartilage was significantly lower in normal BNC than in BNC in which the matrix fixed charges had been reduced by enzymatic digestion or acid neutralization. In normal cartilage, counterion diffusion was not well described by a simple Fickian process, likely owing to the high charge density of the constituent molecules. In contrast, in both digested and acid neutralized BNC, counterion diffusion appeared Fickian. Features of the ion transport process were modeled using a diffusion equation which included a linear sorption term to account for cation binding. The diffusion coefficient of copper in cartilage increased with decreasing matrix fixed charge and was constant for reservoir concentrations up to 30 mM. The activation energy for the diffusion of copper into BNC was determined to be 34.5 kJ/mol.

Assay for bacteria in porous media by diffusion-weighted NMR.

In this work, an NMR technique capable of detecting bacterial cells and measuring the cell density in suspension and in porous media has been developed. It is based on the pulsed-field-gradient technique and relies on the fact that extracellular water diffuses freely while intracellular water is completely restricted by the relatively impermeable cell wall of the bacterium. At high wave vectors, the signal from extracellular water is completely suppressed while the signal from intracellular water is comparatively unaffected. This technique has been applied to the mapping of bacterial distributions in porous media. This method is presented as a non-destructive, real-time technique for biomass characterization within laboratory column and flow cell experiments, and possibly for monitoring in situ bioremediation.

Experimental study of temperature distributions and thermal transport during radiofrequency current therapy of the intervertebral disc.

STUDY DESIGN: The authors measured the temperature changes within the human intervertebral disc during transient intradisc heating with a radiofrequency current lesion generator. OBJECTIVES: The study was undertaken to evaluate the efficacy of thermal denervation of the intervertebral disc from intradisc radiofrequency lesion treatment. SUMMARY OF BACKGROUND DATA: Intradisc radiofrequency heating has emerged recently as a nonoperative treatment for chronic lower back pain. However, no literature exists regarding the temperature distributions within the disc and the consequent efficacy of denervation. METHODS: Vertebral segments obtained at autopsy were instrumented with thermocouples, and intradisc heating was performed according to standard clinical protocols. The tip was maintained at 70 C and the temperature monitored at various distances from the tip. RESULTS: The temperature changes at distances further than 11 mm were insufficient to raise the tissue temperature to the 42 C needed for neuronal cell death. Using the thermal transient data, the authors calculated the thermal diffusivity of the human intervertebral disc and found it to vary from approximately 1.7 x 10(-7) +/- 0.4 x 10(-7) m2/sec in the disc from a 61-year-old man to approximately 4.5 x 10(-7) +/- 1.4 x 10(-7) m2/sec in the disc from a 32-year-old man. CONCLUSIONS: The authors concluded that the mechanism of observed clinical improvement from radiofrequency heating of intervertebral discs is not thermal denervation of the disc and that the physicochemical state of the disc is important to consider when designing a therapeutic heating protocol if thermal denervation is clinically desired.

Ion transport studies in calcium alginate gels by magnetic resonance microscopy.

Polyelectrolyte biopolymers such as calcium alginate are becoming increasingly important for the recovery of heavy metals from aqueous solutions. To understand the mechanism of ion transport in these biopolymer systems, the transport of copper ions into calcium alginate gels was investigated using proton nuclear magnetic resonance (NMR) microscopy. Copper ion transport was imaged using an inversion recovery technique which utilizes the paramagnetic effect of copper on water proton relaxation times. Diffusion experiments were performed in a diffusion cell designed to approximate a semi-infinite slab geometry at temperatures between 278 and 313 K using copper reservoir concentrations between 10 and 60 mM. The diffusion coefficient of copper in these gels was calculated from the NMR data to fit a combined diffusion-reaction model involving a diffusion term (D) and a kinetic binding term (k). At 23 degrees C, the diffusion coefficients in 1, 2, and 3% (w/v) gels were 3.1 x 10(10), 2.0 x 10(10), and 1.4 x 10(10) m2/s, respectively. The activation energy for diffusion in the 2% (w/v) gel was 28 kJ/mol.

Characterization of polyion counterion interactions in cartilage by 23Na NMR relaxation.

