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Proton-electron double-resonance imaging (PEDRI) employs electron paramagnetic resonance irradiation with low-field magnetic resonance imaging so that the electron spin polarization is transferred to nearby protons, resulting in higher signals. PEDRI provides information about free radical distribution and, indirectly, about the local microenvironment such as partial pressure of oxygen (pOHigh-power radio frequency irradiation is needed for optimum signal enhancement, which may be harmful to living tissue by unwanted heat deposition. Free radical probes differ depending on the purpose of PEDRI. Some probes are less effective for enhancing signal than others, which can reduce image quality. It is so far not possible to image endogenous radicals by PEDRI because low concentrations and broad line widths of the radicals lead to negligible signal enhancement.PEDRI has similarities with electron paramagnetic resonance imaging (EPRI) because both techniques observe the EPR signal, directly in the case of EPRI and indirectly with PEDRI. PEDRI provides information that is vital to research on homeostasis, development of diseases, or treatment responses in vivo. It is expected that the development of new EPR techniques will give insights into novel PEDRI applications and vice versa. Antioxid. Redox Signal. 28, 1345-1364.

Magnetisation Transfer Contrast (MTC) is an important MR contrast-generating mechanism to characterise the MR-invisible macromolecular protons using an off-resonance pre-saturation RF irradiation pulse (or MT pulse). MTC MRI is normally implemented at a fixed magnetic field; however, it may be useful to evaluate changes of the MT effect as a function of external magnetic field strength (B₀). In order to conduct field-dependent MTC experiments with a single MR system, two techniques are crucially needed. B₀ should be able to be switched between levels during irradiation of the MT pulse. At the same time, the resonance frequency of the RF coil (f₀) should also be able to be shifted to the corresponding value. Switching B₀ is attained by the fast field-cycling technique, while in order to switch f₀, a specially designed multi-tunable RF coil is required. Here, we designed and constructed an actively frequency-switchable RF coil for frequencies at and below 2.5 MHz. The design employed PIN diodes, and enabled switching f₀ between five different values, with excellent impedance matching (approximately -37 dB S₁₁ reflection) and Q-factor of about 100 at each configuration.

Theoretical Overhauser-detected EPR spectra of 14 N and 15 N nitroxide systems in low magnetic field by field-cycled dynamic nuclear polarization (FC-DNP) were described by a combination of DNP theory and a model of FC-DNP. Spectra were simulated at magnetic fields between 0 and 8 mT. The simulations were able to predict both the EPR peak positions and their amplitudes, corresponding to those from FC-DNP experiments with 14 N and 15 N TEMPOL solutions. EPR irradiation was in the 45–133 MHz range while NMR signal detection occurred at a field of 59 mT. At this frequency range, four π transitions of a 14 N system and three π transitions of a 15 N system were observed. The simulation programmes were also used to predict the spectral amplitudes of the FC-DNP with EPR irradiation power in the 1–15 W range. Theoretical FC-DNP systems were in good agreement with experimental results; however, at low magnetic fields the inhomogeneity of our magnet system resulted in the EPR peaks being left-shifted and somewhat broader than those from the theoretical prediction.

In vitro and in vivo measurements of pH and thiols provide critical information on physiology and pathophysiology of living organisms, particularly related to oxidative stress. Stable nitroxides of imidazoline and imidazolidine types provide the unique possibility of measuring local values of pH and glutathione content in various biological systems, including in vivo studies. The basis for these applications is the observation of specific chemical reactions of these nitroxides with protons or thiols, followed by significant changes in the electron paramagnetic resonance (EPR) spectra of these probes, measured by low-frequency EPR techniques. The applications of some newly developed pH and SH probes in model systems of pharmacological interest, biological fluids, tissues, and cells as well as in vivo studies in isolated hearts and in the gut of living animals are discussed.

