Potentials of radio-frequency field gradient NMR microscopy in environmental science

An understanding of transport, flow, diffusivity and mass transfer processes is of central importance in many fields of environmental biotechnology such as biofilm, bioreactor and membrane engineering, soil and groundwater bioremediation, and wastewater treatment. Owing to its remarkable sensitivity to molecular displacements and to its noninvasive and nondestructive character, pulsed field gradient (PFG) nuclear magnetic resonance (NMR) can be a valuable tool for investigating such processes. In conventional NMR microscopy, spatial encoding is achieved by using static magnetic field gradients (B ₀ gradients). However, an interesting alternative is to use radio-frequency magnetic field gradients (RF or B ₁ gradients). Although the latter are less versatile than the former, RF field gradient microscopy is particularly suitable for dealing with heterogeneous systems such as porous media because of its quasi-immunity to background static magnetic field gradients arising from magnetic susceptibility inhomogeneities, unlike the B ₀ gradients microscopy. Here, we present an overview of basic principles and the main features of this technique, which is still relatively unused. Different examples of diffusion imaging illustrate the potentialities of the method in both micro-imaging and the measurement of global or local diffusion coefficients within membranes and at liquid–solid interfaces. These examples suggest that a number of environmental problems could benefit from this technique. Different future prospects of application of B ₁ gradient NMR microscopy in environmental biotechnology are considered. Journal of Industrial Microbiology & Biotechnology (2001) 26, 53–61. Received 09 February 2000/ Accepted in revised form 07 August 2000     

Nuclear magnetic resonance in environmental engineering: Principles and applications

This paper gives an introduction to nuclear magnetic resonance spectroscopy (NMR) and magnetic resonance imaging (MRI) in relation to applications in the field of environmental science and engineering. The underlying principles of high resolution solution and solid state NMR, relaxation time measurements and imaging are presented. Then, the use of NMR is illustrated and reviewed in studies of biodegradation and biotransformation of soluble and solid organic matter, removal of nutrients and xenobiotics, fate of heavy metal ions, and transport processes in bioreactor systems.     

Diffusional Properties of Methanogenic Granular Sludge: 1H NMR Characterization

The diffusive properties of anaerobic methanogenic and sulfidogenic aggregates present in wastewater treatment bioreactors were studied using diffusion analysis by relaxation time-separated pulsed-field gradient nuclear magnetic resonance (NMR) spectroscopy and NMR imaging. NMR spectroscopy measurements were performed at 22°C with 10 ml of granular sludge at a magnetic field strength of 0.5 T (20 MHz resonance frequency for protons). Self-diffusion coefficients of H₂O in the investigated series of mesophilic aggregates were found to be 51 to 78% lower than the self-diffusion coefficient of free water. Interestingly, self-diffusion coefficients of H₂O were independent of the aggregate size for the size fractions investigated. Diffusional transport occurred faster in aggregates growing under nutrient-rich conditions (e.g., the bottom of a reactor) or at high (55°C) temperatures than in aggregates cultivated in nutrient-poor conditions or at low (10°C) temperatures. Exposure of aggregates to 2.5% glutaraldehyde or heat (70 or 90°C for 30 min) modified the diffusional transport up to 20%. In contrast, deactivation of aggregates by HgCl₂ did not affect the H₂O self-diffusion coefficient in aggregates. Analysis of NMR images of a single aggregate shows that methanogenic aggregates possess a spin-spin relaxation time and self-diffusion coefficient distribution, which are due to both physical (porosity) and chemical (metal sulfide precipitates) factors.     

Intact plant MRI for the study of cell water relations, membrane permeability, cell-to-cell and long distance water transport

Water content and hydraulic conductivity, including transport within cells, over membranes, cell-to-cell, and long-distance xylem and phloem transport, are strongly affected by plant water stress. By being able to measure these transport processes non-invasely in the intact plant situation in relation to the plant (cell) water balance, it will be possible explicitly or implicitly to examine many aspects of plant function, plant performance, and stress responses. Nuclear magnetic resonance imaging (MRI) techniques are now available that allow studying plant hydraulics on different length scales within intact plants. The information within MRI images can be manipulated in such a way that cell compartment size, water membrane permeability, water cell-to-cell transport, and xylem and phloem flow hydraulics are obtained in addition to anatomical information. These techniques are non-destructive and non-invasive and can be used to study the dynamics of plant water relations and water transport, for example, as a function of environmental (stress) conditions. An overview of NMR and MRI methods to measure such information is presented and hardware solutions for minimal invasive intact plant MRI are discussed.     