Nuclear quadrupole relaxation is a sensitive measure of electrolyte environments. We used the relaxation of 23Na to probe mobile ion-matrix interactions and the electrostatic structure of the polyelectrolyte extracellular matrix of cartilage. Specifically, we measured spin-lattice and spin-spin relaxation times of 23Na in bovine nasal cartilage at 132 MHz under several conditions. Matrix fixed charge density was reduced by protonating anionic sites and by matrix digestion with trypsin and the relaxation times compared to controls. Under all conditions studied, measured longitudinal relaxation was monoexponential with values ranging from 16-32 msec. Transverse relaxation exhibited biexponential behavior in all cartilage samples with a fast component in the range of 2 to 5 ms and a slow component between 16 and 53 ms. Reduction in matrix fixed charge density in all cases led to a decrease in the relaxation rates. The results suggest a two-site model for Na+ ions in cartilage and a relaxation mechanism involving both polyion segmental motion and counterion diffusion. In the context of ion condensation theory, the implication of a two-site model is that the mean polyion-polyion spacing may be less than 0.7 nm. The mean polyion-counterion spacings were estimated by calculating correlation times and quadrupole coupling constants. These spacings were found to be 0.5-0.7 nm.

Activity of creatine kinase in a contracting mammalian muscle of uniform fiber type.

We investigated whether the creatine kinase-catalyzed phosphate exchange between PCr and gamma ATP in vivo equilibrated with cellular substrates and products as predicted by in vitro kinetic properties of the enzyme, or was a function of ATPase activity as predicted by obligatory "creatine phosphate shuttle" concepts. A transient NMR spin-transfer method was developed, tested, and applied to resting and stimulated ex vivo muscle, the soleus, which is a cellularly homogeneous slow-twitch mammalian muscle, to measure creatine kinase kinetics. The forward and reverse unidirectional CK fluxes were equal, being 1.6 mM.s-1 in unstimulated muscle at 22 degrees C, and 2.7 mM.s-1 at 30 degrees C. The CK fluxes did not differ during steady-state stimulation conditions giving a 10-fold range of ATPase rates in which the ATP/PCr ratio increased from approximately 0.3 to 1.6. The observed kinetic behavior of CK activity in the muscle was that expected from the enzyme in vitro in a homogeneous solution only if account was taken of inhibition by an anion-stabilized quaternary dead-end enzyme complex: E.Cr.MgADP.anion. The CK fluxes in soleus were not a function of ATPase activity as predicted by obligatory phosphocreatine shuttle models for cellular energetics.

Time-independent point-spread function for NMR microscopy.

The resolution of NMR microscopy is analyzed in terms of the point-spread function, PSF(r), and the equivalent k space modulation transfer function, MTF(k). The analysis is developed for NMR spin warp and projection reconstruction imaging experiments; however, the framework provided is quite general. Incoherent spin motion is analyzed to predict what limits, if any, on spatial resolution are imposed by diffusion. Previous estimates of diffusion limits at 1-5 microns were developed for specific imaging techniques, typically using a mean displacement argument. Although qualitatively correct, the quantitative predictions represent practical rather than fundamental limits. It is shown that diffusion-dependent "blurring" can be made arbitrarily small and that the practical limits are less stringent than previously thought. A major point illustrated by the PSF-MTF formulation is that the irreversible loss of coherence by randomly diffusing spins occurs faster than the physical displacement, thereby reducing their effect considerably on the frequency or phase of the net detected signal. The irreversible loss of signal due to diffusive motion will contribute to and possibly dominate the signal-to-noise limit of resolution. The resolution as measured by the width of the PSF and MTF for diffusion is shown to be independent of the signal acquisition time, and their functional forms allow selection of microscopic imaging parameters. An example of a three-dimensional spin-warp image of a green algae cell is shown with resolution of approximately 16 microns x 13 microns x 10 microns.

Three-dimensional NMR microscopy: improving SNR with temperature and microcoils.

