Overhauser Dynamic Nuclear Polarization Research Articles

Dynamic nuclear polarization-enhanced NMR on aligned lipid bilayers at ambient temperature.

Dynamic nuclear polarization (DNP)-enhanced solid-state NMR spectroscopy has been shown to hold great potential for functional studies of membrane proteins at low temperatures due to its great sensitivity improvement. There are, however, numerous applications for which experiments at ambient temperature are desirable and which would also benefit from DNP signal enhancement. Here, we demonstrate as a proof of concept that a significant signal increase for lipid bilayers under room-temperature conditions can be achieved by utilizing the Overhauser effect. Experiments were carried out on aligned bilayers at 400MHz/263GHz using a stripline structure combined with a Fabry-Perot microwave resonator. A signal enhancement of protons of up to -10 was observed. Our results demonstrate that Overhauser DNP at high field provides efficient polarization transfer within insoluble samples, which is driven by fast local molecular fluctuations. Furthermore, our experimental setup offers an attractive option for DNP-enhanced solid-state NMR on ordered membranes and provides a general perspective toward DNP at ambient temperatures.

Cholesterol enhances surface water diffusion of phospholipid bilayers.

Elucidating the physical effect of cholesterol (Chol) on biological membranes is necessary towards rationalizing their structural and functional role in cell membranes. One of the debated questions is the role of hydration water in Chol-embedding lipid membranes, for which only little direct experimental data are available. Here, we study the hydration dynamics in a series of Chol-rich and depleted bilayer systems using an approach termed (1)H Overhauser dynamic nuclear polarization (ODNP) NMR relaxometry that enables the sensitive and selective determination of water diffusion within 5-10 Å of a nitroxide-based spin label, positioned off the surface of the polar headgroups or within the nonpolar core of lipid membranes. The Chol-rich membrane systems were prepared from mixtures of Chol, dipalmitoyl phosphatidylcholine and/or dioctadecyl phosphatidylcholine lipid that are known to form liquid-ordered, raft-like, domains. Our data reveal that the translational diffusion of local water on the surface and within the hydrocarbon volume of the bilayer is significantly altered, but in opposite directions: accelerated on the membrane surface and dramatically slowed in the bilayer interior with increasing Chol content. Electron paramagnetic resonance (EPR) lineshape analysis shows looser packing of lipid headgroups and concurrently tighter packing in the bilayer core with increasing Chol content, with the effects peaking at lipid compositions reported to form lipid rafts. The complementary capability of ODNP and EPR to site-specifically probe the hydration dynamics and lipid ordering in lipid membrane systems extends the current understanding of how Chol may regulate biological processes. One possible role of Chol is the facilitation of interactions between biological constituents and the lipid membrane through the weakening or disruption of strong hydrogen-bond networks of the surface hydration layers that otherwise exert stronger repulsive forces, as reflected in faster surface water diffusivity. Another is the concurrent tightening of lipid packing that reduces passive, possibly unwanted, diffusion of ions and water across the bilayer.

Probing water density and dynamics in the chaperonin GroEL cavity.

ATP-dependent binding of the chaperonin GroEL to its cofactor GroES forms a cavity in which encapsulated substrate proteins can fold in isolation from bulk solution. It has been suggested that folding in the cavity may differ from that in bulk solution owing to steric confinement, interactions with the cavity walls, and differences between the properties of cavity-confined and bulk water. However, experimental data regarding the cavity-confined water are lacking. Here, we report measurements of water density and diffusion dynamics in the vicinity of a spin label attached to a cysteine in the Tyr71 → Cys GroES mutant obtained using two magnetic resonance techniques: electron-spin echo envelope modulation and Overhauser dynamic nuclear polarization. Residue 71 in GroES is fully exposed to bulk water in free GroES and to confined water within the cavity of the GroEL–GroES complex. Our data show that water density and translational dynamics in the vicinity of the label do not change upon complex formation, thus indicating that bulk water-exposed and cavity-confined GroES surface water share similar properties. Interestingly, the diffusion dynamics of water near the GroES surface are found to be unusually fast relative to other protein surfaces studied. The implications of these findings for chaperonin-assisted folding mechanisms are discussed.

