Use of multi-coil parallel-gap resonators for co-registration EPR/NMR imaging

This article reports experimental investigations on the use of RF resonators for continuous-wave electron paramagnetic resonance (cw-EPR) and proton nuclear magnetic resonance (NMR) imaging. We developed a composite resonator system with multi-coil parallel-gap resonators for co-registration EPR/NMR imaging. The resonance frequencies of each resonator were 21.8MHz for NMR and 670MHz for EPR. A smaller resonator (22mm in diameter) for use in EPR was placed coaxially in a larger resonator (40mm in diameter) for use in NMR. RF magnetic fields in the composite resonator system were visualized by measuring a homogeneous 4-hydroxy-2,2,6,6-tetramethyl-piperidinooxy (4-hydroxy-TEMPO) solution in a test tube. A phantom of five tubes containing distilled water and 4-hydroxy-TEMPO solution was also measured to demonstrate the potential usefulness of this composite resonator system in biomedical science. An image of unpaired electrons was obtained for 4-hydroxy-TEMPO in three tubes, and was successfully mapped on the proton image for five tubes. Technical problems in the implementation of a composite resonator system are discussed with regard to co-registration EPR/NMR imaging for animal experiments.     

In Vivo EPR Imaging of a Perfluoroalkyl Radical in Mouse Abdomen

Although it is thought that perfluoro-2,4-dimethyl-3-isopropyl-3-pentyl (PFR-2) is a candidate for electron paramagnetic resonance (EPR) imaging agents because of its high stability, no study has been made yet on the EPR imaging of PFR-2. In this study, EPR imaging of a phantom including PFR-2 and mice that had received PFR-2 was performed by an in vivo EPR imaging system operating at an EPR frequency of 700 MHz equipped with a bridged loop-gap resonator (inner diameter, 41 mm; axial length, 10 mm). Because PFR-2 is insoluble in water, it was dissolved in perfluorocarbon. The PFR-2 solution was put in cylindrical sample tubes with various inner diameters, and these sample tubes were placed together in a larger cylindrical sample tube filled with a physiological saline solution, which was used as a phantom. The spatial resolution was estimated to be about 3 mm on the basis of EPR imaging of the phantom. EPR images of mice that had received a PFR-2 injection via the intraperitoneal route indicated that PFR-2 remained in the peritoneal cavity even 2 days after the injection. This finding suggests that it is possible to perform EPR imaging of experimental animals using PFR-2 as an imaging agent which persists in a biological system. Authors' address: Hidekatsu Yokoyama, National Institute of Advanced Industrial Science and Technology, 2266-98 Anagahora, Shimoshidami, Moriyama-ku, Nagoya, Aichi 463-8560, Japan     

Direct EPR irradiation of a sample using a quartz oscillator operating at 250 MHz for EPR measurements

Direct irradiation of a sample using a quartz oscillator operating at 250 MHz was performed for EPR measurements. Because a quartz oscillator is a frequency fixed oscillator, the operating frequency of an EPR resonator (loop-gap type) was tuned to that of the quartz oscillator by using a single-turn coil with a varactor diode attached (frequency shift coil). Because the frequency shift coil was mobile, the distance between the EPR resonator and the coil could be changed. Coarse control of the resonant frequency was achieved by changing this distance mechanically, while fine frequency control was implemented by changing the capacitance of the varactor electrically. In this condition, EPR measurements of a phantom (comprised of agar with a nitroxide radical and physiological saline solution) were made. To compare the presented method with a conventional method, the EPR measurements were also done by using a synthesizer at the same EPR frequency. In the conventional method, the noise level increased at high irradiation power. Because such an increase in the noise was not observed in the presented method, high sensitivity was obtained at high irradiation power.     