Biofilm shows spatially stratified metabolic responses to contaminant exposure

Biofilms are core to a range of biological processes, including the bioremediation of environmental contaminants. Within a biofilm population, cells with diverse genotypes and phenotypes coexist, suggesting that distinct metabolic pathways may be expressed based on the local environmental conditions in a biofilm. However, metabolic responses to local environmental conditions in a metabolically active biofilm interacting with environmental contaminants have never been quantitatively elucidated. In this study, we monitored the spatiotemporal metabolic responses of metabolically active Shewanella oneidensis MR‐1 biofilms to U(VI) (uranyl, UO₂ ²⁺) and Cr(VI) (chromate, CrO₄ ^2?) using non‐invasive nuclear magnetic resonance imaging (MRI) and spectroscopy (MRS) approaches to obtain insights into adaptation in biofilms during biofilm‐contaminant interactions. While overall biomass distribution was not significantly altered upon exposure to U(VI) or Cr(VI), MRI and spatial mapping of the diffusion revealed localized changes in the water diffusion coefficients in the biofilms, suggesting significant contaminant‐induced changes in structural or hydrodynamic properties during bioremediation. Finally, we quantitatively demonstrated that the metabolic responses of biofilms to contaminant exposure are spatially stratified, implying that adaptation in biofilms is custom‐developed based on local microenvironments.     

Magnetic Resonance Imaging of Mass Transport and Structure Inside a Phototrophic Biofilm

The aim of this study was to utilize magnetic resonance imaging (MRI) to image structural heterogeneity and mass transport inside a biofilm which was too thick for photon based imaging. MRI was used to map water diffusion and image the transport of the paramagnetically tagged macromolecule, Gd-DTPA, inside a 2.5 mm thick cyanobacterial biofilm. The structural heterogeneity of the biofilm was imaged at resolutions down to 22 × 22 μm, enabling the impact of biofilm architecture on the mass transport of both water and Gd-DTPA to be investigated. Higher density areas of the biofilm correlated with areas exhibiting lower relative water diffusion coefficients and slower transport of Gd-DTPA, highlighting the impact of biofilm structure on mass transport phenomena. This approach has potential for shedding light on heterogeneous mass transport of a range of molecular mass molecules in biofilms.     

MRI of intact plants

Nuclear magnetic resonance imaging (MRI) is a non-destructive and non-invasive technique that can be used to acquire two- or even three-dimensional images of intact plants. The information within the images can be manipulated and used to study the dynamics of plant water relations and water transport in the stem, e.g., as a function of environmental (stress) conditions. Non-spatially resolved portable NMR is becoming available to study leaf water content and distribution of water in different (sub-cellular) compartments. These parameters directly relate to stomatal water conductance, CO₂ uptake, and photosynthesis. MRI applied on plants is not a straight forward extension of the methods discussed for (bio)medical MRI. This educational review explains the basic physical principles of plant MRI, with a focus on the spatial resolution, factors that determine the spatial resolution, and its unique information for applications in plant water relations that directly relate to plant photosynthetic activity.     

Biopolymer and water dynamics in microbial biofilm extracellular polymeric substance

Nuclear magnetic resonance (NMR) is a noninvasive and nondestructive tool able to access several observable quantities in biofilms such as chemical composition, diffusion, and macroscale structure and transport. Pulsed gradient spin echo (PGSE) NMR techniques were used to measure spectrally resolved biomacromolecular diffusion in biofilm biomass, extending previous research on spectrally resolved diffusion in biofilms. The dominant free water signal was nulled using an inversion recovery modification of the traditional PGSE technique in which the signal from free water is minimized in order to view the spectra of components such as the rotationally mobile carbohydrates, DNA, and proteins. Diffusion data for the major constituents obtained from each of these spectral peaks demonstrate that the biomass of the biofilm contains both a fast and slow diffusion component. The dependence of diffusion on antimicrobial and environmental challenges suggests the polymer molecular dynamics measured by NMR are a sensitive indicator of biofilm function.     

