Quantitative MRI analysis of structural changes in tomato tissues resulting from dehydration.

A quantitative magnetic resonance imaging (MRI) analysis at 1.5T of the effects of different dehydration regimes on transverse relaxation parameters measured in tomato tissue is presented. Multi-exponential T2 maps have been estimated for the first time, providing access to spatialized microstructural information at voxel scale. The objective was to provide a better understanding of the changes in the multi-exponential transverse relaxation parameters induced by dehydration in tomato tissues and to unravel the effects of microstructure and composition on relaxation parameters. The results led to the hypothesis that the multi-exponential relaxation signal reflects cell compartmentation and tissue heterogeneity, even at the voxel scale. Multi-exponential relaxation times provided information about water loss from specific cell compartments and seem to indicate that the dehydration process mainly affects large cells. By contrast, total signal intensity showed no sensitivity to variations in water content in the range investigated in the present study (between 95% [fresh tissue] and 90% [after dehydration]). The variation in relaxation times resulting from water loss was due to both changes in solute concentration and compartment size. The comparative analysis of the two contrasted tissues in terms of microporosity demonstrated that magnetic susceptibility effects, caused by the presence of air in the placenta tissue, significantly impact the effective relaxation and might be the dominant effect in the variations observed in relaxation times in this tissue.

NMR study of fresh cut salads: Influence of temperature and storage time on leaf structure and water distribution in escarole.

Consumption of fresh-cut vegetables has rapidly increased over the past decades. Among salads, escarole is one of the most popular varieties. Specific packaging limits gas exchange and consequently water loss and bacterial respiration, increasing the shelf life of salads. Although the major cause of quality loss for minimally processed salads is the leaf textural changes, this aspect has rarely been investigated. Therefore, investigating structural changes of leaves during storage is important in order to understand and minimize quality loss of salads. In this study, we focused on the impact of storage duration and temperature on the escarole leaf structure. The complex leaf structure was investigated by relaxation NMR, via transverse relaxation times, which allows the specific description of vacuolar water compartment of the cell. The storage duration (maximum 12 days) and temperatures (4°C, 7°C, 10°C, and 12°C) have been chosen in order to represent the conditions registered in factory. The results showed that the temperature did not have significant impact on the salad structure during the first week. During the second week, changes in the water distribution and changes in the relaxation time T2 have been observed. The changes in transverse relaxation times associated with vacuolar water are related to lost of cell membrane and wall integrity. The NMR results confirmed the effect of storage temperature on the degradation process of the cell before visual detection of the salad leaf degradation. The present study confirmed the sensibility of NMR relaxometry for monitoring water changes in the leaf.

Assessment of nutrient remobilization through structural changes of palisade and spongy parenchyma in oilseed rape leaves during senescence.

MAIN CONCLUSION: Differential palisade and spongy parenchyma structural changes in oilseed rape leaf were demonstrated. These dismantling processes were linked to early senescence events and associated to remobilization processes. During leaf senescence, an ordered cell dismantling process allows efficient nutrient remobilization. However, in Brassica napus plants, an important amount of nitrogen (N) in fallen leaves is associated with low N remobilization efficiency (NRE). The leaf is a complex organ mainly constituted of palisade and spongy parenchyma characterized by different structures and functions concerning water relations and carbon fixation. The aim of the present study was to demonstrate a specific structural evolution of these parenchyma throughout natural senescence in B. napus, probably linked to differential nutrient remobilization processes. The study was performed on 340 leaves from 32 plants during an 8-week development period under controlled growing conditions. Water distribution and status at the cellular level were investigated by low-field proton nuclear magnetic resonance (NMR), while light and electron microscopy were used to observe cell and plast structure. Physiological parameters were determined on all leaves studied and used as indicators of leaf development and remobilization progress. The results revealed a process of hydration and cell enlargement of leaf tissues associated with senescence. Wide variations were observed in the palisade parenchyma while spongy cells changed only very slightly. The major new functional information revealed was the link between the early senescence events and specific tissue dismantling processes.

Structural changes in senescing oilseed rape leaves at tissue and subcellular levels monitored by nuclear magnetic resonance relaxometry through water status.