Along with significant progress in low-frequency electron spin resonance (ESR, also called electron paramagnetic resonance, EPR), other techniques such as Longitudinally-Detected ESR (LODESR), proton electron double-resonance imaging (PEDRI) and field-cycled dynamic nuclear polarization (FC-DNP) have been developed forin vivoapplications. However their potential is still far from maximally defined, in part, because of the need for new specific function-directed spin probes. An application of stable nitroxides of imidazoline and imidazolidine types provides unique possibility to measure local values of pH and thiol content in various biological systems, includingin vivostudies. These applications are based on the observation of specific chemical reactions of these nitroxides with protons or thiols, followed by significant changes in their EPR spectra. To increase sensitivity of pH probes for low-frequency EPR spectroscopy we evaluated two alternative approaches: (i) application of isotopically substituted labels, and (ii) acquisition of EPR spectra at high modulation amplitude. Spatial and spectral-spatial imaging (pH-mapping) using PEDRI and L-band EPR imagers were performed both on phantom samples andin vivo. The applications of the pH and SH probes in model systems, biological fluids, andin vivoin living animals are discussed.

Proton electron double resonance imaging (PEDRI) measures the spatial distribution of paramagnetic species in biological samples using the Overhauser effect. Triaryl methyl (TAM) free radicals have been developed as a spin probe for PEDRI since they have very long T1e. Therefore, low RF power levels are sufficient to saturate the electron spin system with resultant high NMR enhancement facilitating application of PEDRI in living animals. In this report, PEDRI studies were performed at 0.02 T. The power-dependent image enhancement was studied using phantoms of TAM in saline, then the distribution and pharmacokinetics of TAM in living mice were measured. Following intravenous administration of 0.7 mmol/kg of TAM, enhancements of up to −34 were observed enabling visualization of its distribution within the body. Maximum uptake of TAM in the vascular compartment was seen 1 min postadministration with half-clearance within 5 min. Maximum uptake in the kidneys occurred at 10 min with half-clearance at 26 min and maximum accumulation in the bladder after 50 min. Thus, TAM is initially compartmentalized in the vasculature and this is followed by rapid uptake and excretion by the kidneys. PEDRI enabled rapid imaging of the distribution and clearance of this paramagnetic probe and this information should facilitate the use of TAM as a label for oximetry and other applications. Magn Reson Med 48:530–534, 2002. © 2002 Wiley-Liss, Inc.

A new pulse sequence for rapid imaging of free radicals is presented which combines snapshot imaging methods and conventional field-cycled proton electron double resonance imaging (FC-PEDRI). The new sequence allows the number of EPR irradiation periods to be optimized to obtain an acceptable SNR and spatial resolution of free radical distribution in the final image while reducing the RF power deposition and increasing the temporal resolution. Centric reordered phase encoding has been employed to counter the problem of rapid decay of the Overhauser-enhanced signal. A phase-correction scheme has also been used to correct problems arising from instability of the magnetic field following field-cycling. In vivo experiments were carried out using triaryl methyl free radical contrast agent, injected at a dose of 0.214 mmol kg(-1) body weight in anaesthetized adult male Sprague-Dawley rats. Transaxial images through the abdomen were collected using 1, 2, 4 and 8 EPR irradiation periods. Using 4 EPR irradiation periods it was possible to generate free radical distributions of acceptable SNR and resolution. The EPR power deposition is reduced by a factor of 16 and the acquisition time is reduced by a factor of 4 compared to an acquisition using the conventional FC-PEDRI pulse sequence.

Proton electron double resonance imaging (PEDRI) uses the Overhauser effect to image the distribution of free-radicals in biological samples and animals. Standard MRI hardware and software is used, with the addition of hardware to irradiate the free-radical-of-interest's EPR resonance. For in vivo applications it must be implemented at a sufficiently low magnetic field to result in an EPR irradiation frequency that will penetrate the sample but will not cause excessive nonresonant power deposition therein. Many clinical MRI systems use resistive magnets that are capable of operating at 10-20 mT, and which could thus be used as PEDRI imagers with the addition of a small amount of extra hardware. This article describes the conversion of a 0.38 T whole-body MRI system for operation as a 20.1 mT small-animal PEDRI imager. The magnet power supply control electronics required a small modification to operate at the lower field strength, but no permanent hardware changes to the MRI console were necessary, and no software modification was required. Frequency down- and up-conversion was used on the NMR RF system, together with a new NMR/EPR dual-resonance RF coil assembly. The system was tested on phantoms containing free-radical solution, and was also used to image the distribution of a free-radical contrast agent injected intravenously into anesthetized mice.