Effects of Oxidation on Water Distribution and Physicochemical Properties of Porcine Myofibrillar Protein Gel

The effects of hydroxyl radicals induced oxidation on water distribution of porcine myofibrillar protein (MP) gels were investigated using nuclear magnetic resonance (NMR) and magnetic resonance imaging (MRI), and relationship between MP gel properties and water distribution was analyzed with Pearson’s correlation. Results of NMR spin-spin relaxation time (T₂) and MRI suggested that, comparing with unoxidized MP gel, the population of immobile water of gel (P₂₂) decreased by 18.81 %, while that of free water (P₂₃) increased by 85.56 %, and grey scale value decreased by 18.52 %, after being oxidized with 20 mM H₂O₂, respectively. Images of scanning electron microscope (SEM) showed the highest degree of oxidation led to the most coarse and porous gel-network. Gel properties including WHC, whiteness and strength of MP gels decreased by 14.65 %, 2.83 % and 52.74 % after being oxidized with 20 mM H₂O₂ (p?<?0.05), respectively. Pearson’s correlation analysis showed oxidation, gel-properties and NMR results were highly correlated (p?<?0.05). Therefore, we hypothesized that hydroxyl radicals induced oxidation resulted in immobile water shifting to free water partly, thus reduced the abundance of water and gel properties of gels.     

Measuring local flow velocities and biofilm structure in biofilm systems with magnetic resonance imaging (MRI)

The characterization of substrate transport in the bulk phase and in the biofilm matrix is one of the problems which has to be solved for the verification of biofilm models. Additionally, the surface structure of biofilms has to be described with appropriate parameters. Magnetic resonance imaging (MRI) is one of the promising methods for the investigation of transport phenomena and structure in biofilm systems. The MRI technique allows the noninvasive determination of flow velocities and biofilm structures with a high resolution on the sub-millimeter scale. The presented investigations were carried out for defined heterotrophic biofilms which were cultivated in a tube reactor at a Reynolds number of 2000 and 8000 and a substrate load of 6 and 4 g/m2d glucose. Magnetic resonance imaging provides both structure data of the biofilm surface and flow velocities in the bulk phase and at the bulk/biofilm interface. It is shown that the surface roughness of the biofilms can be determined in one experiment for the complete cross section of the test tubes both under flow and stagnant conditions. Furthermore, the local shear stress was calculated from the measured velocity profiles. In the investigated biofilm systems the local shear stress at the biofilm surface was up to 3 times higher compared to the mean wall shear stress calculated on the base of the mean flow velocity.     

Diffusional properties of methanogenic granular sludge: 1H NMR characterization

The diffusive properties of anaerobic methanogenic and sulfidogenic aggregates present in wastewater treatment bioreactors were studied using diffusion analysis by relaxation time-separated pulsed-field gradient nuclear magnetic resonance (NMR) spectroscopy and NMR imaging. NMR spectroscopy measurements were performed at 22 degrees C with 10 ml of granular sludge at a magnetic field strength of 0.5 T (20 MHz resonance frequency for protons). Self-diffusion coefficients of H(2)O in the investigated series of mesophilic aggregates were found to be 51 to 78% lower than the self-diffusion coefficient of free water. Interestingly, self-diffusion coefficients of H(2)O were independent of the aggregate size for the size fractions investigated. Diffusional transport occurred faster in aggregates growing under nutrient-rich conditions (e.g., the bottom of a reactor) or at high (55 degrees C) temperatures than in aggregates cultivated in nutrient-poor conditions or at low (10 degrees C) temperatures. Exposure of aggregates to 2.5% glutaraldehyde or heat (70 or 90 degrees C for 30 min) modified the diffusional transport up to 20%. In contrast, deactivation of aggregates by HgCl(2) did not affect the H(2)O self-diffusion coefficient in aggregates. Analysis of NMR images of a single aggregate shows that methanogenic aggregates possess a spin-spin relaxation time and self-diffusion coefficient distribution, which are due to both physical (porosity) and chemical (metal sulfide precipitates) factors.     