An Alderman–Grant resonator for S-Band Dynamic Nuclear Polarization

An Alderman–Grant resonator with resonance at 2GHz (S-Band) was simulated, developed and constructed for Dynamic Nuclear Polarization (DNP) experiments at 73mT. The resonator fits into magnet bores with a minimum diameter of 20mm and is compatible with standard 3mm NMR tubes. The compact resonator design achieves good separation of electric and magnetic fields and therefore can be used with comparatively large sample volumes with only small sample heating effects comparable to those obtained with optimized X- and W-Band DNP setups. The saturation efficiency and sample heating effects were investigated for Overhauser DNP experiments of aqueous solutions of TEMPOL radical, showing relative saturation better than 0.9 and sample heating not exceeding a few Kelvin even at high microwave power and long irradiation time. An application is demonstrated, combining the DNP setup with a commercial fast field cycling NMR relaxometer. Using this resonator design at low microwave frequencies can provide DNP polarization for a class of low-field and time-domain NMR experiments and therefore may enable new applications that benefit from increased sensitivity.

Specific Ions Modulate Diffusion Dynamics of Hydration Water on Lipid Membrane Surfaces

Effects of specific ions on the local translational diffusion of water near large hydrophilic lipid vesicle surfaces were measured by Overhauser dynamic nuclear polarization (ODNP). ODNP relies on an unpaired electron spin-containing probe located at molecular or surface sites to report on the dynamics of water protons within ∼10 Å from the spin probe, which give rise to spectral densities for electron–proton cross-relaxation processes in the 10 GHz regime. This pushes nuclear magnetic resonance relaxometry to more than an order of magnitude higher frequencies than conventionally feasible, permitting the measurement of water moving with picosecond to subnanosecond correlation times. Diffusion of water within ∼10 Å of, i.e., up to ∼3 water layers around the spin probes located on hydrophilic lipid vesicle surfaces is ∼5 times retarded compared to the bulk water translational diffusion. This directly reflects on the activation barrier for surface water diffusion, i.e., how tightly water is bound to the hydrophilic surface and surrounding waters. We find this value to be modulated by the presence of specific ions in solution, with its order following the known Hofmeister series. While a molecular description of how ions affect the hydration structure at the hydrophilic surface remains to be answered, the finding that Hofmeister ions directly modulate the surface water diffusivity implies that the strength of the hydrogen bond network of surface hydration water is directly modulated on hydrophilic surfaces.

Tuning Function of the Light-Driven Proteorhodopsin Proton Pump by Formation of Oligomeric and Surfactant-Based Synthetic Complexes

The Proteorhodopsin (PR) membrane protein equips marine bacteria worldwide with the capability of converting light energy into chemical energy. The proton pump absorbs light via an intrinsic retinal molecule, followed by subsequent conformational changes that store energy in the form of a proton gradient across the bacterial cell membrane. Because PR exists in the complex membrane environment, a multitude of factors could contribute to its light absorption and energy-transducing properties, including protein-protein, protein-lipid, and electrostatic interactions. Here we demonstrate by steady-state and time-resolved optical absorption spectroscopy that there are profound functional effects (e.g. altered activity and photocycle kinetics) modulated by the association of PR with other PR molecules to form oligomeric complexes, and by reconstitution in membrane-mimetic surfactant environments with varying chemical and physical characteristics.We study the molecular-level properties accompanying these functional consequences of environment with magnetic resonance techniques, specifically amino acid side-chain dynamics probed by Electron Paramagnetic Resonance (EPR) and local hydration dynamics by Overhauser Dynamic Nuclear Polarization (ODNP). Such information lends insight into the mechanisms tuning PR function and can be applied towards the design of function-enhancing synthetic environments. For example, surface water diffusion is faster in a model lipid bilayer than in surfactant micelles, concurrent with faster conformational motion, and possibly efficiency (Hussain, et.al, Angew. Chem. 52, 2013). We further have developed a synthetic platform for potential device applications by the incorporation of active PR into surfactant-directed mesostructured silica films, in both its hexameric and monomeric forms, and using different surfactants. This provides a unique opportunity to apply results from the biophysical characterization of PR to the design of an optimized solar energy-harvesting biomaterial, tuning molecular interactions to match the desired functional properties.