Nitrogen use efficiency is relatively low in oilseed rape (Brassica napus) due to weak nitrogen remobilization during leaf senescence. Monitoring the kinetics of water distribution associated with the reorganization of cell structures, therefore, would be valuable to improve the characterization of nutrient recycling in leaf tissues and the associated senescence processes. In this study, nuclear magnetic resonance (NMR) relaxometry was used to describe water distribution and status at the cellular level in different leaf ranks of well-watered plants. It was shown to be able to detect slight variations in the evolution of senescence. The NMR results were linked to physiological characterization of the leaves and to light and electron micrographs. A relationship between cell hydration and leaf senescence was revealed and associated with changes in the NMR signal. The relative intensities and the transverse relaxation times of the NMR signal components associated with vacuole water were positively correlated with senescence, describing water uptake and vacuole and cell enlargement. Moreover, the relative intensity of the NMR signal that we assigned to the chloroplast water decreased during the senescence process, in agreement with the decrease in relative chloroplast volume estimated from micrographs. The results are discussed on the basis of water flux occurring at the cellular level during senescence. One of the main applications of this study would be for plant phenotyping, especially for plants under environmental stress such as nitrogen starvation.

Translational and rotational diffusion of flexible PEG and rigid dendrimer probes in sodium caseinate dispersions and acid gels.

The dynamics of rigid dendrimer and flexible PEG probes in sodium caseinate dispersions and acid gels, including both translational diffusion and rotational diffusion, were studied by NMR. Above the onset of the close-packing limit (C ∼ 10 g/100 g H2 O), translational diffusion of the probe depended on its flexibility and on the fluctuations of the matrix chains. The PEG probe diffused more rapidly than the spherical dendrimer probe of corresponding hydrodynamic radius. The greater conformational flexibility of PEG facilitated its motion through the crowded casein matrix. Rotational diffusion was, however, substantially less hindered than the translational diffusion and depended on the local protein-probe friction which became high when the casein concentration increased. The coagulation of the matrix led to the formation of large voids, which resulted in an increase in the translational diffusion of the probes, whereas the rotational diffusion of the probes was retarded in the gel, which could be attributed to the immobilized environment surrounding the probe. Quantitative information from PFG-NMR and SEM micrographs have been combined for characterizing microstructural details in SC acid gels.

Impact of chemical exchange on transverse relaxation at low and moderate magnetic field strengths for sugar solutions representative of fruit tissues analyzed by simulation and MRI experiments.

Proton exchange effects on transverse relaxation rate were studied at low and moderate magnetic fields. Analysis was conducted on low-concentrate simple sugar (fructose and glucose) solutions modeling the vacuolar liquid in fruits. Simulated data obtained from Carver and Richards equations were used to analyze the effects of temperature and pH on parameters involved in the chemical exchange mechanisms. The results have highlighted that measurement conditions and tissue characteristics (echo time, magnetic field strength, temperature, pH, etc.) significantly impact the transverse relaxation rate via chemical exchange, even for low and mid magnetic fields and the narrow echo time ranges of TD-NMR and MRI measurements. This explains some of the divergent relaxation data from plant tissue NMR measurements reported in the literature and emphasizes the importance of taking experimental conditions and tissue characteristics into account when interpreting results. It also clearly demonstrates that the appropriate choice of experimental conditions can emphasize the effects of sugar concentration or pH variations on transverse relaxation in vacuolar liquids, making it possible to monitor these parameters accurately via transverse relaxation measurements. The impact of concentration, solute type, pH and temperature on transverse relaxation of sugar solutions were demonstrated experimentally at 1.5 T with an MRI scanner. These data were interpreted using the Carver-Richards model which was effective in estimating parameters involved in chemical exchange mechanism in the imaging experiment.

Impact of casein gel microstructure on self-diffusion coefficient of molecular probes measured by 1H PFG-NMR.

The translational dynamics of poly(ethylene glycol) (PEG) polymers with molecular weights (Mw) varying from 6x10(2) to 5x10(5) were investigated by pulsed field gradient NMR in casein suspensions and in gels induced by acidification, enzyme action, and a combination of both. For molecules with Mwor=8000, there was strong dependence of diffusion on PEG size and on the casein network structure as revealed by scanning electron microscopy images. The diffusion coefficients of the two largest PEGs were increased after coagulation by amounts that depended on the internal structure of the gel. In addition, the 527,000 g/mol PEG was found to deviate from Gaussian diffusion behavior to greater or lesser extents according to the casein concentration and the sample microstructure. The results are discussed in terms of network rearrangements.

A mobile NMR lab for leaf phenotyping in the field.

