250 MHz Rapid Scan Cross Loop Resonator.

A 25 mm diameter 250 MHz crossed-loop resonator was designed for rapid scan electron paramagnetic resonance imaging. It has a saddle coil for the driven resonator and a fine wire, loop gap resonator for the sample resonator. There is good separation of E and B fields and high isolation between the two resonators, permitting a wide range of sample types to be measured. Applications to imaging of nitroxide, trityl, and LiPc samples illustrate the utility of the resonator. Using this resonator and a trityl sample the signal-to-noise of a rapid scan absorption spectrum is about 20 times higher than for a first-derivative CW spectrum.

Tabletop 700 MHz electron paramagnetic resonance imaging spectrometer.

Low frequency electron paramagnetic resonance imaging is a powerful tool to non-invasively measure the physiological status of tumors. Here, we report on the design and functionality of a rapid scan and pulse table-top imaging spectrometer based around an arbitrary waveform generator and 25mm cross-loop resonator operating at 700 MHz. Two and four-dimensional rapid scan spectral-spatial images are presented. This table-top imager is a prototype for future pre-clinical imagers.

Triarylmethyl Radical OX063d24 Oximetry: Electron Spin Relaxation at 250 MHz and RF Frequency Dependence of Relaxation and Signal-to-Noise.

The triarylmethyl radical OX063d24 is currently used for pulsed electron paramagnetic resonance oximetry at 250 MHz. Both 1/T 1 and 1/T 2 increase with increasing oxygen concentration. The dependence of 1/T 1 on probe concentration is smaller than for 1/T 2. To inform the selection of the optimum frequency for in vivo oximetry 1/T 1, 1/T 2 and signal-to-noise were measured as a function of frequency between 400 and 1000 MHz on a variable-frequency spectrometer with an adjustable-frequency cross-loop resonator. 1/T 1 and 1/T 2 decrease with increasing frequency and signal-to-noise increases with increasing frequency, which are all favourable for imaging at higher frequencies. However, depth of penetration of the radio frequency (RF) into an animal decreases with increasing frequency. Assuming that the RF loss in the animal to be studied determines the resonator Q, our results indicate that the optimum frequency for in vivo imaging will be determined by the desired depth of penetration in the tissue.

An X-Band Crossed-Loop EPR Resonator.

A copper X-band (9.22 GHz) cross loop resonator has been constructed for use with 4 mm sample tubes. The Q for the two resonators are 380 and 350, respectively. The resonator efficiency is about 1 G per square root of watt. Operation has been demonstrated with measurement of T1 by saturation recovery for samples of coal and an immobilized nitroxide radical.

Resonators for In Vivo Imaging: Practical Experience.

Resonators for preclinical electron paramagnetic resonance imaging have been designed primarily for rodents and rabbits and have internal diameters between 16 and 51 mm. Lumped circuit resonators include loop-gap, Alderman-Grant, and saddle coil topologies and surface coils. Bimodal resonators are useful for isolating the detected signal from incident power and reducing dead time in pulse experiments. Resonators for continuous wave, rapid scan, and pulse experiments are described. Experience at the University of Chicago and University of Denver in design of resonators for in vivo imaging is summarized.

Triarylmethyl Radical: EPR Signal to Noise at Frequencies between 250 MHz and 1.5 GHz and Dependence of Relaxation on Radical and Salt Concentration and on Frequency.