Rationalizing Overhauser DNP of nitroxide radicals in water through MD simulations

The recently introduced methodology (Sezer, Phys. Chem. Chem. Phys., 2013, 15, 526) for calculating dynamic nuclear polarization (DNP) coupling factors through synergistic use of molecular dynamics (MD) simulations and the analytical model of diffusing hard spheres with spins at their centers (HSCS) is applied to several nitroxides in water. Computations with one TEMPONE and one TEMPOL in water agree with experiments at 0.35 T and 3.4 T, respectively. At 9.2 T the predicted coupling factors are larger by about 50% than the experimental numbers obtained with 1 M TEMPOL solution. MD simulations at this elevated concentration reveal nanoscopic TEMPOL clusters and qualitatively explain the lower experimental values. Comparing the dynamics from the MD simulations with those of the HSCS model, the assumption of centered spins is shown to be too limiting even for small molecules like TEMPOL and water. Using the available extension of the HSCS model to off-centered spins, the current procedure for analyzing hydration water dynamics from Overhauser DNP measurements on spin-labeled proteins is revisited.

Fluidity and water in nanoscale domains define coacervate hydrogels

Coacervate-based hydrogels, formed in aqueous solution by simple mixing of two oppositely charged ABA block copolyelectrolytes represent a new and versatile approach to the design of bio-inspired gelators. While coacervate-based hydrogels provide high tunability of a range of desirable properties, little is understood about the molecular-level makeup of the nanometer-scale domains. Small angle neutron scattering was employed to quantify the effective polymer density and water content of each domain. Further, electron paramagnetic resonance and Overhauser dynamic nuclear polarization of block-specific spin labels elucidate domain-specific, local, polymer and water dynamics. This unique combination of techniques reveals that the charged A blocks segregate into spherical domains with a radius of 8 nm, and are dispersed in a continuous matrix of water soluble, PEO B blocks. The edges of the spherical A block domains are found to be soft and diffuse, and the B block matrix exhibits higher water and polymer dynamics than the A block domains. The selective measurement of the local water and polymer dynamics shows a viscous and dense, but fluidic environment in the spherical A block domains, thus permitting the designation as a complex coacervate phase. Further, the physical properties of the analogous homopolymers mixed at equal composition to that of the triblock copolyelectrolytes leads to the conclusion that “the whole is greater than the sum of its parts”: nanometer scale complex coacervates only form when the two charged A blocks are covalently linked by a PEO midblock that serves as an intrinsic osmolyte.

High-field liquid state NMR hyperpolarization: a combined DNP/NMRD approach.

Here we show how fast dynamics between radicals and solvent molecules in liquid solutions can be detected by comparison of coupling factors determined by nuclear magnetic relaxation dispersion (NMRD) measurements and dynamic nuclear polarization (DNP) enhancement measurements at high magnetic field (9.2 T). This is important for a theoretical understanding of the Overhauser DNP mechanism at high magnetic fields and thus for optimization of the DNP agent/target system for high resolution liquid state NMR applications. Mixtures of the solution of TEMPOL radicals in water, toluene, acetone and DMSO have been investigated. The results are compared to the classical hard-sphere model and molecular dynamic simulations. Our results clearly indicate that fast sub-ps dynamics, which are not related to classical rotational or translational motion of the molecules, significantly contribute to the Overhauser DNP mechanism at high magnetic fields.

