High Field MicroMRI Velocimetric Measurement of Quantitative Local Flow Curves.

Performing rheo-microMRI velocimetry at a high magnetic field with strong pulsed field gradients has clear advantages in terms of (chemical) sensitivity and resolution in velocities, time, and space. To benefit from these advantages, some artifacts need to be minimized. Significant sources of such artifacts are chemical shift dispersion due to the high magnetic field, eddy currents caused by the pulsed magnetic field gradients, and possible mechanical instabilities in concentric cylinder (CC) rheo-cells. These, in particular, hamper quantitative assessment of spatially resolved velocity profiles needed to construct local flow curves (LFCs) in CC geometries with millimeter gap sizes. A major improvement was achieved by chemical shift selective suppression of signals that are spectroscopically different from the signal of interest. By also accounting for imperfections in pulsed field gradients, LFCs were obtained that were virtually free of artifacts. The approach to obtain quantitative LFCs in millimeter gap CC rheo-MRI cells was validated for Newtonian and simple yield stress fluids, which both showed quantitative agreement between local and global flow curves. No systematic effects of gap size and rotational velocity on the viscosity of a Newtonian fluid and yield stress of a complex fluid could be observed. The acquisition of LFCs during heterogeneous and transient flow of fat crystal dispersion demonstrated that local constitutive laws can be assessed by rheo-microMRI at a high magnetic field in a noninvasive, quantitative, and real-time manner.

Characterizing the structure of aerobic granular sludge using ultra-high field magnetic resonance.

Despite aerobic granular sludge wastewater treatment plants operating around the world, our understanding of internal granule structure and its relation to treatment efficiency remains limited. This can be attributed in part to the drawbacks of time-consuming, labor-intensive, and invasive microscopy protocols which effectively restrict samples sizes and may introduce artefacts. Time-domain nuclear magnetic resonance (NMR) allows non-invasive measurements which describe internal structural features of opaque, complex materials like biofilms. NMR was used to image aerobic granules collected from five full-scale wastewater treatment plants in the Netherlands and United States, as well as laboratory granules and control beads. T1 and T2 relaxation-weighted images reveal heterogeneous structures that include high- and low-density biofilm regions, water-like voids, and solid-like inclusions. Channels larger than approximately 50 µm and connected to the bulk fluid were not visible. Both cluster and ring-like structures were observed with each granule source having a characteristic structural type. These structures, and their NMR relaxation behavior, were stable over several months of storage. These observations reveal the complex structures within aerobic granules from a range of sources and highlight the need for non-invasive characterization methods like NMR to be applied in the ongoing effort to correlate structure and function.

Quantitative permeability imaging of plant tissues.

A method for mapping tissue permeability based on time-dependent diffusion measurements is presented. A pulsed field gradient sequence to measure the diffusion encoding time dependence of the diffusion coefficients based on the detection of stimulated spin echoes to enable long diffusion times is combined with a turbo spin echo sequence for fast NMR imaging (MRI). A fitting function is suggested to describe the time dependence of the apparent diffusion constant in porous (bio-)materials, even if the time range of the apparent diffusion coefficient is limited due to relaxation of the magnetization. The method is demonstrated by characterizing anisotropic cell dimensions and permeability on a subpixel level of different tissues of a carrot (Daucus carota) taproot in the radial and axial directions.

Full-Harmonics Phasor Analysis: Unravelling Multiexponential Trends in Magnetic Resonance Imaging Data.

Phasor analysis is a robust, nonfitting, method for the study of multiexponential decays in lifetime imaging data, routinely used in Fluorescence Lifetime Imaging Microscopy (FLIM) and only recently validated for Magnetic Resonance Imaging (MRI). In the established phasor approach, typically only the first Fourier harmonic is used to unravel time-domain exponential trends and their intercorrelations across image voxels. Here, we demonstrate the potential of full-harmonics (FH) phasor analysis by using all frequency-domain data points in simulations and quantitative MRI (qMRI) T2 measurements of phantoms with bulk liquids or liquid-filled porous particles and of a human brain. We show that FH analysis, while of limited advantage in FLIM due to the correlated nature of shot noise, in MRI outperforms single-harmonic phasor in unravelling multiple physical environments and partial-volume effects otherwise undiscernible. We foresee application of FH phasor to, e.g., big-data analysis in qMRI of biological or other multiphase systems, where multiparameter fitting is unfeasible.