BACKGROUND: Low field NMR has been used to investigate water status in various plant tissues. In plants grown in controlled conditions, the method was shown to be able to monitor leaf development as it could detect slight variations in senescence associated with structural modifications in leaf tissues. The aim of the present study was to demonstrate the potential of NMR to provide robust indicators of the leaf development stage in plants grown in the field, where leaves may develop less evenly due to environmental fluctuations. The study was largely motivated by the need to extend phenotyping investigations from laboratory experiments to plants in their natural environment. METHODS: The mobile NMR laboratory was developed, enabling characterization of oilseed rape leaves throughout the canopy without uprooting the plant. The measurements made on the leaves of plants grown and analyzed in the field were compared to the measurements on plants grown in controlled conditions and analyzed in the laboratory. RESULTS: The approach demonstrated the potential of the method to assess the physiological status of leaves of plants in their natural environment. Comparing changes in the patterns of NMR signal evolution in plants grown under well-controlled laboratory conditions and in plants grown in the field shows that NMR is an appropriate method to detect structural modifications in leaf tissues during senescence progress despite plant heterogeneity in natural conditions. Moreover, the specific effects of the environmental factors on the structural modifications were revealed. CONCLUSION: The present study is an important step toward the selection of genotypes with high tolerance to water or nitrogen depletion that will be enabled by further field applications of the method.

Effects of ionic strength and denaturation time on polyethyleneglycol self-diffusion in whey protein solutions and gels visualized by nuclear magnetic resonance.

Pulsed field gradient NMR spectroscopy was used to determine the poly(ethylene glycol) (PEG) self-diffusion coefficient (D(PEG)) as a function of NaCl concentration (C(NaCl)) and denaturation time (t(D)) in whey protein solutions and gels. D(PEG) in the gel decreased with increasing C(NaCl) concentrations and increased with increasing t(D); the increase ceased for all PEGs when the gel was fixed. This increase was more pronounced for the 82250 g/mol PEG than the 1080 g/mol PEG. Moreover, the diffusion coefficient of nonaggregated whey protein was measured and an increase for longer t(D) was also observed. Scanning electron microscopy images and (1)H spectra demonstrated that D(PEG) were related to the structure changes and to the percentage of beta-lactoglobulin denaturation.

Nitrogen deficiency impacts on leaf cell and tissue structure with consequences for senescence associated processes in Brassica napus.

Improvement of nutrient use efficiency is a major goal for several crop plants, especially Brassica napus. Indeed, the low nitrogen use efficiency (NUE) in this crop results in negative economic and ecological consequences. The low NUE of oilseed rape is mainly due to low remobilization of nitrogen from vegetative parts to growing organs. Remobilization of leaf nitrogen takes place during senescence, a process known to strongly modify cell and tissue structure. This study focused on the impact of moderate N depletion, expected to induce 30 % reduction of seed yield, on these structural modifications. Two genotypes (Aviso and Express) were studied, with different tolerance of nitrogen depletion, evaluated through seed yield and dry mass production. Structural modifications of leaf cells and tissues were investigated through NMR relaxometry and light microscopy. Lower tolerance of N depletion was associated with higher impact on senescence associated structural modification pattern. The link between leaf structure modifications and nutrient remobilization is discussed. It is proposed that leaf structure monitoring during senescence through NMR device could be developed to select genotypes with high NUE.

Diffusion of polyethyleneglycols in casein solutions and gels as studied by pulsed field gradient NMR.

Molecular transport characterized by diffusion coefficients is a key feature of food processes and especially in dairy processes. Caseins represent 80% of the protein content in milk and are directly involved in the formation of dairy gels. Consequently, providing a quantitative description of the solute diffusion in casein gels should contribute significantly to rationalization of the dairy processes. The objectives of this study were to study the self-diffusion coefficient of molecular probes [polyethyleneglycol (PEGs)] in casein solutions and gels, in particular with regard to the effects of the probe molecular size and casein concentration. We have shown that the PEG diffusion was an efficient tool to reveal the structural information relative to the gel matrix structure.

NMR signal analysis to attribute the components to the solid/liquid phases present in mixes and ice creams.

The NMR relaxation signals from complex products such as ice cream are hard to interpret because of the multiexponential behavior of the relaxation signal and the difficulty of attributing the NMR relaxation components to specific molecule fractions. An attribution of the NMR relaxation parameters is proposed, however, based on an approach that combines quantitative analysis of the spin-spin and spin-lattice relaxation times and the signal intensities with characterization of the ice cream components. We have been able to show that NMR can be used to describe the crystallized and liquid phases separately. The first component of the spin-spin and spin-lattice relaxation describes the behavior of the protons of the crystallized fat in the mix. The amount of fat crystals can then be estimated. In the case of ice cream, only the spin-lattice relaxation signal from the crystallized fraction is relevant. However, it enables the ice protons and the protons of the crystallized fat to be distinguished. The spin-lattice relaxation time can be used to describe the mobility of the protons in the different crystallized phases and also to quantify the amount of ice crystals and fat crystals in the ice cream. The NMR relaxation of the liquid phase of the mix has a biexponential behavior. A first component is attributable to the liquid fraction of the fat and to the sugars, while a second component is attributable to the aqueous phase. Overall, the study shows that despite the complexity of the NMR signal from ice cream, a number of relevant parameters can be extracted to study the influence of the formulation and of the process stages on the ice fraction, the crystallized fat fraction, and the liquid aqueous fraction.

