Evaluation of double-tuned single-sided planar microcoils for the analysis of small 13 C enriched biological samples using 1 H-13 C 2D heteronuclear correlation NMR spectroscopy.

Microcoils provide a cost-effective approach to improve detection limits for mass-limited samples. Single-sided planar microcoils are advantageous in comparison to volume coils, in that the sample can simply be placed on top. However, the considerable drawback is that the RF field that is produced by the coil decreases with distance from the coil surface, which potentially limits more complex multi-pulse NMR pulse sequences. Unfortunately, 1 H NMR alone is not very informative for intact biological samples due to line broadening caused by magnetic susceptibility distortions, and 1 H-13 C 2D NMR correlations are required to provide the additional spectral dispersion for metabolic assignments in vivo or in situ. To our knowledge, double-tuned single-sided microcoils have not been applied for the 2D 1 H-13 C analysis of intact 13 C enriched biological samples. Questions include the following: Can 1 H-13 C 2D NMR be performed on single-sided planar microcoils? If so, do they still hold sensitivity advantages over conventional 5 mm NMR technology for mass limited samples? Here, 2D 1 H-13 C HSQC, HMQC, and HETCOR variants were compared and then applied to 13 C enriched broccoli seeds and Daphnia magna (water fleas). Compared to 5 mm NMR probes, the microcoils showed a sixfold improvement in mass sensitivity (albeit only for a small localized region) and allowed for the identification of metabolites in a single intact D. magna for the first time. Single-sided planar microcoils show practical benefit for 1 H-13 C NMR of intact biological samples, if localized information within ~0.7 mm of the 1 mm I.D. planar microcoil surface is of specific interest.

Comprehensive Multiphase NMR Probehead with Reduced Radiofrequency Heating Improves the Analysis of Living Organisms and Heat-Sensitive Samples.

Comprehensive multiphase (CMP) NMR, first described in 2012, combines all of the hardware components necessary to analyze all phases (solid, gel, and solution) in samples in their natural state. In combination with spectral editing experiments, it can fully differentiate phases and study the transfer of chemical species across and between phases, providing unprecedented molecular-level information in unaltered natural systems. However, many natural samples, such as swollen soils, plants, and small organisms, contain water, salts, and ionic compounds, making them electrically lossy and susceptible to RF heating, especially when using high-strength RF fields required to select the solid domains. While dedicated reduced-heating probes have been developed for solid-state NMR, to date, all CMP-NMR probes have been based on solenoid designs, which can lead to problematic sample heating. Here, a new prototype CMP probe was developed, incorporating a loop gap resonator (LGR) for decoupling. Temperature increases are monitored in salt solutions analogous to those in small aquatic organisms and then tested in vivo on Hyalella azteca (freshwater shrimp). In the standard CMP probe (solenoid), 80% of organisms died within 4 h under high-power decoupling, while in the LGR design, all organisms survived the entire test period of 12 h. The LGR design reduced heating by a factor of ∼3, which allowed 100 kHz decoupling to be applied to salty samples with generally ≤10 °C sample heating. In addition to expanding the potential for in vivo research, the ability to apply uncompromised high-power decoupling could be beneficial for multiphase samples containing true crystalline solids that require the strongest possible decoupling fields for optimal detection.

Expanding current applications and permitting the analysis of larger intact samples by means of a 7 mm CMP-NMR probe.