It is widely held that the spatial resolution achievable by NMR microscopic imaging is limited in biological systems by diffusion to approximately 1-5 microns. However, these estimates were developed for specific imaging techniques and represent practical rather than fundamental limits. NMR imaging is limited by the signal-to-noise ratio (SNR). Diffusion effects on spatial resolution can be made arbitrarily small in principle by increasing the gradient strength. The exponential signal attenuation from random spin motion in a gradient, however, will reduce the signal far below the noise level when the voxel size is reduced much below 5 microns. Two factors can be optimized to improve the SNR: (1) the inductive linkage between microscopic samples and the detection apparatus and (2) the temperature of the rf probe. In this work, the filling factor was optimized using inductors with diameters less than 1 mm. It is furthermore shown that probe circuit cooling results in significant improvements in SNR, whereas cooling of the preamplifier is of little value when proper noise matching between the resonant circuit and preamplifier is accomplished. Using three-dimensional Fourier imaging techniques, we have obtained images of single-cell organisms with spatial resolution of approximately 6 microns. Practical limitations include mechanical stability of the apparatus, thermal shielding between the sample and probe, and the magnetic susceptibility of the sample.

Maturational increase in mouse brain creatine kinase reaction rates shown by phosphorus magnetic resonance.

In-vivo phosphorus fluxes in the reaction catalyzed by creatine kinase (CK) were measured in brains of mice from 3 to 40 days of age using high-field (8.45 T) phosphorus magnetic resonance and the saturation transfer technique. This technique gives the ratio of chemical flux to reactant concentration directly and allows the calculation of pseudo-rate constants for the forward direction from PC to ATP (kf) and for the reverse direction (kr). The spin-lattice relaxation times (T1) for phosphocreatine (PC) and for the nucleoside triphosphate (NTP) nuclei, estimated by the progressive saturation technique, did not change during this age period. The PC concentration doubled relative to the NTP concentration over the first month of life. The kf and the flux of phosphorus nuclei in the forward direction increased 2- to 3-fold in the very narrow time period from 12 to 15 days of age. Brain phosphorus flux from PC to ATP thus increased 4- to 6-fold in the first month of life. An increase at least that large occurred in the reverse direction, but the kr could not be measured consistently in the younger animals using the saturation transfer technique. Phosphorus fluxes were equal in the forward and reverse directions in the mature brain. The capacity to increase rates of glycolysis and tissue respiration in response to increased energy demand appears in the same narrow age period as the increase in CK-catalyzed reaction rates in the developing rodent brain. We propose that these coincident changes in brain energy metabolism reflect the maturation of mechanisms for coupling cell energy production to rapid changes in energy requirements.

Chemical exchange magnetic resonance imaging (CHEMI).

Systems investigated with NMR spectroscopy are sometimes heterogeneous with respect to chemical composition, rates of chemical exchange, and other properties influencing magnetic resonance parameters. A method was developed to spatially encode reaction kinetic information and produce NMR images sensitive to chemical exchange. A modified spin-echo pulse sequence was used to allow chemical shift-selective imaging and chemical exchange encoding. 1H and 31P images with microscopic resolution were obtained which yielded chemical exchange as a function of position. Chemical exchange images of the base-catalyzed proton exchange of acetylacetone and of the enzyme-catalyzed 31P transfer between PCr and ATP were obtained at 8.4 T in phantoms at 360 and 146 MHz, respectively. These images demonstrate a means of investigating kinetic heterogeneity and compartmentalization of reactions that are important in the study of both living and non-living systems.

NMR instrumentation and hardware available at present and in the future.

Advances in magnetic resonance hardware and instrumentation have facilitated the rapid development of NMR imaging. Systems based on superconducting, resistive, and permanent magnets have been commercially introduced and are now available in a wide range of field strengths. Excellent images are now routinely obtained in fields from 0.1 to 2.0 Tesla (T). It is now clear, however, that obtaining high-quality images requires much more than a high-strength magnet. Improved radiofrequency transmission and receiving subsystems and new special-purpose coils have been essential for high-sensitivity imaging. User interfaces and computational hardware have borrowed from x-ray CT rapid signal processing and image display capabilities. Developments in systems technology are making possible improved image quality and information content together with increased speed. Presented here is a discussion of the basic function and interconnections of the key elements of a complete NMR imaging system.