In vivo oximetry by pulsed electron paramagnetic resonance is based on measurements of changes in electron spin relaxation rates of probe molecules, such as the triarylmethyl radicals. A series of experiments was performed at frequencies between 250 MHz and 1.5 GHz to assist in the selection of an optimum frequency for oximetry. Electron spin relaxation rates for the triarylmethyl radical OX063 as a function of radical concentration, salt concentration, and resonance frequency were measured by electron spin echo 2-pulse decay and 3-pulse inversion recovery in the frequency range of 250 MHz-1.5 GHz. At constant OX063 concentration, 1/T1 decreases with increasing frequency because the tumbling dependent processes that dominate relaxation at 250 MHz are less effective at higher frequency. 1/T2 also decreases with increasing frequency because 1/T1 is a significant contribution to 1/T2 for trityl radicals in fluid solution. 1/T2-1/T1, the incomplete motional averaging contribution to 1/T2, increases with increasing frequency. At constant frequency, relaxation rates increase with increasing radical concentration due to contributions from collisions that are more effective for 1/T2 than 1/T1. The collisional contribution to relaxation increases as the concentration of counter-ions in solution increases, which is attributed to interactions of cations with the negatively charged radicals that decrease repulsion between trityl radicals. The Signal-to-Noise ratio (S/N) of field-swept echo-detected spectra of OX063 were measured in the frequency range of 400 MHz-1 GHz. S/N values, normalized by √Q, increase as frequency increases. Adding salt to the radical solution decreased S/N because salt lowers the resonator Q. Changing the temperature from 19 to 37 °C caused little change in S/N at 700 MHz. Both slower relaxation rates and higher S/N at higher frequencies are advantageous for oximetry. The potential disadvantage of higher frequencies is the decreased depth of penetration into tissue.

UHF EPR spectrometer operating at frequencies between 400 MHz and 1 GHz.

A spectrometer was designed and constructed to facilitate measurements of T1, T2, spin echo signal-to-noise, and resonator quality factor, Q, between about 400 and 1000 MHz. Pulse patterns are generated at 250 MHz and mixed with the output from a second source to perform excitation and detection. A cross-loop resonator was constructed in which the same sample could be measured in the same resonator over the full range of frequencies. An air-core, 4-coil, water-cooled electromagnet with a large experimental volume was built.

Rapid scan electron paramagnetic resonance at 1.0 GHz of defect centers in γ-irradiated organic solids.

The radicals in six 60Co γ-irradiated solids: malonic acid, glycylglycine, 2,6 di-t-butyl 4-methyl phenol, L-alanine, dimethyl malonic acid, and 2-amino isobutyric acid, were studied by rapid scan electron paramagnetic resonance at L-band (1.04 GHz) using a customized Bruker Elexsys spectrometer and a locally-designed dielectric resonator. Sinusoidal scans with widths up to 18.2 mT were generated with the recently described coil driver and Litz wire coils. Power saturation curves showed that the rapid scan signals saturated at higher powers than did conventional continuous wave signals. The rapid scan data were deconvolved and background subtracted to obtain absorption spectra. For the same data acquisition time the signal-to-noise for the absorption spectra obtained in rapid scans were 23 to 37 times higher than for first-derivative spectra obtained by conventional continuous wave electron paramagnetic resonance.

Imaging Reactive Oxygen Radicals in Excised Mouse Lung Trapped by Reaction with Hydroxylamine Probes Using 1 GHz Rapid Scan Electron Paramagnetic Resonance.

PURPOSE: Oxidative stress is proposed to be critical in acute lung disease, but methods to monitor radicals in lungs are lacking. Our goal is to develop low-frequency electron paramagnetic resonance (EPR) methods to monitor radicals that contribute to the disease. PROCEDURES: Free radicals generated in a lipopolysaccharide-induced mouse model of acute respiratory distress syndrome reacted with cyclic hydroxylamines CPH (1-hydroxy-3-carboxy-2,2,5,5-tetramethylpyrrolidine hydrochloride) and DCP-AM-H (4-acetoxymethoxycarbonyl-1-hydroxy-2,2,5,5-tetramethylpyrrolidine-3-carboxylic acid), which were converted into the corresponding nitroxide radicals, CP• and DCP•. The EPR signals of the nitroxide radicals in excised lungs were imaged with a 1 GHz EPR spectrometer/imager that employs rapid scan technology. RESULTS: The small numbers of nitroxides formed by reaction of the hydroxylamine with superoxide result in low signal-to-noise in the spectra and images. However, since the spectral properties of the nitroxides are known, we can use prior knowledge of the line shape and hyperfine splitting to fit the noisy data, yielding well-defined spectra and images. Two-dimensional spectral-spatial images are shown for lung samples containing (4.5 ± 0.5) ×1014 CP• and (9.9 ± 1.0) ×1014 DCP• nitroxide spins. These results suggest that a probe that accumulates in cells gives a stronger nitroxide signal than a probe that is more easily washed out of cells. CONCLUSION: The nitroxide radicals in excised mouse lungs formed by reaction with hydroxylamine probes CPH and DCP-AM-H can be imaged at 1 GHz.