Comprehensive multiphase NMR combines the ability to study and differentiate all phases (solids, gels, and liquids) using a single NMR probe. The general goal of CMP-NMR is to study intact environmental and biological samples to better understand conformation, organization, association, and transfer between and across phases/interfaces that may be lost with conventional sample preparation such as drying or solubilization. To date, all CMP-NMR studies have used 4 mm probes and rotors. Here, a larger 7 mm probehead is introduced which provides ∼3 times the volume and ∼2.4 times the signal over a 4 mm version. This offers two main advantages: (1) the additional biomass reduces experiment time, making 13C detection at natural abundance more feasible; (2) it allows the analysis of larger samples that cannot fit within a 4 mm rotor. Chicken heart tissue and Hyalella azteca (freshwater shrimp) are used to demonstrate that phase-based spectral editing works with 7 mm rotors and that the additional biomass from the larger volumes allows detection with 13C at natural abundance. Additionally, a whole pomegranate seed berry (aril) and an intact softgel capsule of hydroxyzine hydrochloride are used to demonstrate the analysis of samples too large to fit inside a conventional 4 mm CMP probe. The 7 mm version introduced here extends the range of applications and sample types that can be studied and is recommended when 4 mm CMP probes cannot provide adequate signal-to-noise (S/N), or intact samples are simply too big for 4 mm rotors.

5-Axis CNC Micromilling for Rapid, Cheap, and Background-Free NMR Microcoils.

The superior mass sensitivity of microcoil technology in nuclear magnetic resonance (NMR) spectroscopy provides potential for the analysis of extremely small-mass-limited samples such as eggs, cells, and tiny organisms. For optimal performance and efficiency, the size of the microcoil should be tailored to the size of the mass-limited sample of interest, which can be costly as mass-limited samples come in many shapes and sizes. Therefore, rapid and economic microcoil production methods are needed. One method with great potential is 5-axis computer numerical control (CNC) micromilling, commonly used in the jewelry industry. Most CNC milling machines are designed to process larger objects and commonly have a precision of >25 µm (making the machining of common spiral microcoils, for example, impossible). Here, a 5-axis MiRA6 CNC milling machine, specifically designed for the jewelry industry, with a 0.3 µm precision was used to produce working planar microcoils, microstrips, and novel microsensor designs, with some tested on the NMR in less than 24 h after the start of the design process. Sample wells could be built into the microsensor and could be machined at the same time as the sensors themselves, in some cases leaving a sheet of Teflon as thin as 10 µm between the sample and the sensor. This provides the freedom to produce a wide array of designs and demonstrates 5-axis CNC micromilling as a versatile tool for the rapid prototyping of NMR microsensors. This approach allowed the experimental optimization of a prototype microstrip for the analysis of two intact adult Daphnia magna organisms. In addition, a 3D volume slotted-tube resonator was produced that allowed for 2D 1H-13C NMR of D. magna neonates and exhibited 1H sensitivity (nLODω600 = 1.49 nmol s1/2) close to that of double strip lines, which themselves offer the best compromise between concentration and mass sensitivity published to date.

Soil Organic Matter in Its Native State: Unravelling the Most Complex Biomaterial on Earth.

Since the isolation of soil organic matter in 1786, tens of thousands of publications have searched for its structure. Nuclear magnetic resonance (NMR) spectroscopy has played a critical role in defining soil organic matter but traditional approaches remove key information such as the distribution of components at the soil-water interface and conformational information. Here a novel form of NMR with capabilities to study all physical phases termed Comprehensive Multiphase NMR, is applied to analyze soil in its natural swollen-state. The key structural components in soil organic matter are identified to be largely composed of macromolecular inputs from degrading biomass. Polar lipid heads and carbohydrates dominate the soil-water interface while lignin and microbes are arranged in a more hydrophobic interior. Lignin domains cannot be penetrated by aqueous solvents even at extreme pH indicating they are the most hydrophobic environment in soil and are ideal for sequestering hydrophobic contaminants. Here, for the first time, a complete range of physical states of a whole soil can be studied. This provides a more detailed understanding of soil organic matter at the molecular level itself key to develop the most efficient soil remediation and agricultural techniques, and better predict carbon sequestration and climate change.

From Spill to Sequestration: The Molecular Journey of Contamination via Comprehensive Multiphase NMR.