The NMR Microscope: a Unique and Promising Tool for Plant Science

An outline is given of nuclear magnetic resonance (NMR) microscopyand its application to plant science. An NMR microscope non-destructivelydetects free water in tissues and creates anatomical imagesof the tissues. Since the quantity and mobility of cell-associatedwater is closely related to the condition of the cells,¹H-NMRimages represent physiological maps of the tissue. In addition,the technique locates soluble organic compounds accumulatedin the tissues, such as sugars in vacuoles or fatty acids storedas oil droplets in vesicles.²³Na-NMR imaging is suitable forstudying the physiology of salt-tolerant plants. Diffusion measurementsprovide information about the transport of substances and ionsaccompanied by water movement. The recently developed techniquesof three-dimensional imaging, flow-encoded imaging and spectroscopicimaging open up new opportunities for plant biologists. TheNMR microscope is thus a unique and promising tool for the studyof living plant systems in relation to morphology, the truefeatures of which are often lost during preparation for moreconventional tissue analysis. Copyright 2000 Annals of BotanyCompany Review, NMR microscope,¹H-NMR imaging, non-destructive analysis, anatomy, cell-associated water, relaxation times, soluble compound mapping,²³Na-NMR imaging, physiological mapping, diffusion measurement, flow-encoded imaging     

Population diversity in model potable water biofilms receiving chlorine or chloramine residual

Abstract

Most water utilities use chlorine or chloramine to produce potable water. These disinfecting agents react with water to produce residual oxidants within a water distribution system (WDS) to control bacterial growth. While monochloramine is considered more stable than chlorine, little is known about the effect it has on WDS biofilms. Community structure of 10-week old WDS biofilms exposed to disinfectants was assessed after developing model biofilms from unamended distribution water. Four biofilm types were developed on polycarbonate slides within annular reactors while receiving chlorine, chloramine, or inactivated disinfectant residual. Eubacteria were identified through 16S rDNA sequence analysis. The model WDS biofilm exposed to chloramine mainly contained Mycobacterium and Dechloromonas sequences, while a variety of alpha- and additional beta-proteobacteria dominated the 16S rDNA clone libraries in the other three biofilms. Additionally, bacterial clones distantly related to Legionella were found in one of the biofilms receiving water with inactivated chlorine residual. The biofilm reactor receiving chloraminated water required increasing amounts of disinfectant after 2 weeks to maintain chlorine residual. In contrast, free chlorine residual remained steady in the reactor that received chlorinated water. The differences in bacterial populations of potable water biofilms suggest that disinfecting agents can influence biofilm development. These results also suggest that biofilm communities in distribution systems are capable of changing in response to disinfection practices.     

Non-invasive histochemistry of plant materials by magnetic resonance microscopy

Summary We have combined nuclear magnetic resonance (NMR) imaging on the microscopic scale with chemical shift selection to demonstrate the application of magnetic resonance imaging (MRI) to plant histochemistry. As an example of the method we have obtained separate images of the distribution of reserve oil and anethole in dried fennel mericarps. The technique can be employed to separately image the distribution of aromatics, carbohydrates, oils, water and possibly fatty acids in suitable plant materials.Abbreviations NMR nuclear magnetic resonance - MRI magnetic resonance imaging - COSY correlation spectroscopy - TMS tetramethylsilane     

NMR methods for in situ biofilm metabolism studies

Novel procedures and instrumentation are described for nuclear magnetic resonance (NMR) spectroscopy and imaging studies of live, in situ microbial films. A perfused NMR/optical microscope sample chamber containing a planar biofilm support was integrated into a recirculation/dilution flow loop growth reactor system and used to grow in situ Shewanella oneidensis strain MR-1 biofilms. Localized NMR techniques were developed and used to non-invasively monitor time-resolved metabolite concentrations and to image the biomass volume and distribution. As a first illustration of the feasibility of the methodology an initial 13C-labeled lactate metabolic pathway study was performed, yielding results consistent with existing genomic data for MR-1. These results represent progress toward our ultimate goal of correlating time- and depth-resolved metabolism and mass transport with gene expression in live in situ biofilms using combined NMR/optical microscopy techniques.     

Solid-state 13C NMR spectroscopic, chemolytic and biological assessment of pretreated municipal solid waste

In Central Europe, composting and anaerobic digestion of municipal solid waste (MSW) is used as pretreatment before landfilling to reduce landfill emissions. MSW samples were analyzed before, during, and after pretreatment to assess the stability of the organic matter. Chemolytic, nuclear magnetic resonance (NMR) spectroscopic, and respiration parameters were correlated to evaluate a substitution of the time-consuming respiration analysis by chemical parameters. ¹³C cross polarization magic angle spinning (CPMAS) NMR spectroscopy showed a preferential biodegradation of O-alkyl carbon (carbohydrates) and a selective accumulation of plastics during all pretreatments, confirming findings from chemolytic analyses. Principal component analysis exhibited a strong association between the respiration rate, the carbohydrate content, and the O-alkyl C content, corroborating that carbohydrates are the most important compounds of MSW with regard to the emission potential. Rank correlation (Spearman) also showed strong relationships between the respiration rate and the content of carbohydrates (r=0.75) and of O-alkyl C (r=0.72). Journal of Industrial Microbiology & Biotechnology (2001) 26, 83–89. Received 20 April 2000/ Accepted in revised form 21 July 2000     