There is growing interest in the use of magnetic resonance imaging (MRI) to examine solid materials where the restricted motion of the probed spins leads to broad lines and short T2 values, rendering many interesting systems invisible to conventional 2DFT pulsed imaging methods. In EPR T2 seldom exceeds 0.1 μs and continuous-wave methods are adopted for spectroscopy and imaging. In this paper we demonstrate the use of continuous-wave MRI to obtain 2-dimensional images of short T2 samples. The prototype system can image samples up to 50 mm in diameter by 60 mm long and has been used to image polymers and water penetration in porous media. Typical acquisition times range between 10 and 40 min. Resolution of 1 to 2 mm has been achieved for samples with T2 values ranging from 38 to 750 μs. There is the possibility of producing image contrast that is determined by the material properties of the sample.

The nitrogen monoxide radical (NO ) forms paramagnetic mono- and dinitrosyl–iron complexes in biologic tissues. To establish a noninvasive technique for in vivo NO imaging, we evaluated the suitability of these complexes as magnetic resonance (MR) contrast agents, making use of the ability of the unpaired electrons of the complexes to enter into dynamic nuclear polarization with water protons and hence produce enhancement on images generated by the technique of proton-electron–double-resonance imaging (PEDRI). Phantom solutions of synthetic nitrosyl–iron complexes (NICs) altered the signal intensity of PEDRI images. The dinitrosyl–iron complex (DNIC) with serum albumin induced a significantly larger signal alteration than the mononitrosyl–iron complex (MNIC) with dithiocarbamate. Exposure of rat liver to sodium nitroprusside (SNP) by ex vivo and in situ perfusion induced a composite X-band electron spin resonance (ESR) spectrum of the isolated liver characteristic of a MNIC and DNIC. On storage of the tissue, the MNIC signal disappeared and the DNIC signal intensity increased. Correspondingly, in cross-sectional PEDRI images taken at room temperature, the SNP-exposed livers initially exhibited a weak signal that strongly increased with time. In conclusion, NICs can be detected using PEDRI and could be exploited for in vivo NO imaging.

We describe a submicrosecond resonator and receiver system for use in pulsed magnetic resonance at 220 MHz. This new resonator and receiver system design enables a reduction of the dead time, and in principle its complete elimination. We show experimentally that the resonator and receiver system permits the detection of free induction decay signals 300 ns from the end of the transmitting pulse, even with large (55-ml) saline samples. This apparatus was specifically developed for in vivo Fourier transform electron paramagnetic resonance detection of free radicals; however, it can also be used in pulsed nuclear magnetic resonance imaging of solids with applications in materials research and the polymer industry.

We report the modification of a low-field whole-body NMR imager to allow radio frequency EPR spectroscopy. The instrument is designed to give optimum sensitivity for in vivo detection of free radicals. The RF circuit is able to operate over a wide frequency range (240 - 320 MHz) and is designed to handle input power levels of up to 12.5 W. The EPR resonator is of the loop - gap type suitable for samples up to 400 ml. A remote resonator coupling method has been developed enabling convenient matching adjustment. An automatic frequency control circuit is able to adjust for frequency deviations caused by animal motion. Where possible, existing imager hardware and commercially available instruments have been used. The instrument is controlled from a central computer via an IEEE 488 instrumentation bus. Here we present sensitivity measurements obtained from a variety of large aqueous samples containing nitroxide free radicals. We show that the instrument is suitable for the detection of exogenous free radicals in 200 g rats.

The birdcage resonator, well appreciated for its high signal-to-noise ratio and its magnetic field uniformity characteristics, operates efficiently in mid- to high-field MRI systems but, unfortunately not for low-field (

We have used tuned receiver coils coupled to a dc SQUID preamplifier in a small scale magnetic resonance imaging (MRI) system operating at 425 kHz (B/sub 0/=0.01 T). The coil and SQUID are cooled to 4.2 K in a modified biomagnetic cryostat. The modifications provide transparency to rf signals originating outside the cryostat while maintaining an acceptably low liquid helium boiloff rate. By applying negative feedback we have damped low loss, high Q-factor SQUID input circuits to achieve a useful imaging bandwidth of over 2 kHz. >

Proton—electron double-resonance imaging (PEDRI) has been developed recently for imaging free radicals in biological samples and small animals. This article summarizes the techniques of PEDRI and the related field-cycled method, FC-PEDRI, and discusses the difficulties in scaling the techniques up to whole-body size. Imaging free radicals with broad EPR lines in humans would require excessive radiofrequency (RF) power, but the use of magnetic field cycling alleviates this problem and improves the signal-to-noise ratio. The results of computer simulations of field-cycled PEDRI are presented, which show that optimum EPR irradiation frequencies exist, depending on the free radical's electron relaxation times and on the applied RF power.