Comprehensive multiphase NMR is a novel NMR technique that permits all components (solutions, gels, and solids) to be studied in unaltered natural samples. In this study a wide range of CMP-NMR interaction and editing-based experiments are combined to follow contaminants (pentafluorophenol (PFP) and perfluorooctanoic acid (PFOA)) from the solution state (after a spill) through the gel-state and finally into the true solid-state (sequestered) in an intact water-swollen soil. Kinetics experiments monitoring each phase illustrate PFOA rapidly transfers from solution to the solid phase while for PFP the process is slower with longer residence times in the solution and gel phase. Interaction-based experiments reveal that PFOA enters the soil via its hydrophobic tails and selectively binds to soil microbial protein. PFP sorption shows less specificity exhibiting interactions with a range of gel and solid soil components with a preference toward aromatics (mainly lignin). The results indicate that in addition to more traditional measurements such as Koc, other factors including the influence of the contaminant on the soil-water interface, specific biological interactions, soil composition (content of lignin, protein, etc.) and physical accessibility/swellability of soil organic components will likely be central to better explaining and predicting the true behavior of contaminants in soil.

Comprehensive multiphase NMR: a promising technology to study plants in their native state.

Nuclear magnetic resonance (NMR) spectroscopy is arguably one the most powerful tools to study the interactions and molecular structure within plants. Traditionally, however, NMR has developed as two separate fields, one dealing with liquids and the other dealing with solids. Plants in their native state contain components that are soluble, swollen, and true solids. Here, a new form of NMR spectroscopy, developed in 2012, termed comprehensive multiphase (CMP)-NMR is applied for plant analysis. The technology composes all aspects of solution, gel, and solid-state NMR into a single NMR probe such that all components in all phases in native unaltered samples can be studied and differentiated in situ. The technology is evaluated using wild-type Arabidopsis thaliana and the cellulose-deficient mutant ectopic lignification1 (eli1) as examples. Using CMP-NMR to study intact samples eliminated the bias introduced by extraction methods and enabled the acquisition of a more complete structural and metabolic profile; thus, CMP-NMR revealed molecular differences between wild type (WT) and eli1 that could be overlooked by conventional methods. Methanol, fatty acids and/or lipids, glutamine, phenylalanine, starch, and nucleic acids were more abundant in eli1 than in WT. Pentaglycine was present in A. thaliana seedlings and more abundant in eli1 than in WT.

High-resolution paramagnetically enhanced solid-state NMR spectroscopy of membrane proteins at fast magic angle spinning.

Magic angle spinning nuclear magnetic resonance (MAS NMR) is well suited for the study of membrane proteins in membrane mimetic and native membrane environments. These experiments often suffer from low sensitivity, due in part to the long recycle delays required for magnetization and probe recovery, as well as detection of low gamma nuclei. In ultrafast MAS experiments sensitivity can be enhanced through the use of low power sequences combined with paramagnetically enhanced relaxation times to reduce recycle delays, as well as proton detected experiments. In this work we investigate the sensitivity of (13)C and (1)H detected experiments applied to 27 kDa membrane proteins reconstituted in lipids and packed in small 1.3 mm MAS NMR rotors. We demonstrate that spin diffusion is sufficient to uniformly distribute paramagnetic relaxation enhancement provided by either covalently bound or dissolved CuEDTA over 7TM alpha helical membrane proteins. Using paramagnetic enhancement and low power decoupling in carbon detected experiments we can recycle experiments ~13 times faster than under traditional conditions. However, due to the small sample volume the overall sensitivity per unit time is still lower than that seen in the 3.2 mm probe. Proton detected experiments, however, showed increased efficiency and it was found that the 1.3 mm probe could achieve sensitivity comparable to that of the 3.2 mm in a given amount of time. This is an attractive prospect for samples of limited quantity, as this allows for a reduction in the amount of protein that needs to be produced without the necessity for increased experimental time.

NMR flow tube for online NMR reaction monitoring.