NMR relaxation and water self-diffusion studies in whey protein solutions and gels.

The changes in water proton transverse relaxation behavior induced by aggregation of whey proteins are explained in terms of the simple molecular processes of diffusion and chemical exchange. The water self-diffusion coefficient was measured in whey protein solutions and gels by the pulsed field gradient NMR method. As expected, water self-diffusion was reduced with increased protein concentrations. Whatever the concentration, the water molecules were free to diffuse over distances varying from 15 to 47 mum. Water diffusion was constant over these distances, demonstrating that no restrictions were found to explain the water hindrance. The modification in protein structure by gelation induced a decrease in water diffusion. The effects of protein concentration on water diffusion are discussed and modeled. Two approaches were compared, the obstruction effect induced by a spherical particle and the cell model, which considered two water compartments with specific self-diffusion coefficients.

MRI investigation of subcellular water compartmentalization and gas distribution in apples.

Water status and distribution at subcellular level in whole apple fruit were evaluated by Magnetic Resonance Imaging (MRI) measurement of the multi-exponential transverse (T2) relaxation of water protons. Apparent microporosity, also estimated by MRI, provided mapping of gas distribution in fruit tissues. Measuring for the first time the multi-exponential relaxation of water and apparent tissue microporosity in whole fruit and combining these with histological measurements provided a more reliable interpretation of the origins of variations in the transverse relaxation time (T2) and better characterization of the fruit tissue. Measurements were performed on 54 fruits from 3 different cultivars. Fruits of different sizes were selected for each cultivar to provide tissues with cells of different dimensions. Macrovision measurements were carried out on parenchymal tissue from all fruits to investigate the impact of cell size on T2 value. The results showed that the MRI transverse relaxation signal is well fitted by a tri-exponential decay curve that reflects cell compartmentalization. Variations in cell size partially explained the different T2 observed. This study highlighted the heterogeneity of apple tissues in terms of relaxation parameters, apparent microporosity and cell morphology and in relation to specific variations between fruit of different cultivars.

Determination of water self-diffusion coefficient in complex food products by low field 1H PFG-NMR: comparison between the standard spin-echo sequence and the T1-weighted spin-echo sequence.

In 1990, Van Den Enden et al. proposed a method for the determination of water droplet size distributions in emulsions using a pulsed-field-gradient nuclear magnetic resonance (PFG-NMR) T1-weighted stimulated-echo technique. This paper describes both the T1-weighted spin-echo sequence, an improved method based on this earlier work, and, the standard PFG spin-echo sequence. These two methods were compared for water self-diffusion coefficient measurement in the fatty protein concentrate sample used as a 'cheese model.' The transversal and longitudinal relaxation parameters T1 and T2 were determined according to the temperature and investigated for each sample; fat-free protein concentrate sample, pure anhydrous milk fat, and fatty protein concentrate sample. The water self-diffusion in fat-free protein concentrate samples followed a linear behavior. Consequently, the water self-diffusion coefficient could be easily characterized for fat-free protein concentrate samples. However, it seemed more complicated to obtain accurate water self-diffusion in fatty protein concentrate samples since the diffusion-attenuation data were fitted by a bi-exponential function. This paper demonstrates that the implementation of the T1-weighted spin-echo sequence, using the different T1 properties of water and fat phases, allows the accurate determination of water self-diffusion coefficient in a food product. To minimize the contribution of the 1H nuclei in the fat phase on the NMR echo signal, the fat protons were selectively eliminated by an additional 180 degrees pulse. This new method reduces the standard errors of diffusion data obtained with a basic spin-echo technique, by a factor of 10. The effectiveness of the use of the T1-weighted spin-echo sequence to perform accurate water self-diffusion coefficients measurement in fatty products is thus demonstrated.

Effects of casein and fat content on water self-diffusion coefficients in casein systems: a pulsed field gradient nuclear magnetic resonance study.