Dynamic nuclear polarization (DNP) is an NMR-based technique which enables detection and spectral characterization of endogenous and exogenous paramagnetic substances measured via transfer of polarization from the saturated unpaired electron spin system to the NMR active nuclei. A variable field system capable of performing DNP spectroscopy with NMR detection at any magnetic field in the range 0–0.38 T is described. The system is built around a clinical open-MRI system. To obtain EPR spectra via DNP, partial cancellation of the detection field B 0 NMR is required to alter the evolution field B 0 EPR at which the EPR excitation is achieved. The addition of resistive actively shielded field cancellation coils in the gap of the primary magnet provides this field offset in the range of 0–100 mT. A description of the primary magnet, cancellation coils, power supplies, interfacing hardware, RF electronics and console are included. Performance of the instrument has been evaluated by acquiring DNP spectra of phantoms with aqueous nitroxide solutions (TEMPOL) at three NMR detection fields of 97 G, 200 G and 587 G corresponding to 413 kHz, 851.6 kHz and 2.5 MHz respectively and fixed EPR evolution field of 100 G corresponding to an irradiation frequency of 282.3 MHz. This variable-field DNP system offers great flexibility for the performance of DNP spectroscopy with independent optimum choice of EPR excitation and NMR detection fields.

The technique of Field-Cycled Dynamic Nuclear Polarization (FC-DNP) involves the EPR irradiation of a free radical solution and the subsequent observation of the NMR signal, the experiment being carried out at a range of magnetic field strengths in order to measure the free radical’s EPR spectrum. In this work FC-DNP has been used to study the EPR spectrum of DMPO spin-trapped hydroxyl free radicals at magnetic field strengths between 0.5 mT and 13.0 mT (5–130 Gauss). The low-field EPR spectrum contains six separate EPR lines, in contrast to the well-known X-band spectrum where only four are seen. Knowledge of the spin-adduct’s EPR spectrum will be of use to workers involved in low-field EPR, especially those conducting biological or in-vivo spin-trapping experiments.

The techniques of proton-electron double-resonance imaging (PEDRI) ( I ) and field-cycled PEDRI (FC-PEDRI) (2) have recently been developed to allow the distribution of free radicals in large aqueous samples to be imaged. In this Communication we describe a development of FC-PEDRI which enables spatially resolved EPR spectral information to be obtained from a series of FC-PEDRI images, in a manner analogous to chemical-shift NMR imaging. We call this development spectral FC-PEDRI. PEDRI relies on dynamic nuclear polarization: a proton NMR image of the sample is collected while an EPR resonance of a free radical solute is irradiated. Under favorable conditions, the NMR signal is enhanced in those parts of the sample where the free radical is influencing the proton relaxation rate, and these regions exhibit greater intensity in the final image. In FC-PEDRI the applied magnetic field B0 is switched rapidly between three values during the course of the pulse sequence, which comprises three distinct periods. The field starts off at a high value of Bz during the polarization period and is then lowered to BE for the evolution period, during which the EPR irradiation is applied, usually for a time of the order of the proton T, . The field is then increased to Bg for the detection period, during which the NMR signal is read out by a 90” pulse and the imaging gradients are applied (2). The enhancement of the NMR signal upon irradiation of the solute’s EPR may be defined as