A Wire Crossed-Loop-Resonator for Rapid Scan EPR.

A crossed-loop (orthogonal mode) resonator (CLR) was constructed of fine wire to achieve design goals for rapid scan in vivo EPR imaging at VHF frequencies (in practice, near 250 MHz). This application requires the resonator to have a very open design to facilitate access to the animal for physiological support during the image acquisition. The rapid scan experiment uses large amplitude magnetic field scans, and sufficiently large resonator and detection bandwidths to record the rapidly-changing signal response. Rapid-scan EPR is sensitive to RF/microwave source noise and to baseline changes that are coherent with the field scan. The sensitivity to source noise is a primary incentive for using a CLR to isolate the detected signal from the RF source noise. Isolation from source noise of 44 and 47 dB was achieved in two resonator designs. Prior results showed that eddy currents contribute to background problems in rapid scan EPR, so the CLR design had to minimize conducting metal components. Using fine (AWG 38) wire for the resonators decreased eddy currents and lowered the resonator Q, thus providing larger resonator bandwidth. Mechanical resonances at specific scan frequencies are a major contributor to rapid scan backgrounds.

Background correction in rapid scan EPR spectroscopy.

In rapid scan EPR the rapidly-changing magnetic field induces a background signal that may be larger than the EPR signal. A method has been developed to correct for that background signal by acquiring two sets of data, denoted as scan 1 and scan 2. In scan 2 the external field B0 is reversed and the data acquisition trigger is offset by one half cycle of the scan field relative to the settings used in scan 1. For data acquired with a cross-loop resonator subtraction of scan 2 from scan 1 cancels the background and enhances the EPR signal. Experiments were performed at an EPR frequency of about 258 MHz, which is in the range that is commonly used for in vivo imaging. Samples include nitroxide radicals, a trityl radical, a dinitroxide, and a nitroxide in the presence of a magnetic field gradient. This method has the advantage that no assumption is made about the shape of the background signal, and it provides an approach to automating the background correction.

Rapid-scan EPR imaging.

In rapid-scan EPR the magnetic field or frequency is repeatedly scanned through the spectrum at rates that are much faster than in conventional continuous wave EPR. The signal is directly-detected with a mixer at the source frequency. Rapid-scan EPR is particularly advantageous when the scan rate through resonance is fast relative to electron spin relaxation rates. In such scans, there may be oscillations on the trailing edge of the spectrum. These oscillations can be removed by mathematical deconvolution to recover the slow-scan absorption spectrum. In cases of inhomogeneous broadening, the oscillations may interfere destructively to the extent that they are not visible. The deconvolution can be used even when it is not required, so spectra can be obtained in which some portions of the spectrum are in the rapid-scan regime and some are not. The technology developed for rapid-scan EPR can be applied generally so long as spectra are obtained in the linear response region. The detection of the full spectrum in each scan, the ability to use higher microwave power without saturation, and the noise filtering inherent in coherent averaging results in substantial improvement in signal-to-noise relative to conventional continuous wave spectroscopy, which is particularly advantageous for low-frequency EPR imaging. This overview describes the principles of rapid-scan EPR and the hardware used to generate the spectra. Examples are provided of its application to imaging of nitroxide radicals, diradicals, and spin-trapped radicals at a Larmor frequency of ca. 250MHz.

Rapid Scan Electron Paramagnetic Resonance Opens New Avenues for Imaging Physiologically Important Parameters In Vivo.

We demonstrate a superior method of 2D spectral-spatial imaging of stable radical reporter molecules at 250 MHz using rapid-scan electron-paramagnetic-resonance (RS-EPR), which can provide quantitative information under in vivo conditions on oxygen concentration, pH, redox status and concentration of signaling molecules (i.e., OH•, NO•). The RS-EPR technique has a higher sensitivity, improved spatial resolution (1 mm), and shorter acquisition time in comparison to the standard continuous wave (CW) technique. A variety of phantom configurations have been tested, with spatial resolution varying from 1 to 6 mm, and spectral width of the reporter molecules ranging from 16 µT (160 mG) to 5 mT (50 G). A cross-loop bimodal resonator decouples excitation and detection, reducing the noise, while the rapid scan effect allows more power to be input to the spin system before saturation, increasing the EPR signal. This leads to a substantially higher signal-to-noise ratio than in conventional CW EPR experiments.