Liquid state dynamic nuclear polarization of ethanol at 3.4 T (95 GHz)

Dynamic Nuclear Polarization (DNP) in the liquid state has become the focus of attention to improve the NMR sensitivity of mass limited samples. The Overhauser model predicts a fast reduction in DNP enhancement at high magnetic fields where the electron Larmor frequency exceeds the typical inverse correlation time of the magnetic interaction between an unpaired electron spin of a radical and proton spins of the solvent molecules. The Overhauser hard sphere model is able to predict quantitatively the DNP enhancement for water TEMPOL solutions. The increase in temperature due to dielectric heating of the sample acts to reduce the correlation times and allows a substantial Overhauser DNP. In this paper we extend the work done on water towards other small molecules, such as ethanol. Experimentally we observe a similar enhancement for all three proton groups in the ethanol molecule. The classical interpretation of the low field Overhauser experiments on ethanol invokes a very fast anisotropic rotation of the hydrogen bonded TEMPOL-ethanol complex to explain the fast relaxation of the OH proton. Here we will discuss W-band relaxation and DNP enhancement within this classical model. Although the description can be made quantitative, the invoked parameters do not seem to be realistic. We will propose an alternative model based on the dynamic interaction both in free collision and due to modulation of the hydrogen bond length of the complex.

Overhauser dynamic nuclear polarization-enhanced NMR relaxometry

We present a new methodological basis for selectively illuminating a dilute population of fluid within a porous medium. Specifically, transport in porous materials can be analyzed by now-standard nuclear magnetic resonance (NMR) relaxometry and NMR pulsed field gradient (PFG) diffusometry methods in combination with the prominent NMR signal amplification tool, dynamic nuclear polarization (DNP). The key components of the approach introduced here are (1) to selectively place intrinsic or extrinsic paramagnetic probes at the site or local volume of interest within the sample, (2) to amplify the signal from the local solvent around the paramagnetic probes with Overhauser DNP, which is performed in situ and under ambient conditions, and (3) to observe the ODNP-enhanced solvent signal with 1D or 2D NMR relaxometry methods, thus selectively amplifying only the relaxation dynamics of the fluid that resides in or percolates through the local porous volume that contains the paramagnetic probe. Here, we demonstrate the proof of principle of this approach by selectively amplifying the NMR signal of only one solvent population, which is in contact with a paramagnetic probe and occluded from a second solvent population. An apparent one-component T2 relaxation decay is shown to actually contain two distinct solvent populations. The approach outlined here should be universally applicable to a wide range of other 1D and 2D relaxometry and PFG diffusometry measurements, including T1–T2 or T1–D correlation maps, where the occluded population containing the paramagnetic probes can be selectively amplified for its enhanced characterization.

Asymmetric Collapse in Biomimetic Complex Coacervates Revealed by Local Polymer and Water Dynamics

Complex coacervation is a phenomenon characterized by the association of oppositely charged polyelectrolytes into micrometer-scale liquid condensates. This process is the purported first step in the formation of underwater adhesives by sessile marine organisms, as well as the process harnessed for the formation of new synthetic and protein-based contemporary materials. Efforts to understand the physical nature of complex coacervates are important for developing robust adhesives, injectable materials, or novel drug delivery vehicles for biomedical applications; however, their internal fluidity necessitates the use of in situ characterization strategies of their local dynamic properties, capabilities not offered by conventional techniques such as X-ray scattering, microscopy, or bulk rheological measurements. Herein, we employ the novel magnetic resonance technique Overhauser dynamic nuclear polarization enhanced nuclear magnetic resonance (DNP), together with electron paramagnetic resonance (EPR) line shape analysis, to concurrently quantify local molecular and hydration dynamics, with species- and site-specificity. We observe striking differences in the structure and dynamics of the protein-based biomimetic complex coacervates from their synthetic analogues, which is an asymmetric collapse of the polyelectrolyte constituents. From this study we suggest charge heterogeneity within a given polyelectrolyte chain to be an important parameter by which the internal structure of complex coacervates may be tuned. Acquiring molecular-level insight to the internal structure and dynamics of dynamic polymer complexes in water through the in situ characterization of site- and species-specific local polymer and hydration dynamics should be a promising general approach that has not been widely employed for materials characterization.