New improved pH-sensitive nitroxides were applied for in vivo studies. An increased stability of the probes towards reduction was achieved by the introduction of the bulky ethyl groups in the vicinity of the paramagnetic N O fragment. In addition, the range of pH sensitivity of the approach was extended by the synthesis of probes with two ionizable groups, and, therefore, with two p K a values. Stability towards reduction and spectral characteristics of the three new probes were determined in vitro using 290 MHz radiofrequency (RF)- and X-band electron paramagnetic resonance (EPR), longitudinally detected EPR (LODEPR), and field-cycled dynamic nuclear polarization (FC-DNP) techniques. The newly synthesized probe, 4-[bis(2-hydroxyethyl)amino]-2-pyridine-4-yl-2,5,5-triethyl-2,5-dihydro-1 H -imidazol-oxyl, was found to be the most appropriate for the application in the stomach due to both higher stability and convenient pH sensitivity range from pH 1.8 to 6. LODEPR, FC-DNP and proton–electron double resonance imaging (PEDRI) techniques were used to detect the nitroxide localization and acidity in the rat stomach. Improved probe characteristics allowed us to follow in vivo the drug-induced perturbation in the stomach acidity and its normalization afterwards during 1 h or longer period of time. The results show the applicability of the techniques for monitoring drug pharmacology and disease in the living animals.

In this work we show the feasibility of sequential, co-registered fluorine and proton field-cycled Overhauser imaging at a detection field of 59 mT. To this purpose we have built an RF coil assembly comprising an Alderman-Grant resonator for EPR irradiation at 127.7 MHz (evolution field of 4.5 mT) and a solenoidal coil for (19)F or (1)H MRI acquisition at the detection field of 59 mT. A removable tuning/matching circuit that allows the solenoid to be tuned to the (19)F frequency (2.346 MHz, FEDRI) or the (1)H frequency (2.494 MHz, PEDRI) without removing the sample was built and tested. Switching of the solenoid between the (19)F and (1)H frequency is thus achieved in less than 1 min. The co-registered FC-FEDRI and FC-PEDRI images show higher enhancement in the sample regions with higher free radical concentration. This work is the first methodological step towards the development of an MRI scanner capable of acquiring morphological ((1)H) and physiological ((19)F) images in animal models at very low fields.

The development of a 3-D, multi-nuclear continuous wave NMR imaging (CW-NMRI) system is described and its imaging capability is demonstrated on a range of materials exhibiting extremely short T2 relaxation values. A variety of radiofrequency resonators were constructed and incorporated into a new gradient and field offset coil assembly, while the overall system design was modified to minimise microphonic noise which was present in an earlier prototype system. The chemically combined 27Al in a high temperature refractory cement was imaged, and the CW-NMRI system was found to be sensitive to small differences in 27Al content in these samples. The penetration of 23Na in salt water into samples of ordinary Portland cement (OPC) was investigated, with enhanced uptake observed for samples with larger pore size distributions. The solid 13C component in a carbonated cement sample was also imaged, as were the 7Li nuclei in a sample of powdered Li2CO3. A spatial resolution of 1 mm was measured in an image of a rigid polymeric material exhibiting a principal T 2 ∗ value of 16.3 μs. Finally, a high-resolution 3-D image of this rigid polymer is presented.

This paper describes the design, construction and use of a field-cycled proton-electron double-resonance imaging (FC-PEDRI) system for the detection and imaging of free radicals. The unique feature of this imager is its use of a 450-mT detection magnetic field in order to achieve good image quality and sensitivity. The detection magnetic field is provided by a superconducting magnet, giving high stability and homogeneity. Field cycling is implemented by switching on and off the current in an internal, coaxial, resistive secondary magnet that partially cancels the superconducting magnet's field at the sample; the secondary magnet is actively shielded to avoid eddy currents. EPR irradiation takes place at approximately 5 mT, following which the field is switched to 450 mT in 40 ms for NMR signal detection. Full details of the imager's subsystems are given, and experiments to image the distribution of stable free radical contrast agents in phantoms and in anesthetized rats are described.

Electron Paramagnetic Resonance (EPR, or ESR) is a powerful non-invasive spectroscopic tool that can be used to monitor drug release processes in vitro and in vivo. Furthermore, spatial dissolution can be achieved by means of EPR-Imaging. The article introduces the basics of EPR and EPR-imaging. It discusses also the challenges of in vivo spectroscopy and imaging and presents information about new developments such as longitudinally detected EPR (LODEPR) and Proton Electron Double Resonance Imaging ((PEDRI). Examples of the usefulness of EPR in the field of drug delivery include the measurement of microviscosity and micropolarity, the direct detection of drug release mechanisms in vitro and in vivo, the monitoring of microacidity in biodegradable polymers and the characterisation of colloidal drug carriers.