Assessing spatial resolution, acquisition time and signal-to-noise ratio for commercial microimaging systems at 14.1, 17.6 and 22.3 T.

This work provides a systematic comparison of the signal-to-noise ratio (SNR), spatial resolution, acquisition time and metabolite limits-of-detection for magnetic resonance microscopy and spectroscopy at three different magnetic field strengths of 14.1 T, 17.6 T and 22.3 T (the highest currently available for imaging), utilizing commercially available hardware. We find an SNR increase of a factor 5.9 going from 14.1 T to 22.3 T using 5 mm radiofrequency (saddle and birdcage) coils, which results in a 24-fold acceleration in acquisition time and deviates from the theoretically expected increase of factor 2.2 due to differences in hardware. This underlines the importance of not only the magnetic field strengths but also hardware optimization. In addition, using a home-built 1.5 mm solenoid coil, we can achieve an isotropic resolution of (5.5 µm)3 over a field-of-view of 1.58 mm × 1.05 mm × 1.05 mm with an SNR of 12:1 using 44 signal averages in 58 h 34 min acquisition time at 22.3 T. In light of these results, we discuss future perspectives for ultra-high field Magnetic Resonance Microscopy and Spectroscopy.

Magnetic Resonance Microscopy at Cellular Resolution and Localised Spectroscopy of Medicago truncatula at 22.3 Tesla.

Interactions between plants and the soil's microbial & fungal flora are crucial for the health of soil ecosystems and food production. Microbe-plant interactions are difficult to investigate in situ due to their intertwined relationship involving morphology and metabolism. Here, we describe an approach to overcome this challenge by elucidating morphology and the metabolic profile of Medicago truncatula root nodules using Magnetic Resonance (MR) Microscopy, at the highest magnetic field strength (22.3 T) currently available for imaging. A home-built solenoid RF coil with an inner diameter of 1.5 mm was used to study individual root nodules. A 3D imaging sequence with an isotropic resolution of (7 µm)3 was able to resolve individual cells, and distinguish between cells infected with rhizobia and uninfected cells. Furthermore, we studied the metabolic profile of cells in different sections of the root nodule using localised MR spectroscopy and showed that several metabolites, including betaine, asparagine/aspartate and choline, have different concentrations across nodule zones. The metabolite spatial distribution was visualised using chemical shift imaging. Finally, we describe the technical challenges and outlook towards future in vivo MR microscopy of nodules and the plant root system.

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, CO(2) 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.

3D biofilm visualization and quantification on granular bioanodes with magnetic resonance imaging.

The use of microbial fuel cells (MFCs) for wastewater treatment fits in a circular economy context, as they can produce electricity by the removal of organic matter in the wastewater. Activated carbon (AC) granules are an attractive electrode material for bioanodes in MFCs, as they are cheap and provide electroactive bacteria with a large surface area for attachment. The characterization of biofilm growth on AC granules, however, is challenging due to their high roughness and three-dimensional structure. In this research, we show that 3D magnetic resonance imaging (MRI) can be used to visualize biofilm distribution and determine its volume on irregular-shaped single AC granules in a non-destructive way, while being combined with electrochemical and biomass analyses. Ten AC granules with electroactive biofilm (i.e. granular bioanodes) were collected at different growth stages (3 to 21 days after microbial inoculation) from a multi-anode MFC and T1-weighted 3D-MRI experiments were performed for three-dimensional biofilm visualization. With time, a more homogeneous biofilm distribution and an increased biofilm thickness could be observed in the 3D-MRI images. Biofilm volumes varied from 0.4 µL (day 4) to 2 µL (day 21) and were linearly correlated (R2 = 0.9) to the total produced electric charge and total nitrogen content of the granular bioanodes, with values of 66.4 C µL-1 and 17 µg N µL-1, respectively. In future, in situ MRI measurements could be used to monitor biofilm growth and distribution on AC granules.

Magnetic resonance imaging suggests functional role of previous year vessels and fibres in ring-porous sap flow resumption.