Nonlinear Scaling of Surface Water Diffusion with Bulk Water Viscosity of Crowded Solutions

The translational hydration dynamics within 0.5-1.5 nm of the surface of a DPPC liposome, a model biomacromolecular surface, is analyzed by the recently developed Overhauser dynamic nuclear polarization (ODNP) technique. We find that dramatic changes to the bulk solvent cause only weak changes in the surface hydration dynamics. Specifically, both a >10-fold increase in bulk viscosity and the restriction of diffusion by confinement on a multiple nm length-scale change the local translational diffusion coefficient of the surface water surrounding the lipid bilayer by <2.5-fold. By contrast, previous ODNP studies have shown that changes to the biomacromolecular surface induced by folding, binding, or aggregation can cause local hydration dynamics to vary by factors of up to 30. We suggest that the surface topology and chemistry at the ≤1.5 nm scale, rather than the characteristics of the solvent, nearly exclusively determine the macromolecule's surface hydration dynamics.

Dynamic Nuclear Polarization Methods in Solids and Solutions to Explore Membrane Proteins and Membrane Systems

Membrane proteins regulate vital cellular processes, including signaling, ion transport, and vesicular trafficking. Obtaining experimental access to their structures, conformational fluctuations, orientations, locations, and hydration in membrane environments, as well as the lipid membrane properties, is critical to understanding their functions. Dynamic nuclear polarization (DNP) of frozen solids can dramatically boost the sensitivity of current solid-state nuclear magnetic resonance tools to enhance access to membrane protein structures in native membrane environments. Overhauser DNP in the solution state can map out the local and site-specific hydration dynamics landscape of membrane proteins and lipid membranes, critically complementing the structural and dynamics information obtained by electron paramagnetic resonance spectroscopy. Here, we provide an overview of how DNP methods in solids and solutions can significantly increase our understanding of membrane protein structures, dynamics, functions, and hydration in complex biological membrane environments.

Hydration Dynamics as a Molecular Ruler for Refining Protein Structure at Lipid Membrane Interfaces

Although the 3D protein structure provides the molecular basis for unraveling its unique function, the structural characterization of protein at lipid membrane interfaces is experimentally challenging under physiological conditions. Here, we demonstrate the feasibility of using the long-range and distinct gradient in hydration dynamics along the bilayer normal as a molecular ruler to determine the secondary structure, orientation, and immersion depth of a membrane-associating protein. It was made possible through the quantification of translational diffusion of loosely bound hydration water at the specific sites of protein and lipid bilayer using our newly developed method, Overhauser Dynamic Nuclear Polarization (ODNP)-1H NMR spectroscopy. This study is demonstrated on α-synuclein (αS), a protein associated with Parkinson's disease, free in solution vs. bound on an overall negatively charged phospholipid vesicle. ODNP exhibits that the membrane-bound αS forms an α-helix from residue-76 to 90, where the center of the α-helix is positioned ∼1 below the lipid phosphate. Our data extends the current understanding by showing that residues 90 to 96 of bound αS form a transition segment that links the α-helical domain and the C-terminus with a larger turn than an idealized α-helix, and that the unstructured C-terminus gradually threads outward through a distinct hydration dynamics gradient spanning 10-20 between the water-membrane interface and bulk solvent. Remarkably, ODNP enables to resolve the structural nature of protein functional domains residing 10-20 above the lipid phosphate, which it is difficult to access experimentally. This study debuts the hydration dynamics gradient at protein-lipid interfaces as a high-resolution molecular ruler for probing the structure of membrane proteins and peptides, whose membrane-bound conformation, location and hydration signature are critical to understanding their biological functions.