A study was made of the in vivo detectability of a pH-sensitive, imidazolidine spin probe, and the efficacy of low-frequency electron spin resonance (ESR)-based techniques for pH measurement in vitro and in vivo in rats. The techniques used were longitudinally-detected ESR (LODESR) and field-cycled dynamic nuclear polarization (FC-DNP) for in vitro and in vivo measurements, and radiofrequency (RF)- and X-band ESR for comparisons in vitro. The spin probe was hexamethyl imidazolidine (HMI) with a pK of 4.6. All techniques detected HMI. Detection by FC-DNP implies coupling between the free radical and solvent water spins. Separations between the three spectral lines of the nitroxide radical, relative to measurement frequency, were consistent with theory. The overall spectrum width from unprotonated HMI (pH > pK) was greater than that from protonated agent (pH < pK). This was observed in vitro and in vivo. Longer-term studies showed that HMI is detectable and has the same spectral width (i.e., is at the same pH) up to 2 hr after gavage into the stomach, although the magnitude of the signal decreases rapidly during the first hour. These findings demonstrate the suitability of LODESR and FC-DNP for monitoring HMI and measuring pH in vivo. These techniques would be useful for monitoring disease and drug pharmacology in the living system.

Proton-electron double-resonance imaging (PEDRI) is an alternative method to EPR for detecting and imaging free radicals in biological samples or animals. PEDRI uses the Overhauser effect, and involves collecting a proton NMR image while irradiating the EPR of the free radical under study. The spatial resolution in PEDRI is not affected by the linewidth of the free radical’s EPR resonance. An important development of the technique is field-cycled PEDRI (FC-PEDRI), in which the applied magnetic field is switched between two levels during the pulse sequence in order to decrease non-resonant RF power deposition and to increase sensitivity. A spectroscopic version of FC-PEDRI, called field-cycled dynamic nuclear polarization (FC-DNP) is useful for studying low concentrations of free radicals and enables EPR spectra to be obtained via the Overhauser effect. This Chapter describes the techniques and practical implementation of PEDRI and related techniques. Applications of the methods are described, and likely future uses of the techniques are discussed.

PUBLISHED, A prototype continuous wave MRI system operating at 7T has been used successfully to study a variety of heterogeneous materials exhibiting T2 relaxation values ranging from 10 ls to 50 ms. Two-dimensional images of a poly(methly methacrylate) (PMMA) resolution phantom (T2 ?38 ls) exhibited a spatial resolution of approximately 1mm at a magneticfield gradient strength of 200 mT/m. The technique was used to study the hydration, drying, and subsequent water penetration properties of cement samples made from ordinary Portland cement, and revealed inhomogeneities arising from the cure conditions. Sandstone samples from an oil reservoir in the North Sea were also studied; structure within these materials, arising from the sedimentary bed layering in the reservoir, was found to have an effect on their water transport properties. A section from a confectionery bar (T#2;2 approximately 50?60 ms) was also imaged, and its internal structure could be clearly discerned., This work was funded by the UK Engineering and Physical Sciences Research Council (Grant No. GR/R02269/01). The authors thank Dr. H. Seton, Department of Biomedical Physics, for help with the measurements on the 4.7 T system, and Dr. S. Ogilvie, Department of Geology, University of Aberdeen, for supplying the sandstone samples.

A numerical model of the behaviour of the magnetization in a field-cycled dynamic nuclear polarization (DNP) experiment is presented, with the aim of optimizing pulse sequence parameters in field-cycled proton–electron double-resonance free radical imaging. The model is used to predict the observed enhancement of the NMR signal as a function of the magnetic field strength, EPR irradiation frequency and pulse sequence timing, as well as the properties of the sample including the NMR and EPR relaxation times. The model allowed optimization of parameters in the field-cycled DNP experiment, in particular the EPR irradiation frequency, to find the value which would give the largest difference between NMR signals recorded with and without EPR irradiation. Experiments to verify the model were carried out using aqueous solutions of TEMPOL, which exhibits three hyperfine lines in its EPR spectrum and triarylmethyl (TAM), which has a single, narrow line. It was found that the model predicted very well the variation in DNP enhancement with EPR irradiation power for both samples. The behaviour of the NMR signal with EPR irradiation frequency in studies using TEMPOL was also accurately modelled, with the optimum frequency lying between 60 and 80 MHz, depending on the EPR irradiation power. The optimum frequency obtained from the model also agreed with the experimental data obtained using the TAM free radical, but with this sample the theoretical curves tended to deviate from the experimental data at irradiation frequencies below 70 MHz.