In this paper we describe the development of a 5 mm NMR flow tube that can be used in a standard 5 mm NMR probe, enabling the user to conduct experiments on flowing samples or, more specifically, on flowing reaction mixtures. This enables reaction monitoring or kinetic experiments to be conducted by flowing reaction mixtures from a reaction vessel to detection in the coil area of the NMR, without the need for a specialized flow NMR probe. One of the key benefits of this flow tube is that it provides flexibility to be used across a range of available spectrometers of varying magnetic field strengths with a standard 5 mm probe setup. The applicability of this flow tube to reaction monitoring is demonstrated using the reaction of p-phenylenediamine and isobutyraldehyde to form the diimine product.

Rapid parameter optimization of low signal-to-noise samples in NMR spectroscopy using rapid CPMG pulsing during acquisition: application to recycle delays.

A method is presented that combines Carr-Purcell-Meiboom-Gill (CPMG) during acquisition with either selective or nonselective excitation to produce a considerable intensity enhancement and a simultaneous loss in chemical shift information. A range of parameters can theoretically be optimized very rapidly on the basis of the signal from the entire sample (hard excitation) or spectral subregion (soft excitation) and should prove useful for biological, environmental, and polymer samples that often exhibit highly dispersed and broad spectral profiles. To demonstrate the concept, we focus on the application of our method to T(1) determination, specifically for the slowest relaxing components in a sample, which ultimately determines the optimal recycle delay in quantitative NMR. The traditional inversion recovery (IR) pulse program is combined with a CPMG sequence during acquisition. The slowest relaxing components are selected with a shaped pulse, and then, low-power CPMG echoes are applied during acquisition with intervals shorter than chemical shift evolution (RCPMG) thus producing a single peak with an SNR commensurate with the sum of the signal integrals in the selected region. A traditional (13)C IR experiment is compared with the selective (13)C IR-RCPMG sequence and yields the same T(1) values for samples of lysozyme and riverine dissolved organic matter within error. For lysozyme, the RCPMG approach is ~70 times faster, and in the case of dissolved organic matter is over 600 times faster. This approach can be adapted for the optimization of a host of parameters where chemical shift information is not necessary, such as cross-polarization/mixing times and pulse lengths.

Magnetic resonance microscopy of human and porcine neurons and cellular processes.

With its unparalleled ability to safely generate high-contrast images of soft tissues, magnetic resonance imaging (MRI) has remained at the forefront of diagnostic clinical medicine. Unfortunately due to resolution limitations, clinical scans are most useful for detecting macroscopic structural changes associated with a small number of pathologies. Moreover, due to a longstanding inability to directly observe magnetic resonance (MR) signal behavior at the cellular level, such information is poorly characterized and generally must be inferred. With the advent of the MR microscope in 1986 came the ability to measure MR signal properties of theretofore unobservable tissue structures. Recently, further improvements in hardware technology have made possible the ability to visualize mammalian cellular structure. In the current study, we expand upon previous work by imaging the neuronal cell bodies and processes of human and porcine α-motor neurons. Complimentary imaging studies are conducted in pig tissue in order to demonstrate qualitative similarities to human samples. Also, apparent diffusion coefficient (ADC) maps were generated inside porcine α-motor neuron cell bodies and portions of their largest processes (mean=1.7 ± 0.5 µm²/ms based on 53 pixels) as well as in areas containing a mixture of extracellular space, microvasculature, and neuropil (0.59 ± 0.37 µm²/ms based on 33 pixels). Three-dimensional reconstruction of MR images containing α-motor neurons shows the spatial arrangement of neuronal projections between adjacent cells. Such advancements in imaging portend the ability to construct accurate models of MR signal behavior based on direct observation and measurement of the components which comprise functional tissues. These tools would not only be useful for improving our interpretation of macroscopic MRI performed in the clinic, but they could potentially be used to develop new methods of differential diagnosis to aid in the early detection of a multitude of neuropathologies.