Reactivation of axial water flow in ring-porous species is a complex process related to stem water content and developmental stage of both earlywood-vessel and leaf formation. Yet empirical evidence with non-destructive methods on the dynamics of water flow resumption in relation to these mechanisms is lacking. Here we combined in vivo magnetic resonance imaging and wood-anatomical observations to monitor the dynamic changes in stem water content and flow during spring reactivation in 4-year-old pedunculate oaks (Quercus robur L.) saplings. We found that previous year latewood vessels and current year developing earlywood vessels form a functional unit for water flow during growth resumption. During spring reactivation, water flow shifted from latewood towards the new earlywood, paralleling the formation of earlywood vessels and leaves. At leaves' full expansion, volumetric water content of previous rings drastically decreased due to the near-absence of water in fibre tissue. We conclude (i) that in ring-porous oak, latewood vessels play an important hydraulic role for bridging the transition between old and new water-conducting vessels and (ii) that fibre and parenchyma provides a place for water storage.

Correlated displacement-T2 MRI by means of a Pulsed Field Gradient-Multi Spin Echo Method.

A method for correlated displacement-T2 imaging is presented. A Pulsed Field Gradient-Multi Spin Echo (PFG-MSE) sequence is used to record T2 resolved propagators on a voxel-by-voxel basis, making it possible to perform single voxel correlated displacement-T2 analyses. In spatially heterogeneous media the method thus gives access to sub-voxel information about displacement and T2 relaxation. The sequence is demonstrated using a number of flow conducting model systems: a tube with flowing water of variable intrinsic T2's, mixing fluids of different T2's in an "X"-shaped connector, and an intact living plant. PFG-MSE can be applied to yield information about the relation between flow, pore size and exchange behavior, and can aid volume flow quantification by making it possible to correct for T2 relaxation during the displacement labeling period Delta in PFG displacement imaging methods. Correlated displacement-T2 imaging can be of special interest for a number of research subjects, such as the flow of liquids and mixtures of liquids or liquids and solids moving through microscopic conduits of different sizes (e.g., plants, porous media, bioreactors, biomats).

Multi-component quantitative magnetic resonance imaging by phasor representation.

Quantitative magnetic resonance imaging (qMRI) is a versatile, non-destructive and non-invasive tool in life, material, and medical sciences. When multiple components contribute to the signal in a single pixel, however, it is difficult to quantify their individual contributions and characteristic parameters. Here we introduce the concept of phasor representation to qMRI to disentangle the signals from multiple components in imaging data. Plotting the phasors allowed for decomposition, unmixing, segmentation and quantification of our in vivo data from a plant stem, a human and mouse brain and a human prostate. In human brain images, we could identify 3 main T 2 components and 3 apparent diffusion coefficients; in human prostate 5 main contributing spectral shapes were distinguished. The presented phasor analysis is model-free, fast and accurate. Moreover, we also show that it works for undersampled data.

Modelling of self-diffusion and relaxation time NMR in multicompartment systems with cylindrical geometry.

Multicompartment characteristics of relaxation and diffusion in a model for (plant) cells and tissues have been simulated as a means to test separating the signal into a set of these compartments. A numerical model of restricted diffusion and magnetization relaxation behavior in PFG-CPMG NMR experiments, based on Fick's second law of diffusion, has been extended for two-dimensional diffusion in systems with concentric cylindrical compartments separated by permeable walls. This model is applicable to a wide range of (cellular) systems and allows the exploration of temporal and spatial behavior of the magnetization with and without the influence of gradient pulses. Numerical simulations have been performed to show the correspondence between the obtained results and previously reported studies and to investigate the behavior of the apparent diffusion coefficients for the multicompartment systems with planar and cylindrical geometry. The results clearly demonstrate the importance of modelling two-dimensional diffusion in relation to the effect of restrictions, permeability of the membranes, and the bulk relaxation within the compartments. In addition, the consequences of analysis by multiexponential curve fitting are investigated.

Influence of water content and drying on the physical structure of native hyaluronan.