Imaging of free radicals in vivo using an interleaved field-cycled proton-electron double-resonance imaging (FC-PEDRI) pulse sequence has recently been investigated. In this work, in order to reduce the EPR (electron paramagnetic resonance) irradiation power required and the imaging time, a centric reordered snapshot FC-PEDRI pulse sequence has been implemented. This is based on the FLASH pulse sequence with a very short repetition time and the use of centric reordering of the phase-encoding gradient, allowing the most significant free induction decay (FID) signals to be collected before the signal enhancement decays significantly. A new technique of signal phaseshift correction was required to eliminate ghost artefacts caused by the instability of the main magnetic field after field cycling. An FID amplitude correction scheme has also been implemented to reduce edge enhancement artefacts caused by the rapid change of magnetization population before reaching the steady state. Using the rapid pulse sequence, the time required for acquisition of a 64 x 64 pixel FC-PEDRI image was reduced to 6 s per image compared with about 2.5 min with the conventional pulse sequence. The EPR irradiation power applied to the sample was reduced by a factor of approximately 64. Although the resulting images obtained by the rapid pulse sequence have a lower signal to noise than those obtained by a normal interleaved FC-PEDRI pulse sequence, the results show that rapid imaging of free radicals in vivo using snapshot FC-PEDRI is possible.

EPR irradiation by a train of inverting pulses has potential advantages over continuous-wave EPR irradiation in DNP applications; however, it has previously been used only at high field (5 T). This paper presents the design and testing of an apparatus for performing pulsed DNP experiments at 10 mT with large samples (17 ml). Experimental results using pulsed DNP with an aqueous solution of a narrow-linewidth paramagnetic probe are presented. A maximum DNP enhancement of about −36 with a train of inverting pulses (width 500 ns, repetition time 4 μs) was measured. A preliminary comparison showed that, when the same enhancement value is considered, the pulsed DNP technique requires an average power that is about three times higher than that required with the CW irradiation. However, for in vivo DNP applications it is very important to minimize the average power deposited in the sample. From the experimental results reported in this work, when considering the maximum enhancement, the pulsed technique requires only 2% of the average power necessary with the CW DNP technique. We believe that this reduction in the average power can be important for future DNP studies with large biological samples.

The use of RF (100 to 300 MHz) PEDRI and CW-EPR techniques allows the in vivo study of large animals such as whole rats and rabbits. Recently a PEDRI instrument was modified to also allow CW-EPR spectroscopy with samples of similar size and under the same experimental conditions. In the present study, this CW-EPR and PEDRI apparatus was used to assess the feasibility of the detection of a pyrrolidine nitroxide free radical (2,2,5,5,-tetramethylpyrrolidine-1-oxyl-3-carboxylic acid, PCA) in the abdomen of rats. In particular, we have shown that after the PCA administration (4 mmol kg(-1) b.w.): (i) the PCA EPR linewidth does not show line broadening due to concentration effects; (ii) a similar PCA up-take phase is observed by EPR and PEDRI; and (iii) the PCA half-lives in the whole abdomen of rats measured with the CW-EPR (T1/2=26+/-4 min, mean+/-sd, n=10) and PEDRI (T1/2=29+/-4 min, mean+/-sd, n=4) techniques were not significantly different (p0.05). These results show, for the first time, that information about PCA pharmacokinetics obtained by CW-EPR is the same as that from PEDRI under the same experimental conditions.

