Portable, low-cost NMR with laser-lathe lithography produced microcoils

Nuclear Magnetic Resonance (NMR) is unsurpassed in its ability to non-destructively probe chemical identity. Portable, low-cost NMR sensors would enable on-site identification of potentially hazardous substances, as well as the study of samples in a variety of industrial applications. Recent developments in RF microcoil construction (i.e. coils much smaller than the standard 5mm NMR RF coils), have dramatically increased NMR sensitivity and decreased the limits-of-detection (LOD). We are using advances in laser pantographic microfabrication techniques, unique to LLNL, to produce RF microcoils for field deployable, high sensitivity NMR-based detectors. This same fabrication technique can be used to produce imaging coils for MRI as well as for standard hardware shimming or "ex-situ" shimming of field inhomogeneities typically associated with inexpensive magnets. This paper describes a portable NMR system based on the use of a 2 kg hand-held permanent magnet, laser-fabricated microcoils, and a compact spectrometer. The main limitations for such a system are the low resolution and sensitivity associated with the low field values and quality of small permanent magnets, as well as the lack of large amounts of sample of interest in most cases. The focus of the paper is on the setting up of this system, initial results, sensitivity measurements, discussion of the limitations and future plans. The results, even though preliminary, are promising and provide the foundation for developing a portable, inexpensive NMR system for chemical analysis. Such a system will be ideal for chemical identification of trace substances on site.     

Three-dimensional microscopy with phase-shifting digital holography

We applied phase-shifting digital holography to microscopy by deriving the complex amplitude of light scattered from microscopic three-dimensional objects through a microscope objective by video camera recording, phase-shifting analysis, and computer reconstruction. This method requires no mechanical movement and provides a flexible display and quantitative evaluation of the reconstructed images. A theory of image formation and experimental verification with specimens are described.     

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 in vivo scanning microscopy with inertia-free focus control

The acquisition of high-resolution images in three dimensions is of utmost importance for the morphological and functional investigation of biological tissues. Here, we present a laser scanning two-photon microscope with remote and motionless control of the focus position. The movement of the excitation spot along the propagation direction is achieved by shaping the laser wavefront with a spatial light modulator. Depending on the optical properties of the objective in use, this approach allows z movements in a range of tens to hundreds of micrometers with small changes of the point spread function. We applied this technique for the three-dimensional (3D) imaging of fluorescent cells in the mouse neocortex in vivo. The presented system bypasses the limitations of microscopes based on moving objectives, enabling high-resolution inertia-free 3D imaging.     

Determination of sample temperature and temperature stability with 129Xe NMR

The (129)Xe chemical shift of xenon dissolved in isotropic liquids is very sensitive to solvent density, which in turn is dependent on the sample temperature. Therefore, the (129)Xe chemical shift can be used as the basis of a thermometer for measuring actual sample temperatures in NMR experiments. Good accuracy can be achieved, but the thermometer is particularly useful in monitoring temperature stability. In the present case, carbon tetrachloride (CCl(4)), ethylbromide (C(2)H(5)Br), and deuterated chloroform (CDCl(3)) were chosen as solvents because of their large thermal expansion coefficient.     

Short-TE projection reconstruction NMR microscopy of trabecular bone.

The aim of this study was to assess the potential of projection reconstruction (PR) NMR microscopy in the quantitative evaluation of trabecular bone architecture. Short-TE PR spin-echo microimages were acquired at 7.05 T on normal bone explants. The main structural parameters such as bone volume fraction (BVF), trabecular thickness (Tb.Th.) and trabecular separation (Tb.Sp.) were obtained from the 3D microimages using the method of directed secants. Quantitative structural data were then compared with those derived from conventional spin-echo microimages. Our study indicates that projection reconstruction NMR microscopy promises to be more accurate than the conventional FTI method in the analysis of trabecular bone.     

Three-dimensional triple-resonance NMR Spectroscopy of isotopically enriched proteins. 1990

Four new and complementary three-dimensional triple-resonance experiments are described for obtaining complete backbone ¹H, ¹³C, and ¹⁵N resonance assignments of proteins uniformly enriched with ¹³C and ¹⁵N. The new methods all rely on ¹H detection and use multiple magnetization transfers through well-resolved one-bond J couplings. Therefore, the 3D experiments are sensitive and permit relatively rapid recording of 3D spectra (l–2 days) for protein concentrations on the order of 1 mM. One experiment (HNCO) correlates the amide ¹H and ¹⁵N shifts with the ¹³C shift of the carbonyl resonance of the preceding amino acid. A second experiment (HNCA) correlates the intraresidue amide ¹H and ¹⁵N shifts with the Cα chemical shift. This experiment often also provides a weak correlation between the amide NH and ¹⁵N resonances of one amino acid and the Ca resonance of the preceding amino acid. A third experiment (HCACO) correlates the Hα and Cα shifts with the intraresidue carbonyl shift. Finally, a 3D relay experiment, HCA(CO)N, correlates Ha and Cal resonances of one residue with the ¹⁵N frequency of the succeeding residue. The principles of these experiments are described in terms of the operator formalism. To optimize spectral resolution, special attention is paid to removal of undesired J splittings in the 3D spectra. Technical details regarding the implementation of these triple-resonance experiments on a commercial spectrometer are also provided. The experiments are demonstrated for the protein calmodulin (16.7 kDa).     