Hydration properties of semi-diluted hyaluronan were studied by means of time domain nuclear magnetic resonance. Based on the transverse proton relaxation times T2, the plasticization of hyaluronan which was precipitated by isopropylalcohol and dried in the oven have been determined at water content 0.4 g of water per g of hyaluronan. Above this water content, the relaxation times increased and levelled off around 0.8 g of water per g of hyaluronan which agrees well with values determined earlier by differential scanning calorimetry and dielectric relaxometry. The freeze dried and oven dried samples showed differences in their physical structure such as glass transition, plasticization concentration and sample topography which influenced their kinetics and mechanisms of hydration. Results confirmed earlier hypothesis that some native biopolymer structures can be easily modified by manipulation of preparation conditions, e.g. drying, giving fractions with specific physicochemical properties without necessity of their chemical modification.

Controlled mixing of lanthanide(III) ions in coacervate core micelles.

This article presents a facile strategy to combine Eu(3+) and Gd(3+) ions into coacervate core micelles in a controlled way with a statistical distribution of the ions. Consequently, the formed micelles show a high tunability between luminescence and relaxivity. These highly stable micelles present great potential for new materials, e.g. as bimodal imaging probes.

Anomalies in moisture transport during broccoli drying monitored by MRI?

Magnetic resonance imaging (MRI) offers unique opportunities to monitor moisture transport during drying or heating of food, which can render unexpected insights. Here, we report about MRI observations made during the drying of broccoli stalks indicating anomalous drying behaviour. In fresh broccoli samples the moisture content in the core of the sample increases during drying, which conflicts with Fickian diffusion. We have put the hypothesis that this increase of moisture is due to the stress diffusion induced by the elastic impermeable skin. Pre-treatments that change skin and bulk elastic properties of broccoli show that our hypothesis of stress-diffusion is plausible.

¹H-NMR study of the impact of high pressure and thermal processing on cell membrane integrity of onions.

Proton nuclear magnetic resonance (¹H-NMR) relaxometry was used to study the effects of high pressure and thermal processing on membrane permeability and cell compartmentalization, important components of plant tissue texture. High pressure treated onions were subjected to pressure levels from 20 to 200 MPa at 5 min hold time at initial temperatures of 5 and 20 °C. Thermally treated onions were exposed for 30 min at temperatures from 40 to 90 °C. Loss of membrane integrity was clearly shown by changes in transverse relaxation time (T(2)) of water at temperatures of 60 °C and above. Destabilization effects on membranes exposed to high pressure were observed at 200 MPa as indicated by T(2) measurements and cryo-scanning electron microscopy (Cryo-SEM). T(2) relaxation successfully discriminated different degrees of membrane damage based on the T(2) shift of the vacuolar component. Analyses of the average water self-diffusion coefficient indicated less restricted diffusion after membrane rupture occurred in cases of severe thermal treatments. Milder processing treatments yielded lower average diffusion coefficients than the controls. ¹H-NMR proved to be an effective method for quantification of cell membrane damage in onions and allowed for the comparison of different food processes based on their impact on tissue integrity.

Magnetic resonance microscopy of iron transport in methanogenic granules.

Interactions between anaerobic biofilms and heavy metals such as iron, cobalt or nickel are largely unknown. Magnetic resonance imaging (MRI) is a non-invasive method that allows in situ studies of metal transport within biofilm matrixes. The present study investigates quantitatively the penetration of iron (1.7 5mM) bound to ethylenediaminetetraacetate (EDTA) into the methanogenic granules (spherical biofilm). A spatial resolution of 109x109x218 microm(3) and a temporal resolution of 11 min are achieved with 3D Turbo Spin Echo (TSE) measurements. The longitudinal relaxivity, i.e. the slope the dependence of the relaxation rate (1/T(1)) on the concentration of paramagnetic metal ions, was used to measure temporal changes in iron concentration in the methanogenic granules. It took up to 300 min for the iron-EDTA complex ([FeEDTA](2-)) to penetrate into the methanogenic granules (3-4mm in diameter). The diffusion was equally fast in all directions with irregularities such as diffusion-facilitating channels and diffusion-resistant zones. Despite these irregularities, the overall process could be modeled using Fick's equations for diffusion in a sphere, because immobilization of [FeEDTA](2-) in the granular matrix (or the presence of a reactive barrier) was not observed. The effective diffusion coefficient (D(ejf)) of [FeEDTA](2-) was found to be 2.8x10(-11)m(2)s(-1), i.e. approximately 4% of D(ejf) of [FeEDTA](2-) in water. The Fickian model did not correspond to the processes taking place in the core of the granule (3-5% of the total volume of the granule), where up to 25% over-saturation by iron (compare to the concentration in the bulk solution) occurred.