Electron spin resonance (ESR) was first demonstrated in 1945, the same year that the first NMR experiments were carried out. The earliest publication on twodimensional ESR imaging (ESRI) appeared in 1979, around the same time as the first good quality, wholebody NMR images were presented. Fundamentally, ESR and NMR differ only in the fact that one method involves a magnetic resonance experiment on the unpaired electron, while the other uses the atomic nucleus. Why, then, have the rates of progress in the two fields diverged so significantly? Many thousands of MRI machines are now installed worldwide, and there are several hundred active research groups developing new hardware, methodologies and applications. In contrast, there are less than 20 research groups worldwide working on ESRI for biological and medical applications. Is it a lost cause, or is its day still to come? This article will summarise the current status of ESRI and related techniques, and will indicate how, in the author's opinion, the field is likely to develop over the next decade. ESR is able to detect moieties which possess unpaired electrons. These include paramagnetic metal ions, certain solid materials with trapped electrons in crystal defects, as well as free radicals in which the unpaired electron is associated with a particular bond. ESR relies on the fact that the electron has a magnetic moment, by virtue of its charge and quantum-mechanical spin. When a magnetic field is applied, unpaired electrons in the sample tend to align themselves with the field, and will respond when irradiated with electromagnetic radiation at the "resonant" frequency. As in NMR, the frequency is directly proportional to the applied magnetic field strength, although the constants of proportionality are very different. In a 1 tesla magnetic field the proton NMR frequency would be 42.6 MHz, while the ESR frequency for a typical free radical would be 28 GHz, 660 times higher! A practical consequence is that biological ESR cannot be carried out on a clinical MRI machine, as the microwave radiation would not penetrate sufficiently into a conducting sample. Most biomedical ESRI is carried out at fields of around 10 mT, giving a resonant frequency of approximately 300 MHz, or at 30-40 mT, with a resonant frequency of about 1 GHz [1]. The former allows biological samples up to about 70 mm in size to be studied (whole rats), while the latter can be used to examine biological samples with dimensions up to 20 mm (mice, excised organs or tissue samples). Another difference between the two techniques is that NMR relaxation times (7^ and T2) are measured in tens or hundreds of milliseconds in the body, while in ESR they are usually in the 100-1000 ns range. One result of this is that although pulsed ESR is possible with some samples, it is much more difficult to implement than the pulsed NMR techniques which have enabled the rapid development of MRI. To date, all bio-medical ESR and ESRI have used continuous-wave (CW) detection of the ESR signals, involving continuous irradiation of the sample with radiowaves while the magnetic field is increased (swept) slowly past the ESR resonances. As with MRI, ESRI is implemented by applying magnetic field gradients across the sample. The field gradient is applied continuously during the field-sweep. Images are built up by repeating the sweep many times, with the gradient applied in different directions. Very strong magnetic field gradients must be used in order to encode spatially the broad ESR resonances. This, coupled with the fact that the gradients are continuous rather than pulsed, means that ESRI is difficult to apply to large samples, and no animals larger than rats have so far been studied by this technique. ESRI is inherently much slower than MRI. The broad ESR resonances also result in rather poor spatial resolution compared with MRI images. While there is still scope for improvement, it is very unlikely that a whole-body, human-sized ESR imager is possible. Although surface-coil imaging of localized regions of the body is feasible, this will always be more limiting than a true whole-body imaging approach. Proton-electron double-resonance imaging (PEDRI) has been developed with the ultimate intention of imaging free radicals in humans, with superior spatial resolution to ESRI. PEDRI is a combination of standard (albeit low-field) MRI with ESR. The ESR of a free radical of interest is irradiated periodically during the collection of an NMR image. The Overhauser effect causes an increase in the NMR signal amplitude in parts of the sample containing free radicals, revealing the spatial distribution of the free radical. Strong field gradients are not required, and MRI-like spatial resolution is achieved. A development, called field-cycled PEDRI (FC-PEDRI) allows large samples to be imaged by

We have recently published a new method of imaging free radicals in aqueous solutions called proton-electron double-resonance imaging (PEDRI) ( I ). In this technique a conventional proton NMR image is collected while the EPR resonance of a free radical solute is irradiated. If the EPR irradiation has sufficient power, the NMR signal from those protons being relaxed by the paramagnetic solute is enhanced, and the parts of the sample containing free radical exhibit greater intensity in the final image. Unlike EPR imaging (2) the sample size in PEDRI is not constrained by magnetic field gradient requirements. In this Communication we present the first results of an extension of PEDRI which uses magnetic field cycling, allowing larger samples to be imaged with lower levels of applied radiofrequency power. PEDRI is an imaging version of a dynamic nuclear polarization experiment (35). The enhancement of the NMR signal upon irradiation of the EPR of the solute may be written empirically as