Rapid-scan EPR with triangular scans and fourier deconvolution to recover the slow-scan spectrum.

Direct-detected rapid-scan EPR signals were recorded using triangular field scan rates between 1.7 and 150 kG/s for deoxygenated samples of lithium phthalocyanine (LiPc) and Nycomed trityl-CD3. These scan rates are rapid relative to the reciprocals of the electron spin relaxation times and cause characteristic oscillations in the signals. Fourier deconvolution with an analytical function permitted recovery of lineshapes that are in good agreement with experimental slow-scan spectra. Unlike slow-scan EPR, direct detection rapid-scan EPR does not use phase sensitive detection and records the absorption signal directly instead of the first derivative of the absorption signal. The amplitude of the signal decreases approximately linearly with applied magnetic field gradient. Images of phantoms constructed from samples of LiPc and trityl-CD3 were reconstructed by filtered back-projection from data sets with a missing angle. The lineshapes in spectral slices from the image are in good agreement with slow-scan spectra and the spacing between sample tubes matches well with the known sample geometry.

Field-stepped direct detection electron paramagnetic resonance.

The widest scan that had been demonstrated previously for rapid scan EPR was a 155G sinusoidal scan. As the scan width increases, the voltage requirement across the resonating capacitor and scan coils increases dramatically and the background signal induced by the rapidly changing field increases. An alternate approach is needed to achieve wider scans. A field-stepped direct detection EPR method that is based on rapid-scan technology is now reported, and scan widths up to 6200G have been demonstrated. A linear scan frequency of 5.12kHz was generated with the scan driver described previously. The field was stepped at intervals of 0.01 to 1G, depending on the linewidths in the spectra. At each field data for triangular scans with widths up to 11.5G were acquired. Data from the triangular scans were combined by matching DC offsets for overlapping regions of successive scans. This approach has the following advantages relative to CW, several of which are similar to the advantages of rapid scan. (i) In CW if the modulation amplitude is too large, the signal is broadened. In direct detection field modulation is not used. (ii) In CW the small modulation amplitude detects only a small fraction of the signal amplitude. In direct detection each scan detects a larger fraction of the signal, which improves the signal-to-noise ratio. (iii) If the scan rate is fast enough to cause rapid scan oscillations, the slow scan spectrum can be recovered by deconvolution after the combination of segments. (iv) The data are acquired with quadrature detection, which permits phase correction in the post processing. (v) In the direct detection method the signal typically is oversampled in the field direction. The number of points to be averaged, thereby improving the signal-to-noise ratio, is determined in post processing based on the desired field resolution. A degased lithium phthalocyanine sample was used to demonstrate that the linear deconvolution procedure can be employed with field-stepped direct detection EPR signals. Field-stepped direct detection EPR spectra were obtained for Cu(2+) doped in Ni(diethyldithiocarbamate)2, Cu(2+) doped in Zn tetratolylporphyrin, perdeuterated tempone in sucrose octaacetate, vanadyl ion doped in a parasubstituted Zn tetratolylporphyrin, Mn(2+) impurity in CaO, and an oriented crystal of Mn(2+) doped in Mg(acetylacetonate)2(H2O)2.

Rapid-scan coherence signals in X-band EPR spectra of semiquinones with small hyperfine splittings.

Rapid-scan EPR signals for semiquinones with very-small well-resolved hyperfine splittings exhibit coherence signals at a time after passing through the EPR line that is proportional to the reciprocal of the hyperfine splitting. Such coherences are a general phenomenon due to constructive interference of the responses to transient excitation of spins by rapid scan of the magnetic field across equally spaced spin packets. Examples are shown for 2,3,5,6-tetramethoxy-1,4-benzosemiquinone with aH=46 mG for 12 protons and for 2,5-di-t-butyl-1,4-benzosemiquinone with aH=59 mG for 18 protons.

