Levels of mineral nutrients in fresh- and frozen bulk hydrated biological specimens: A comparison of EDS data collected in the environmental SEM and a conventional cryo-SEM

Energy-dispersive X-ray microanalysis (EDS) was compared in fresh- and frozen bulk hydrated tissues using the Environmental SEM (ESEM) and conventional cryo-SEM, respectively. Analyses of globoid inclusions of Eucalyptus calophylla seed from two soil types demontsrated that higher levels of cations (K, Ca, Mg, Al, Fe, Mn, Cu, Zn) occurred in seeds from soils containing higher levels of Al, while EDS-detectable levels of S and P were dependent upon the techniques utilised. Cumulative changes in ESEM-EDS-detectable levels of S and P were characterised by collecting cumulative spectra from nutrient standards and compared with those for K. Progressive increases in K occurred and were consistent with an enriching effect. Levels of S and P increased during early analysis (40–60 sec live time) and decreased thereafter. The semi-conductive nature of biological samples, the loss of anions and gain of cations from the net negatively-charged electron interaction volume contributed to an electrochemical bias. These local modifications in fluid chemistry were reversible. Dehydration effects also occurred in stable, “wet‘ samples. These differences indicated that EDS in ESEM may be limited to cations rather than anions, and that changes in fluid electrochemistry and dehydration may affect the level and distribution of elements.     

Dynamic nuclear polarization at 9T using a novel 250 GHz gyrotron microwave source. 2003

In this communication, we report enhancements of nuclear spin polarization by dynamic nuclear polarization (DNP) in static and spinning solids at a magnetic field strength of 9 T (250 GHz for g = 2 electrons, 380 MHz for ¹H). In these experiments, ¹H enhancements of up to 170 ± 50 have been observed in 1-¹³C-glycine dispersed in a 60:40 glycerol/water matrix at temperatures of 20 K; in addition, we have observed significant enhancements in ¹⁵N spectra of unoriented pf1-bacteriophage. Finally, enhancements of ∼17 have been obtained in two-dimensional ¹³C–¹³C chemical shift correlation spectra of the amino acid U–¹³C, ¹⁵N-proline during magic angle spinning (MAS), demonstrating the stability of the DNP experiment for sustained acquisition and for quantitative experiments incorporating dipolar recoupling. In all cases, we have exploited the thermal mixing DNP mechanism with the nitroxide radical 4-amino-TEMPO as the paramagnetic dopant. These are the highest frequency DNP experiments performed to date and indicate that significant signal enhancements can be realized using the thermal mixing mechanism even at elevated magnetic fields. In large measure, this is due to the high microwave power output of the 250 GHz gyrotron oscillator used in these experiments.     

1H detected 1H,15N correlation spectroscopy in rotating solids

We describe new correlation experiments suitable for determining long-range 1H-1H distances in 2H,15N-labeled peptides and proteins. The approach uses perdeuteration together with back substitution of exchangeable protons during sample preparation as a means of attenuating the strong 1H-1H dipolar couplings that broaden 1H magic angle spinning (MAS) spectra of solids. In the approach described here, we retain 100% of the 1H sensitivity by labeling and detecting all exchangeable sites. This is in contrast to homonuclear multiple pulse decoupling sequences that are applied during detection and that compromise sensitivity because of the requirement of sampling between pulses. As a result 1H detection provides a gain in sensitivity of >5 compared to the 15N detected version of the experiment (at a MAS frequency of 13.5kHz). The pulse schemes make use of the favorable dispersion of the amide 15Ns resonances in the protein backbone. The experiments are demonstrated on a sample of the uniformly 2H,15N-labeled dipeptide N-Ac-Val-Leu-OH and are analogous to the solution-state suite of HSQC-NOESY experiments. In this compound the 1H amide linewidths at 750MHz vary from approximately 0.67 ppm at omega(r)/2pi approximately 5kHz to approximately 0.20 ppm at omega(r)/2pi approximately 30kHz, indicating that useful resolution is available in the 1H spectrum via this approach. Since the experiments circumvent the problem of dipolar truncation in the 1H-1H spin system, they should make it possible to measure long-range distances in a uniformly labeled environment. Thus, we expect the experiments to be useful in constraining the global fold of a protein.     

Deterministic schedules for robust and reproducible non-uniform sampling in multidimensional NMR

We show that a simple, general, and easily reproducible method for generating non-uniform sampling (NUS) schedules preserves the benefits of random sampling, including inherently reduced sampling artifacts, while removing the pitfalls associated with choosing an arbitrary seed. Sampling schedules are generated from a discrete cumulative distribution function (CDF) that closely fits the continuous CDF of the desired probability density function. We compare random and deterministic sampling using a Gaussian probability density function applied to 2D HSQC spectra. Data are processed using the previously published method of Spectroscopy by Integration of Frequency and Time domain data (SIFT). NUS spectra from deterministic sampling schedules were found to be at least as good as those from random schedules at the SIFT critical sampling density, and significantly better at half that sampling density. The method can be applied to any probability density function and generalized to greater than two dimensions.     

Continuous-Wave Operation of a 460-GHz Second Harmonic Gyrotron Oscillator

We report the regulated continuous-wave (CW) operation of a second harmonic gyrotron oscillator at output power levels of over 8 W (12.4 kV and 135 mA beam voltage and current) in the TE(0,6,1) mode near 460 GHz. The gyrotron also operates in the second harmonic TE(2,6,1) mode at 456 GHz and in the TE(2,3,1) fundamental mode at 233 GHz. CW operation was demonstrated for a one-hour period in the TE(0,6,1) mode with better than 1% power stability, where the power was regulated using feedback control. Nonlinear simulations of the gyrotron operation agree with the experimentally measured output power and radio-frequency (RF) efficiency when cavity ohmic losses are included in the analysis. The output radiation pattern was measured using a pyroelectric camera and is highly Gaussian, with an ellipticity of 4%. The 460-GHz gyrotron will serve as a millimeter-wave source for sensitivity-enhanced nuclear magnetic resonance (dynamic nuclear polarization) experiments at a magnetic field of 16.4 T.     

Modelling resonances of the standing body exposed to vertical whole-body vibration: Effects of posture

Lumped parameter mathematical models representing anatomical parts of the human body have been developed to represent body motions associated with resonances of the vertical apparent mass and the fore-and-aft cross-axis apparent mass of the human body standing in five different postures: ‘upright’, ‘lordotic’, ‘anterior lean’, ‘knees bent’, and ‘knees more bent’. The inertial and geometric parameters of the models were determined from published anthropometric data. Stiffness and damping parameters were obtained by comparing model responses with experimental data obtained previously.The principal resonance of the vertical apparent mass, and the first peak in the fore-and-aft cross-axis apparent mass, of the standing body in an upright posture (at 5–6 Hz) corresponded to vertical motion of the viscera in phase with the vertical motion of the entire body due to deformation of the tissues at the soles of the feet, with pitch motion of the pelvis out of phase with pitch motion of the upper body above the pelvis. Upward motion of the body was in phase with the forward pitch motion of the pelvis. Changing the posture of the upper body had minor effects on the mode associated with the principal resonances of the apparent mass and cross-axis apparent mass, but the mode changed significantly with bending of the legs. In legs-bent postures, the principal resonance (at about 3 Hz) was attributed to bending of the legs coupled with pitch motion of the pelvis in phase with pitch motion of the upper body. In this mode, extension of the legs was in phase with the forward pitch motion of the upper body and the upward vertical motion of the viscera.