MRI of long-distance water transport: a comparison of the phloem and xylem flow characteristics and dynamics in poplar, castor bean, tomato and tobacco.

We used dedicated magnetic resonance imaging (MRI) equipment and methods to study phloem and xylem transport in large potted plants. Quantitative flow profiles were obtained on a per-pixel basis, giving parameter maps of velocity, flow-conducting area and volume flow (flux). The diurnal xylem and phloem flow dynamics in poplar, castor bean, tomato and tobacco were compared. In poplar, clear diurnal differences in phloem flow profile were found, but phloem flux remained constant. In tomato, only small diurnal differences in flow profile were observed. In castor bean and tobacco, phloem flow remained unchanged. In all plants, xylem flow profiles showed large diurnal variation. Decreases in xylem flux were accompanied by a decrease in velocity and flow-conducting area. The diurnal changes in flow-conducting area of phloem and xylem could not be explained by pressure-dependent elastic changes in conduit diameter. The phloem to xylem flux ratio reflects what fraction of xylem water is used for phloem transport (Münch's counterflow). This ratio was large at night for poplar (0.19), castor bean (0.37) and tobacco (0.55), but low in tomato (0.04). The differences in phloem flow velocity between the four species, as well as within a diurnal cycle, were remarkably small (0.25-0.40 mm s(-1)). We hypothesize that upper and lower bounds for phloem flow velocity may exist: when phloem flow velocity is too high, parietal organelles may be stripped away from sieve tube walls; when sap flow is too slow or is highly variable, phloem-borne signalling could become unpredictable.

Lipid bilayer topology of the transmembrane alpha-helix of M13 Major coat protein and bilayer polarity profile by site-directed fluorescence spectroscopy.

This article presents a new formalism to perform a quantitative fluorescence analysis using the Stokes shift of AEDANS-labeled cysteine mutants of M13 major coat protein incorporated in lipid bilayers. This site-directed fluorescence spectroscopy approach enables us to obtain the topology of the bilayer-embedded transmembrane alpha-helix from the orientation and tilt angles, and relative bilayer location. Both in pure dioleoylphosphatidylcholine and dioleoylphosphatidylcholine/dioleoylphosphatidylglycerol (4:1 mol/mol) bilayers, which have a similar bilayer thickness, the tilt angle of the transmembrane helix of the coat protein turns out to be 23 degrees +/- 4. Upon decreasing the hydrophobic thickness on going from dieicosenoylphosphatidylcholine to dimyristoylphosphatidylcholine, the tilt angle and orientation angle of the transmembrane alpha-helix change. The protein responds to an increase of hydrophobic stress by increasing the tilt angle so as to keep much of its hydrophobic part inside the bilayer. At the same time, the transmembrane helix rotates at its long axis so as to optimize the hydrophobic and electrostatic interactions of the C-terminal phenylalanines and lysines, respectively. The increase of tilt angle cannot completely keep the hydrophobic protein section within the bilayer, but the C-terminal part remains anchored at the acyl-chain/glycerol backbone interface at the cost of the N-terminal section. In addition, our analysis results in the profile of the dielectric constant of the hydrophobic domain of the bilayer. For all phospholipid bilayers studied the profile has a concave shape, with a value of the dielectric constant of 4.0 in the center of the bilayer. The dielectric constant increases on approaching the headgroup region with a value of 12.4 at the acyl-chain/glycerol backbone interface for the various phosphatidylcholines with different chain lengths. For dioleoylphosphatidylcholine/dioleoylphosphatidylglycerol (4:1 mol/mol) bilayers the value of the dielectric constant at the acyl-chain/glycerol backbone interface is 18.6. In conclusion, the consistency of our analysis shows that the applied cysteine-scanning mutagenesis method with AEDANS labeling of a helical transmembrane protein in combination with a quantitative formalism offers a reliable description of the lipid bilayer topology of the protein and bilayer properties. This also indicates that the spacer link between the protein and AEDANS label is long enough to monitor the local polarity of the lipid environment and not that of the amino-acid residues of the protein, and short enough to have the topology of the protein imposing on the fluorescence properties of the AEDANS label.