Assessment of an anti-scale low-frequency electromagnetic field device on drinking water biofilms

Abstract

Low intensity and very low-frequency electromagnetic fields (EMF) used for preventing scaling in water distribution systems were tested for the first time for their potential impact on drinking water biofilms. The assays were carried out in laboratory-scale flow-through reactors that mimic water distribution systems. The drinking water biofilms were not directly exposed to the core of the EMF generator and only subjected to waterborne electromagnetic waves. The density and chlorine susceptibility of nascent or mature biofilms grown under exposure to EMF were evaluated in soft and hard water. This EMF treatment was able to modify CaCO₃ crystallization but it did not significantly affect biofilms. Indeed, over all the tested conditions, there was no significant change in cell number, or in the integrity of the cells (membrane, culturability), and no measurable effect of chlorine on the biofilm.     

Application of Paramagnetically Tagged Molecules for Magnetic Resonance Imaging of Biofilm Mass Transport Processes

Molecules become readily visible by magnetic resonance imaging (MRI) when labeled with a paramagnetic tag. Consequently, MRI can be used to image their transport through porous media. In this study, we demonstrated that this method could be applied to image mass transport processes in biofilms. The transport of a complex of gadolinium and diethylenetriamine pentaacetic acid (Gd-DTPA), a commercially available paramagnetic molecule, was imaged both in agar (as a homogeneous test system) and in a phototrophic biofilm. The images collected were T₁ weighted, where T₁ is an MRI property of the biofilm and is dependent on Gd-DTPA concentration. A calibration protocol was applied to convert T₁ parameter maps into concentration maps, thus revealing the spatially resolved concentrations of this tracer at different time intervals. Comparing the data obtained from the agar experiment with data from a one-dimensional diffusion model revealed that transport of Gd-DTPA in agar was purely via diffusion, with a diffusion coefficient of 7.2 × 10⁻¹⁰ m² s⁻¹. In contrast, comparison of data from the phototrophic biofilm experiment with data from a two-dimensional diffusion model revealed that transport of Gd-DTPA inside the biofilm was by both diffusion and advection, equivalent to a diffusion coefficient of 1.04 × 10⁻⁹ m² s⁻¹. This technology can be used to further explore mass transport processes in biofilms, either by using the wide range of commercially available paramagnetically tagged molecules and nanoparticles or by using bespoke tagged molecules.Biofilms are utilized in a wide range of biotechnological processes, such as cleansing municipal and industrial wastewater, bioremediation of hazardous waste sites, biofuel production, and the generation of electricity in microbial fuel cells (20, 31, 35). They also play an important role in mediating the geochemistry of the natural environment (35). Critically, our growing understanding of the biology, physics, and chemistry of biofilms is allowing us to manipulate biofilms and enhance their performance in a variety of biotechnologies (33). The optimization of biofilm processes is, however, hindered when a lack of quantitative measurements of critical biofilm parameters exists.For the biofilm to function, the relevant substrates must be transported through the biofilm matrix, where they are metabolized. The rate at which these metabolites are transported through the biofilm can be critical in controlling the performance of the biofilm (5, 8, 13, 31). Equally, the rate at which the biofilm can sequester nonmetabolizable pollutants, such as nonmetabolizable heavy metals and recalcitrant organics, is also mediated by the transport rate (9, 28). Previous studies of mass transport inside biofilms show that transport occurs not only by diffusion but also by advection if the biofilm contains interconnected channels (5, 9, 13, 19, 39, 40, 45). When transported by diffusion, the mass of the diffusing solute plays a key role in mediating the transport rate. That is, the higher the molecular mass of the solute, the lower its diffusion coefficient (7, 39). Moreover, the molecular masses and diffusion rates of these solutes vary considerably, ranging from low-mass, fast-diffusing metabolites, such as H₂ and O₂, to large, slowly diffusing organic macromolecules tens to hundreds of kDa in size. Indeed, high-molecular-mass molecules and nanoparticles are an important part of the substrate and pollutant load in both wastewater treatment and natural aquatic systems (21). At a certain size, large macromolecules and nanoparticles become too large to diffuse into the dense extracellular polymeric substance (EPS) matrix, although they still can be transported deep into the biofilm along open channels (9, 39).Moreover, due to the heterogeneous nature of biofilms, substrates can also display significant spatial variation in mass transport rates, such as a decrease in transport rate with biofilm depth (4). As attempts to understand biofilm function or enhance biofilm performance are dependent upon accurate mass transport data sets, quantifying the transport behaviors of different-molecular-mass molecules in different biofilms is key to allowing us to model real biofilm systems more accurately.Recognizing the importance of mass transport, researchers have already used a variety of methods, such as microelectrodes, confocal laser scanning microscopy (CLSM), fluorescence recovery after photobleaching (FRAP), and two-photon excitation microscopy to obtain mass transport data from biofilms (7, 11, 12). These approaches have provided invaluable data on mass transport within biofilms. However, as with any method, each has certain limitations. For example, microelectrodes are used to measure the mass transport of low-molecular-mass molecules; particulates and high-molecular-mass molecules are undetectable by this method. Moreover, the insertion of a probe is invasive and thus has potential to disrupt the surrounding material, altering results. This could be problematic when numerous insertions must be made, such as during spatial mapping of diffusion coefficients in heterogeneous biofilms. Conversely, CLSM is noninvasive. However, small molecules such as H₂ or O₂ cannot be labeled with the fluorescent probe, and thus only the transport of higher-molecular-weight compounds can be determined. This method, which relies on photons penetrating the biofilm, is limited both to biofilm thickness (<100 μm) and to its density due to optical scattering effects (26, 43). Although the two-photon excitation method can overcome the depth penetration limitation of CLSM by approximately four times (26), it is not suitable where biofilms exceed these thicknesses. FRAP also suffers similar thickness limitations and light-scattering effects. However, the capacity of magnetic resonance imaging (MRI) for completely noninvasive measurement of the transport of both low- and high-molecular-mass compounds and its ability to image inside hydrated biological matrices (1, 30), no matter what thickness, means that it has significant potential for mass transport analysis of biofilms and can thus be an invaluable additional tool in this research field.Researchers have already used MRI to examine flow dynamics over biofilm surfaces (22, 37), metabolite consumption and production (23), the flux of heavy metals in metal-immobilizing bioreactors (15, 25), water diffusion in biofilms (28, 44), and the transport and fate of metals both in natural and artificial biofilms (28, 29) and in real methanogenic granules which are employed in anaerobic wastewater treatment (2).     