The water self-diffusion coefficients in casein matrixes were measured using a pulsed field gradient spin-echo nuclear magnetic resonance technique (PFG-SE NMR). The dependence of the water self-diffusion coefficient on the casein concentration and the aqueous phase composition is reported in both a rehydrated native phosphocaseinate dispersion and a concentrated casein retentate. A model has been proposed to explain the different behavior of the water self-diffusion coefficient in the two casein systems. This model demonstrates that the water self-diffusion cannot be simply explained by the water content only. So, taking into account the specific effect of each constituent of the aqueous dispersing phase, the water self-diffusion reduction induced by the casein micelle can be modeled. The effect of fat on the water self-diffusion coefficients was investigated. Anhydrous milk fat-reconstituted retentate samples were used in order to estimate the obstruction effect of fat globules in the modeling process. The dependence of the self-diffusion coefficient of water on the fat and casein content is reported. A general model included the effect of the aqueous phase composition, and the obstruction effects of casein micelles and fat globules were proposed. This model was validated for water self-diffusion coefficients in industrial fatty retentates.

1H nuclear magnetic resonance relaxometry study of water state in milk protein mixtures.

1H NMR signal was used to characterize highly hydrated milk protein dispersions (3-20% dry matter) with various micellar casein concentrations (3-15%), whey protein concentrations (0-3%), lactose concentrations (0-7.5%), CaCl(2) concentrations (0-2 mM), and pH (6.2-6.6). The results showed the predominant effect of micellar casein concentration on water state and were consistent with the three-site relaxation model in the absence of lactose. The relaxation rates observed for these dispersions were explained by the free water relaxation rate, the hydration water relaxation rate, and the exchangeable proton relaxation rate. Hydration water was found to be mainly influenced by casein micelle concentration and structure. The variations in hydration with pH were consistent with those observed for classical measurement of voluminosity observed at this range of pH. The effects of lactose and whey protein content are discussed.

1H NMR diffusometry study of water in casein dispersions and gels.

The self-diffusion coefficients of water in casein solutions and gels were measured using a pulsed-gradient spin-echo nuclear magnetic resonance technique (PGSE NMR). The dependence of the self-diffusion coefficient of water on the concentration and structure of casein is reported. The results were analyzed using a cell model. It was found that the water self-diffusion coefficient is insensitive to the structure of the casein in solution or in a gelled state. The influence of casein concentration on the water self-diffusion coefficient could be explained by obstruction from the casein molecule. Assuming a simple model with two water regions, each characterized by a specific water concentration and value of the water diffusion coefficient, the water mobility reduction induced by the casein can be rationalized.

Probe mobility in native phosphocaseinate suspensions and in a concentrated rennet gel: effects of probe flexibility and size.

Pulsed field gradient nuclear magnetic resonance and proton nuclear magnetic resonance relaxometry were used to study the self-diffusion coefficients and molecular dynamics of linear (PEGs) and spherical probes (dendrimers) in native phosphocaseinate suspensions and in a concentrated rennet gel. It was shown that both the size and the shape of the diffusing molecules and the matrix topography affected the diffusion and relaxation rates. In suspensions, both translational and rotational diffusion decreased with increasing casein concentrations due to increased restriction in the freedom of motion. Rotational diffusion was, however, less hindered than translational diffusion. After coagulation, translational diffusion increased but rotational diffusion decreased. Analysis of the T2 relaxation times obtained for probes of different sizes distinguished the free short-chain relaxation formed from a few monomeric units from (i) the relaxation of protons attached to long polymer chains and (ii) the short-chain relaxation attached to a rigid dendrimer core.

MSE-MRI sequence optimisation for measurement of bi- and tri-exponential T2 relaxation in a phantom and fruit.

The transverse relaxation signal from vegetal cells can be described by multi-exponential behaviour, reflecting different water compartments. This multi-exponential relaxation is rarely measured by conventional MRI imaging protocols; mono-exponential relaxation times are measured instead, thus limiting information about of the microstructure and water status in vegetal cells. In this study, an optimised multiple spin echo (MSE) MRI sequence was evaluated for assessment of multi-exponential transverse relaxation in fruit tissues. The sequence was designed for the acquisition of a maximum of 512 echoes. Non-selective refocusing RF pulses were used in combination with balanced crusher gradients for elimination of spurious echoes. The study was performed on a bi-compartmental phantom with known T2 values and on apple and tomato fruit. T2 decays measured in the phantom and fruit were analysed using bi- and tri-exponential fits, respectively. The MRI results were compared with low field non-spatially resolved NMR measurements performed on the same samples. The results demonstrated that the MSE-MRI sequence can be used for up to tri-exponential T2 quantification allowing for estimation of relaxation times from a few tens of milliseconds to over a second. The effects of the crusher moment and the TE value on T2 measurements were studied both on the bi-compartmental phantom and on the fruit tissues. It was demonstrated that the sequence should be optimised with regard to the characteristics of the tissue to be examined by considering the effects of water molecular diffusion in the presence of both imaging gradients and gradients produced by susceptibility inhomogeneities.