In-situ molecular-level elucidation of organofluorine binding sites in a whole peat soil.

The chemical nature of xenobiotic binding sites in soils is of vital importance to environmental biogeochemistry. Interactions between xenobiotics and the naturally occurring organic constituents of soils are strongly correlated to environmental persistence, bioaccessibility, and ecotoxicity. Nevertheless, because of the complex structural and chemical heterogeneity of soils, studies of these interactions are most commonly performed indirectly, using correlative methods, fractionation, or chemical modification. Here we identify the organic components of an unmodified peat soil where some organofluorine xenobiotic compounds interact using direct molecular-level methods. Using (19)F→(1)H cross-polarization magic angle spinning (CP-MAS) nuclear magnetic resonance (NMR) spectroscopy, the (19)F nuclei of organofluorine compounds are used to induce observable transverse magnetization in the (1)H nuclei of organic components of the soil with which they interact after sorption. The observed (19)F→(1)H CP-MAS spectra and dynamics are compared to those produced using model soil organic compounds, lignin and albumin. It is found that lignin-like components can account for the interactions observed in this soil for heptafluoronaphthol (HFNap) while protein structures can account for the interactions observed for perfluorooctanoic acid (PFOA). This study employs novel comprehensive multi-phase (CMP) NMR technology that permits the application of solution-, gel-, and solid-state NMR experiments on intact soil samples in their swollen state.

Diffusion tensor microscopy in human nervous tissue with quantitative correlation based on direct histological comparison.

Thanks to its proven utility in both clinical and research applications, diffusion tensor tractography (DTT) is regularly employed as a means of delineating white-matter tracts. While successful efforts have been made to validate tractographic predictions, comparative methods which would permit the validation of such predictions at microscopic resolutions in complex biological tissues have remained elusive. In a previous study, we attempted to validate for the first time such predictions at microscopic resolutions in rat and pig spinal cords using a semi-quantitative analysis method. In the current study, we report improved quantitative analysis methods that can be used to determine the accuracy of DTT through comparative histology and apply these techniques for the first time to human tissue (spinal cord) samples. Histological images are down-sampled to resolutions equivalent to our magnetic resonance microscopy (MRM) and converted to binary maps using an automated thresholding tool. These maps (n=3) are co-registered to the MRM allowing us to quantify the agreement based on the number of pixels which contain tracts common to both imaging datasets. In our experiments, we find that-on average-89% of imaging pixels predicted by DTT to contain in-plane white-matter tract structure correspond to physical tracts identified by histology. In addition, angular analysis comparing the orientation of fiber tracts measured in histology to their corresponding in-plane primary eigenvector components is presented. Thus, as well as demonstrating feasibility in human tissue, we report a robust agreement between imaging datasets taken at microscopic resolution and confirm the primary eigenvector's role as a fundamental parameter with clear physical correlates in the microscopic regime.

Cellular-level diffusion tensor microscopy and fiber tracking in mammalian nervous tissue with direct histological correlation.

Magnetic resonance imaging techniques have literally revolutionized neuroimaging with an unprecedented ability to explore tissue structure and function. Over the last three decades, the sensitivity and array of imaging techniques available have improved providing ever finer structural information and more sensitive functional techniques. Among these methods, diffusion imaging techniques have facilitated the generation of fiber-tract maps of the brain enabling an examination of issues related to brain structure and neural connectivity. Despite the potential utility of the techniques described, validation has not yet been achieved on biological samples. Recently, using newly developed surface microcoils on small samples at high magnetic fields, we demonstrated the ability of MR microscopy to image individual neurons in mammalian brain tissue. In the present work, we combine MR microscopy with the highest resolution (15microm) fiber tracking yet reported and demonstrate the accuracy of the fiber tract maps with direct histological validation. Thus it becomes possible to delineate fiber structure in tissues at the cellular level. A semi-quantitative approach was used to estimate the cell overlap fraction (cOF) and fiber tract overlap fraction (tOF), with cOFs of 94%, 92% and 100%, and tOFs of 84%, 86% and 100%, in rat cervical, rat lumbar, and pig spinal cord tissue, respectively. These methods provide a way to directly validate fiber tracking techniques with histology so that contemporary tracking techniques may be compared and refined using the microstructural details of a biological template as a ground truth.