Mathematical modelling of biofilm structures

The morphology of biofilms received much attention in the last years. Several concepts to explain the development of biofilm structures have been proposed. We believe that biofilm structure formation depends on physical as well as general and specific biological factors. The physical factors (e.g. governing substrate transport) as well as general biological factors such as growth yield and substrate conversion rates are the basic factors governing structure formation. Specific strain dependent factors will modify these, giving a further variation between different biofilm systems. Biofilm formation seems to be primarily dependent on the interaction between mass transport and conversion processes. When a biofilm is strongly diffusion limited it will tend to become a heterogeneous and porous structure. When the conversion is the rate-limiting step, the biofilm will tend to become homogenous and compact. On top of these two processes, detachment processes play a significant role. In systems with a high detachment (or shear) force, detachment will be in the form of erosion, giving smoother biofilms. Systems with a low detachment force tend to give a more porous biofilm and detachment occurs mainly by sloughing. Biofilm structure results from the interplay between these interactions (mass transfer, conversion rates, detachment forces) making it difficult to study systems taking only one of these factors into account. This revised version was published online in August 2006 with corrections to the Cover Date.     

Nuclear magnetic resonance in plant science research

Summary

In vivo nuclear magnetic resonance (NMR) spectroscopy and NMR imaging are noninvasive techniques with the potential to investigate a wide range of biochemical and physiological problems in living systems. The extent to which this potential has been realized in plant tissues is discussed with reference to recent applications to a number of systems, including root tissues and plant cell suspensions. It is concluded that while the potential of NMR imaging has yet to be fully realized in plants, in vivo NMR spectroscopy has matured into a problem-solving technique that can be used to test metabolic hypotheses directly.