Three-dimensional fourier transform NMR imaging. High-resolution chemical-shift-resolved planar imaging

A conventional high-resolution NMR spectrometer has been adapted for a three-dimensional imaging experiment which involves two spatial coordinates plus chemical shift. One dimension has far greater digital resolution than the other two, and when it is used to encode the chemical shifts it is possible to obtain separate slice images showing the distribution of each chemical species within the imaging plane. The method is illustrated using a tube phantom containing ethanol and water, in this case the ethanol gives sufficiently narrow finewidths that it is possible to obtain a separate image from the individual transitions of each multiplet.     

Fluorescence confocal polarizing microscopy: Three-dimensional imaging of the director

Much of the modern understanding of orientational order in liquid crystals (LCs) is based on polarizing microscopy (PM). A PM image bears only two-dimensional (2D) information, integrating the 3D pattern of optical birefringence over the path of light. Recently, we proposed a technique to image 3D director patterns by fluorescence confocal polarizing microscopy (FCPM). The technique employs the property of LC to orient the fluorescent dye molecules of anisometric shape, added in small quantities to the LC. In LC, smooth director deformations do not alter mass density of the material. Thus the density of dye is also uniform across the sample, except, perhaps, near the surfaces or at the cores of topological defects. In polarized light, the measured fluorescence signal is determined by the spatial orientation of the molecules rather than by dye concentration (as in regular biological samples stained with tissue-specific dyes). The contrast is enhanced when both excitation and detection of fluorescence light are performed in polarized light. This short review describes the essence of FCPM technique and illustrates some of its applications, including imaging of Frederiks electric-field induced effect in a nematic LC and defects such as dislocations in cholesteric LCs.     

Velocity and diffusion imaging in dynamic NMR microscopy

The imposition of resolution gradients in a pulsed-gradient spin-echo (PGSE) NMR sequence induces motionally dependent phase and amplitude modulation in the image, a technique which we have termed dynamic NMR microscopy. Fourier analysis of this modulation gives a dynamic displacement profile for each pixel which can then be analyzed to obtain velocity and diffusion maps. The application of this method at high spatial resolution is motivated by a desire to measure vascular flow in living plants and variations in molecular self-diffusion under the influence of velocity shear in narrow capillaries. The theory of dynamic NMR microscopy is presented and potential artifacts discussed, including the effect of slice selection gradients, PGSE gradient nonuniformity, and specific problems associated with the measurement of self-diffusion in the presence of velocity gradients. It is demonstrated that a double-echo PGSE pulse sequence can be used to restore coherent phase shifts associated with steady-state flow, and examples of self-diffusion maps and signed velocity maps from sequences of phase-encoded images obtained by projection reconstruction are given. This method has been applied at 20,um transverse resolution in laminar capillary flow.     

Purpose-designed probes and their applications for dynamic NMR microscopy in an electromagnet.

The electromagnet provides a favorable environment for certain applications of NMR microscopy. These include plant imaging experiments and measurements of slow molecular diffusion, where high magnetic field gradients for the pulsed gradient spin echo (PGSE) technique are required. In this paper, two probes designed specifically for these two applications are described. In the first case, the open space within the probe has been maximized in order to incorporate environmental support systems for the plant, while in the second the smallest possible PGSE gradient coil former has been used to maximize the gradient strength. Examples are given of Dynamic NMR Microscopy experiments on a castor bean stem and on poly(ethylene oxide)/water solutions under shear thinning conditions.     

A novel application of NMR microscopy: measurement of water diffusion inside bioadhesive bonds.

The self-diffusion coefficient of water (D) inside bioadhesive bonds formed by dry and prehydrated hydrophilic matrices has been spatially resolved using nuclear magnetic resonance (NMR) microscopy. One-dimensional profiles showing the variation of D inside bioadhesive bonds were calculated from nine diffusion-weighted profiles obtained immediately after bond formation and every 5 min for 30 min. The resulting data indicated that the hydration state of a hydrophilic matrix can significantly and dramatically influence the dynamics of water movement inside a bioadhesive bond.     