Ex vivo Comprehensive Multiphase NMR of whole organisms: A complementary tool to in vivo NMR.

Nuclear Magnetic Resonance (NMR) spectroscopy is a non-invasive analytical technique which allows for the study of intact samples. Comprehensive Multiphase NMR (CMP-NMR) combines techniques and hardware from solution state and solid state NMR to allow for the holistic analysis of all phases (i.e. solutions, gels and solids) in unaltered samples. This study is the first to apply CMP-NMR to deceased, intact organisms and uses 13C enriched Daphnia magna (water fleas) as an example. D. magna are commonly used model organisms for environmental toxicology studies. As primary consumers, they are responsible for the transfer of nutrients across trophic levels, and a decline in their population can potentially impact the entire freshwater aquatic ecosystem. Though in vivo research is the ultimate tool to understand an organism's most biologically relevant state, studies are limited by conditions (i.e. oxygen requirements, limited experiment time and reduced spinning speed) required to keep the organisms alive, which can negatively impact the quality of the data collected. In comparison, ex vivo CMP-NMR is beneficial in that; organisms do not need oxygen (eliminating air holes in rotor caps and subsequent evaporation); samples can be spun faster, leading to improved spectral resolution; more biomass per sample can be analyzed; and experiments can be run for longer. In turn, higher quality ex vivo NMR, can provide more comprehensive NMR assignments, which in many cases could be transferred to better understand less resolved in vivo signals. This manuscript is divided into three sections: 1) multiphase spectral editing techniques, 2) detailed metabolic assignments of 2D NMR of 13C enriched D. magna and 3) multiphase biological changes over different life stages, ages and generations of D. magna. In summary, ex vivo CMP-NMR proves to be a very powerful approach to study whole organisms in a comprehensive manner and should provide very complementary information to in vivo based research.

Magnetic resonance microscopy of mammalian neurons.

Magnetic resonance imaging (MRI) is now a leading diagnostic technique. As technology has improved, so has the spatial resolution achievable. In 1986 MR microscopy (MRM) was demonstrated with resolutions in the tens of micrometers, and is now an established subset of MRI with broad utility in biological and non-biological applications. To date, only large cells from plants or aquatic animals have been imaged with MRM limiting its applicability. Using newly developed microsurface coils and an improved slice preparation technique for correlative histology, we report here for the first time direct visualization of single neurons in the mammalian central nervous system (CNS) using native MR signal at a resolution of 4-8 microm. Thus MRM has matured into a viable complementary cellular imaging technique in mammalian tissues.

MRI of human hair.

INTRODUCTION: Hair care for humans is a major world industry with specialised tools, chemicals and techniques. Studying the effect of hair care products has become a considerable field of research, and besides mechanical and optical testing numerous advanced analytical techniques have been employed in this area. In the present work, another means of studying the properties of hair is added by demonstrating the feasibility of magnetic resonance imaging (MRI) of the human hair. MATERIALS AND METHODS: Established dedicated nuclear magnetic resonance microscopy hardware (solenoidal radiofrequency microcoils and planar field gradients) and methods (constant time imaging) were adapted to the specific needs of hair MRI. RESULTS: Images were produced at a spatial resolution high enough to resolve the inner structure of the hair, showing contrast between cortex and medulla. Quantitative evaluation of a scan series with different echo times provided a T*(2) value of 2.6 ms for the cortex and a water content of about 90% for hairs saturated with water. CONCLUSION: The demonstration of the feasibility of hair MRI potentially adds a new tool to the large variety of analytical methods used nowadays in the development of hair care products.