A comparative study of 1H and 19F Overhauser DNP in fluorinated benzenes

Hyperpolarization techniques, such as Overhauser dynamic nuclear polarization (DNP), can provide a dramatic increase in the signal obtained from nuclear magnetic resonance experiments and may therefore enable new applications where sensitivity is a limiting factor. In this contribution, studies of the (1)H and (19)F Overhauser dynamic nuclear polarization enhancements at 345 mT are presented for three different aromatic solvents with the TEMPO radical for a range of radical concentrations. Furthermore, nuclear magnetic relaxation dispersion measurements of the same solutions are analyzed, showing contributions from dipolar and scalar coupling modulated by translational diffusion and different coupling efficiency for different solvents and nuclei. Measurements of the electron paramagnetic resonance linewidth are included to support the analysis of the DNP saturation factor for varying radical concentration. The results of our study give an insight into the characteristics of nitroxide radicals as polarizing agents for (19)F Overhauser DNP of aromatic fluorinated solvents. Furthermore, we compare our results with the findings of the extensive research on Overhauser DNP that was conducted in the past for a large variety of other radicals.

Protein-Protein and Protein-Lipid Interactions Tune Proteorhodopsin Function by Altering Water Dynamics

Currently, the role of water and the membrane assembly in tuning the function of seven-helical transmembrane (7TM) proteins is not well-understood. Here, we focus on the light activation and functional properties of a prototypical example, the Proteorhodopsin (PR) proton pump from marine bacteria, observing how the protein and surrounding hydration water rearrange upon activation. This is made possible by the application of the powerful residue-specific magnetic resonance methods of electron paramagnetic resonance (EPR), which measures protein segment mobility, and Overhauser dynamic nuclear polarization (ODNP), as recently developed for probing local water diffusivity within 10 Å of a nitroxide spin-label. We investigate further how these dynamics are affected by the surrounding environment, encompassing both protein-protein and protein-lipid interactions. With these techniques together with optical absorption spectroscopy, we find that water dynamics (both ps scale translational motion and ns scale “bound” water) at the membrane protein surface is dramatically affected by the lipid bilayer or surfactant micelle environment. Furthermore, hydration is correlated to functional changes such that water could modulate the timescale of conformational motion. Specifically, the slowdown of translational water motion at the membrane surface, coupled to a lack of bound waters, may facilitate proton uptake by PR. The association of PR with other PR molecules within the membrane, or oligomerization, has similar functional consequences in addition to effects on the protonation properties of key residues for ion transport (pKa of D97). The implication of our study is that PR-PR association alters the hydrogen bond network within the channel, possibly mediated by an altered interaction with the surfactant and surface hydration water upon oligomerization. This result, combined with the homology of PR with sensory receptors, elicits the intriguing possibility that PR has a functional flexibility mediated by oligomerization.