Three-dimensional color photon counting microscopy using Bayesian estimation with adaptive priori information

In this Letter, we propose a novel three-dimensional(3D) color microscopy for microorganisms under photonstarved conditions using photon counting integral imaging and Bayesian estimation with adaptive priori information. In photon counting integral imaging, 3D images can be visualized using maximum likelihood estimation(MLE). However, since MLE does not consider a priori information of objects, the visual quality of 3D images may not be accurate. In addition, the only grayscale image can be reconstructed. Therefore, to enhance the visual quality of 3D images, we propose photon counting microscopy using maximum a posteriori with adaptive priori information. In addition, we consider a wavelength of each basic color channel to reconstruct 3D color images. To verify our proposed method, we carry out optical experiments.     

Three-dimensional molecular imaging using mass spectrometry and atomic force microscopy

We combine imaging ToF-SIMS depth profiling and wide area atomic force microscopy to analyze a test structure consisting of a 300 nm trehalose film deposited on a Si substrate and pre-structured by means of a focused 15-keV Ga⁺ ion beam. Depth profiling is performed using a 40-keV C₆₀⁺ cluster ion beam for erosion and mass spectral data acquisition. A generic protocol for depth axis calibration is described which takes into account both lateral and in-depth variations of the erosion rate. By extrapolation towards zero analyzed lateral area, an “intrinsic” depth resolution of about 8 nm is found which appears to be characteristic of the cluster-surface interaction process.     

Improving NMR sensitivity in room temperature and cooled probes with dipolar ions

The response of inverse triple resonance cold and conventional probes to ionic strength has been compared under a variety of conditions relevant to protein NMR. Increasing the salt concentration degrades probe performance in terms of sensitivity, and the effect is more severe for cold probes and with increasing magnetic field strength. This is especially noticeable for experiments that involve a spin lock or decoupling, where sensitivity losses compared with pure water can be more than 2-fold. We have investigated the use of glycine as a substitute for salt as a supporting solute for proteins, and we show that it has a minimal effect on probe tuning or performance. Readily available d5-Gly is a useful co-solute for protein NMR, especially at high magnetic field strengths and on cold probes, as it maintains solubility while not degrading probe performance.     

Variable magnetic field and temperature magnetic force microscopy

Magnetic force microscopy (MFM) studies of epitaxial MnAs films on GaAs(001) have been performed as a function of the applied magnetic field and the sample temperature. For this purpose, we combined a stable variable-temperature sample stage with a compact magnet assembly to fit a commercial magnetic force microscope. In order to keep the thermal drift that affects MFM measurements low, we employed a permanent magnet that can be rotated in a yoke assembly guiding the magnetic flux to the sample. PACS 68.37.Rt; 68.35.Rh; 75.70.-i; 75.70.Kw     

Hadamard encoding with surface coils for high SNR MR spectroscopy.

The advantages of Hadamard over phase encoding in magnetic resonance spectroscopy (MRS) applied with surface coils in the direction perpendicular to the coil are demonstrated experimentally. With the recently introduced, time-shifted adiabatic pulses, the application of Hadamard encoding with surface coils results with almost ideal point spread function for pixels up to a distance of a radius from the coil. Comparison to phase encoding with equal region of interest size shows the significant advantage of Hadamard encoding in slice sharpness, overlapping, and spatial contamination. In addition, since there is no aliasing in Hadamard space, the total experimental time for the same region of interest is much shorter. We conclude that the hybrid of Hadamard encoding in the direction perpendicular to the coil and phase encoding in other directions is the method of choice to obtain reliable high signal to noise ratio MRS in vivo.     

High temperature NMR in vanadium: Electron paramagnetism and atomic diffusion effects

We present preliminary results on Knight shift T₁ and T₂ measurements between 300 and 1700 K. We have observed the motional narrowing due to the self diffusion as well as a strong quadrupolar contribution to T₁ due to the diffusion of interstitial gases. K varies from 0.58 to 0.63%. This temperature dependence is shown to be mainly due to an intrinsic temperature effect. The high temperature Korringa like behaviour of T₁ with T₁T = 1.05 sec. K is explained in terms of the temperature dependence of the d spin contribution.     

Three-dimensional mapping of cholinergic molecules by confocal laser scanning microscopy in sea urchin larvae.

Confocal laser scanning microscopy (CLSM) was used to examine molecules related to the cholinergic neurotransmission system and detected at all the larval stages of Paracentrotus lividus, by histochemical and immunohistochemical methods. CLSM, providing spatial resolution of the cells located both at the larval surface and at depth, allows a complete mapping in a three-dimensional volumetric frame. At early larval stages acetylcholinesterase- as well as choline acetyltransferase-like molecules were found mainly in the gut wall cells, and along the ciliary bands of the arms, together with muscarinic acetylcholine receptors. At perimetamorphic stages, cholinergic molecules were present in the ciliate strands along the arms, in the larval body and in the rudiment. At metamorphosis, positivity to cholinergic molecules translocated to the juvenile, where a high frequency of mAChR- and ChAT-like positive cells was found.