A closer look into DESIRE for NMR microscopy.

The major challenge of nuclear magnetic resonance (NMR) microscopy at a spatial resolution of a few micrometers is to obtain a sufficiently high signal-to-noise-ratio (SNR) within a reasonable measurement time. As a particular difficulty, molecular self-diffusion poses a serious limitation to true spatial resolution and SNR if conventional Fourier encoding techniques are used. Opposed to that, the alternative DESIRE (Diffusion Enhancement of SIgnal and REsolution) approach to NMR microscopy utilises diffusion to increase the SNR. Being a real-space imaging method, spatial localisation is accomplished by saturation pulses while diffusion continuously replaces the saturated by unsaturated spins. For this technique a signal enhancement of up to three orders of magnitude has been predicted and initial experimental data have provided a proof of principle. In the present work, a detailed investigation of one-dimensional (1D) DESIRE is presented including simulations of a real implementation of the method, a quantitative experimental analysis, and basic 1D imaging. The simulations reveal the importance and provide the means of ensuring the true spatial resolution for this particular way of localisation, enable the selection of useful experimental parameters, and predict the specific image contrast to be expected around barriers restricting diffusion. Experimental data are presented with resolutions down to 3 microm and DESIRE enhancement up to 25 that are in good agreement with the simulation results. In particular, 1D DESIRE imaging in a phantom confirms the expected signal drop close to barriers due to spatially restricted diffusion.

Gradient shimming with spectrum optimisation.

Shimming, i.e. homogenising the unavoidable distortion of the static magnetic field B0 in NMR spectroscopy, is still an annoying, time consuming task. Although compared with conventional manual or computerised search methods gradient shimming initiated a new era in terms of operation and efficiency, there remain aspects that inhibit fully automated shimming with a result of guaranteed quality. The major reason for this limitation is that the judgement of the quality of the B0 homogeneity takes place in the spatial domain, although the actual objects of interest are the lines in the spectral domain. In this work, this restriction is removed by the introduction of a new framework for gradient shimming that enables to directly aim at the spectrum quality. Based on the mapped B0 field shimming is simulated and spectra are calculated for the virtual residual inhomogeneity. Using a suitable criterion to judge the spectrum quality an optimisation is performed, thus providing the predicted optimum spectrum and the corresponding residual B0 field. This target field is then aimed at during the real, iterative shimming procedure. For the widely applicable case of optimising the shape of a single line a powerful quality criterion was developed using an envelope of the calculated lineshape spectrum. The whole procedure is demonstrated for adjusting the on-axis shim functions based on one-dimensional field map data and for both on- and off-axis shimming using three-dimensional data. The results are verified with 1H NMR spectra acquired on standard NMR test samples.

Comprehensive multiphase NMR applied to a living organism.

Comprehensive multiphase (CMP) NMR is a novel technology that integrates all the hardware from solution-, gel- and solid-state into a single NMR probe, permitting all phases to be studied in intact samples. Here comprehensive multiphase (CMP) NMR is used to study all components in a living organism for the first time. This work describes 4 new scientific accomplishments summarized as: (1) CMP-NMR is applied to a living animal, (2) an effective method to deliver oxygen to the organisms is described which permits longer studies essential for in-depth NMR analysis in general, (3) a range of spectral editing approaches are applied to fully differentiate the various phases solutions (metabolites) through to solids (shell) (4) 13C isotopic labelling and multidimensional NMR are combined to provide detailed assignment of metabolites and structural components in vivo. While not explicitly studied here the multiphase capabilities of the technique offer future possibilities to study kinetic transfer between phases (e.g. nutrient assimilation, contaminant sequestration), molecular binding at interfaces (e.g. drug or contaminant binding) and bonding across and between phases (e.g. muscle to bone) in vivo. Future work will need to focus on decreasing the spinning speed to reduce organism stress during analysis.