Stressing Lipid Membranes: Effects of Polymers on Membrane Structural Integrity

Cell membrane dysfunction due to loss of structure integrity is the pathology of tissue death in trauma, muscular dystrophies, reperfusion injuries and some common diseases. It is now established that certain poly(ethylene oxide) (PEO)-based biocompatible polymers, such as Poloxamer 188, Poloxamine 1107, and PEO homopolymers, are effective in sealing of injured cell membranes, and thus prevent acute necrosis. Despite the highly potential application of PEO-based polymers for these medical problems, the fundamental mechanism of how these polymers interact with cell membranes are still under debate. Here, the effects of these polymers on structural integrity of lipid vesicles were explored under osmotic and oxidative stress. Through fluorescence leakage assays, time-lapse fluorescence microscopy, dynamic light scattering and isothermal titration calorimetry, we identified that the surface-adsorbed hydrophilic polymers efficiently inhibits the loss of structural integrity of lipid vesicles under external stimuli, while the insertion of the hydrophobic polymers increases membrane permeability. To elucidate the mechanism by which hydrophilic polymers help restore membrane integrity while their hydrophobic counterparts disrupt it, 1H Overhauser Dynamic Nuclear Polarization (ODNP)-NMR spectroscopy, a newly developed NMR technique that is highly effective in differentiating weak surface adsorption versus translocation of polymers to membranes, was employed to detect polymer-lipid membrane interactions through the modulation of local hydration dynamics in lipid membranes. Our study shows that P188, the most hydrophilic poloxamer known as a membrane sealant, weakly adsorbs onto the membrane surface, yet effectively retards membrane hydration dynamics. Contrarily, P181, the most hydrophobic poloxamer known as a membrane permeabilizer, initially penetrates past lipid headgroups and enhances intrabilayer water diffusivity. Consequently, our results illustrate that the relative hydrophilic/hydrophobic ratio of the polymer dictates its functions.

Structural Insight into Proteorhodopsin Oligomers

Oligomerization has important functional implications for many membrane proteins. However, obtaining structural insight into oligomeric assemblies is challenging, as they are large and resist crystallization. We focus on proteorhodopsin (PR), a protein with seven transmembrane α-helices that was found to assemble to hexamers in densely packed lipid membrane, or detergent-solubilized environments. Yet, the structural organization and the subunit interface of these PR oligomers were unknown. We used site-directed spin-labeling together with electron spin-resonance lineshape and Overhauser dynamic nuclear polarization analysis to construct a model for the specific orientation of PR subunits within the hexameric complex. We found intersubunit distances to average 16 Å between neighboring 55 residues and that residues 177 are >20 Å apart from each other. These distance constraints show that PR has a defined and radial orientation within a hexamer, with the 55-site of the A-B loop facing the hexamer core and the 177-site of the E-F loop facing the hexamer exterior. Dynamic nuclear polarization measurements of the local solvent dynamics complement the electron spin-resonance-based distance analysis, by resolving whether protein surfaces at positions 55, 58, and 177 are exposed to solvent, or covered by protein-protein or protein-detergent contacts.

Hyperpolarized Water as an MR Imaging Contrast Agent: Feasibility of in Vivo Imaging in a Rat Model

To assess the feasibility of a perfusion magnetic resonance (MR) imaging technique that uses Overhauser dynamic nuclear polarization (DNP) to provide contrast during the continuous delivery of hyperpolarized water in rats. Protocols approved by the local institutional animal care and use committees were followed. Twelve male Wistar rats were anesthetized and prepared by placing injection tubing in the subcutaneous layer (n=3), peritoneum (n=3), aorta (n=3), or carotid artery (n=3). Water was hyperpolarized by means of Overhauser DNP in the 0.35-T fringe field of a 1.5-T MR imaging magnet by using a custom-built system to continuously deliver radical-free hyperpolarized water to the subject. Fast gradient-echo and spoiled gradient-recalled-echo MR imaging sequences were used. The signal-to-noise ratio (SNR) of the images was calculated and compared. Images showed greatly altered SNR and enhanced flow contrast at all injection locations. For subcutaneous and intraperitoneal injections, the water perfusion trajectory was observed for approximately 5 seconds after injection. Flow through a 4.2-cm length of artery was seen during intra-aortic injection. The right hemisphere of the brain was seen during injection into the right carotid artery. Images with hyperpolarized water had greatly altered SNR compared with images without injection or with the injection of nonhyperpolarized water, with a range of 13%-27% for the carotid and 444%-2900% for the other regions. Perfusion contrast for MR imaging can be obtained by continuously infusing hyperpolarized water, providing localized angiography or brain perfusion information in vivo for rat models.