Imaging disulfide dinitroxides at 250 MHz to monitor thiol redox status.

Measurement of thiol-disulfide redox status is crucial for characterization of tumor physiology. The electron paramagnetic resonance (EPR) spectra of disulfide-linked dinitroxides are readily distinguished from those of the corresponding monoradicals that are formed by cleavage of the disulfide linkage by free thiols. EPR spectra can thus be used to monitor the rate of cleavage and the thiol redox status. EPR spectra of (1)H,(14)N- and (2)H,(15)N-disulfide dinitroxides and the corresponding monoradicals resulting from cleavage by glutathione have been characterized at 250 MHz, 1.04 GHz, and 9 GHz and imaged by rapid-scan EPR at 250 MHz.

Frequency dependence of EPR signal intensity, 248 MHz to 1.4 GHz.

The electron paramagnetic resonance pulsed free induction decay (FID) of a degassed solution of a triaryl methyl radical, methyl tris(8-carboxy-2,2,6,6-tetramethyl(-d3)-benzo[1,2-d:4,5-d']bis(1,3)dithiol-4-yl) tripotassium salt, 0.2 mM in H2O, was measured at VHF (247.5 MHz) and L-band (1.40 GHz). The calculated and observed FID signal amplitudes (in millivolts) agreed within 1 and 6%, and the ratio of the normalized FID signals at the two frequencies agreed within 5%. The FID decay time constant was 2.7 micros at both frequencies.

Enhanced signal intensities obtained by out-of-phase rapid-passage EPR for samples with long electron spin relaxation times.

To understand the signals that are observed under rapid-passage conditions for samples with long electron spin relaxation times, the E' defect in irradiated vitreous SiO(2) was studied. For these samples at room temperature, T(1) is 200 mciro s and T(2) ranged from 35 to 200 micro s, depending on spin concentration. At X band with 100-kHz modulation frequency and 1-G modulation amplitude there was minimal lineshape difference between the low-power, in-phase spectra and high-power spectra detected 90 degrees out-of-phase with respect to the magnetic field modulation. Signal enhancement, defined as the ratio of the intensities of the out-of-phase to the in-phase signals when B(1) for both observation modes is adjusted to give maximum signal, was 3.4 to 9.5 at room temperature. The origin of the out-of-phase signal was modeled by numerical integration of the Bloch equations including magnetic field modulation. The waveforms for the E' signal, prior to phase sensitive detection, were simulated by summing the contributions of many individual spin packets. Good agreement was obtained between experimental and calculated waveforms. At low B(1) the experimental values of T(1) and T(2) were used in the simulations. However, at higher B(1), T(2) was adjusted to match the experimental signal intensity and increased with increasing B(1). At high B(1), T(2)=T(1), consistent with Redfield's and Hyde's models. For the spin concentrations examined, the out-of-phase signals at very high power (B(1) approximately 0.33 G) displayed a linear relationship between peak-to-peak signal amplitude and spin concentration. Under the conditions used for spin quantitation the signal-to-noise for these spectra was up to 5 times higher than for the in-phase signal, which greatly facilitates quantitation for these types of samples. For samples in which T(2) is dominated by electron spin-spin interaction, lower spin concentration results in longer T(2) and the enhancement is increased.

Frequency dependence of EPR signal intensity, 250 MHz to 9.1 GHz.

Experimental EPR signal intensities at 250 MHz, 1.5 GHz, and 9.1 GHz agree within experimental error with predictions from first principles. When both the resonator size and the sample size are scaled with the inverse of RF/microwave frequency, omega, the EPR signal at constant B(1) scales as omega(-1/4). Comparisons were made for three different samples in two pairs of loop gap resonators. Each pair was geometrically scaled by a factor of 6. One pair of resonators was scaled from 250 MHz to 1.5 GHz, and the other pair was scaled from 1.5 GHz to 9 GHz. All terms in the comparison were measured directly, and their uncertainties estimated. The theory predicts that the signal at the lower frequency will be larger than the signal at the higher frequency by the ratio 1.57. For 250 MHz to 1.5 GHz, the experimental ratio was 1.52 and for the 1.5-GHz to 9-GHz comparison the ratio was 1.14.