Determination of absolute concentration of nitroxide radical by radio-frequency EPR imaging

The absolute concentrations of a nitroxide radical in samples in a loop-gap resonator (LGR) were determined by using a radio-frequency (about 720 MHz) electron paramagnetic resonance (EPR) imaging system. EPR imaging of phantoms containing a nitroxide radical, 3-carbamoyl-2,2,5,5-tetramethylpyrrolidin-1-yloxy (carbamoyl-PROXYL), dissolved in various concentrations of an aqueous sodium chloride solution was made to investigate the influence of dielectric losses and sample position within the LGR. As it was found that these influences on the signal intensity were sufficiently small (less than 6%), it is possible to use identical radical solutions in which the radical is dissolved in a known concentration as an internal marker. Two phantoms containing aqueous solutions of 3 mM (as a marker) and 1, 2, 3, 4, or 5 mM (as a sample) carbamoyl-PROXYL were placed together in the LGR. From EPR images of these phantoms, the absolute concentration of the sample could be calculated by using the gray-scale value (i.e., the signal intensity) of the marker and sample within a small margin of error (about 4%).     

Vertical input optical ring resonator with differential operation for optical sensor

A novel waveguide ring resonator optical sensor with two resonant wavelength channels is proposed for a refractive index measurement of a test sample placed on the sensor substrate and its performance characteristics are investigated analytically and numerically. The waveguide device consists of a ring resonator, a split-ring-shaped loop waveguide, and a vertical input/output grating coupler, in which the loop waveguide acts as an additional resonator and provides another output wavelength channel of the sensor. The differential detection between the two wavelength channels enables the highly sensitive detection with temperature compensation. A numerical simulation based on a finite difference time domain (FDTD) method shows that a precise index change detection with a resolution of 10⁻⁶ can be achieved using of the proposed device.     

1.1-GHz continuous-wave EPR spectroscopy with a frequency modulation method

Continuous-wave EPR spectroscopy using a frequency modulation (FM) scheme was developed. An electronically tunable resonator and an automatic tuning control (ATC) system were used. Using the FM scheme instead of magnetic field modulation, we detected EPR absorption at the first derivative mode. We used a microwave frequency of 1.1 GHz in the present experiment. Similar signal-to-noise ratios were obtained with conventional field modulation and the FM method, and a low-quality factor EPR resonator was not necessary to suppress the significant microwave reflection from the resonator. The FM method with a tunable resonator may be an alternative solution to achieving phase-sensitive detection, when the side-effects of magnetic field modulation, such as microphonic noise and mechanical vibration, are detrimental for EPR detection.     

Rectangular loop-gap resonator with the light access to the sample

A modified rectangular loop-gap resonator for X-band electron paramagnetic resonance (EPR) studies of aqueous samples, enabling the light access, is described. Changes introduced into rectangular resonator geometry, previously presented in Piasecki et al. (1998) [1], and redesigned coupling structure lead to the better thermal and mechanical stability. The modified structure makes provision for the controlled light access to the sample placed in a flat cell during an EPR experiment. The sensitivity of the resonator for aqueous samples as well as an experimentally tested microwave magnetic field homogeneity are presented. Results of simulations and experimental tests indicate that the presence of light access holes in the resonator's front side does not disturb the uniformity of microwave magnetic field distribution in the nodal plane. The optimal flat cell thickness for unsaturable and saturable aqueous samples has been calculated for this new structure. A modified rectangular geometry of the loop-gap resonator ensures a good performance for aqueous samples allowing its convenient and efficient light illumination during EPR signal recording .     

Single loop multi-gap resonator for whole body EPR imaging of mice at 1.2 GHz

For whole body EPR imaging of small animals, typically low frequencies of 250-750 MHz have been used due to the microwave losses at higher frequencies and the challenges in designing suitable resonators to accommodate these large lossy samples. However, low microwave frequency limits the obtainable sensitivity. L-band frequencies can provide higher sensitivity, and have been commonly used for localized in vivo EPR spectroscopy. Therefore, it would be highly desirable to develop an L-band microwave resonator suitable for in vivo whole body EPR imaging of small animals such as living mice. A 1.2 GHz 16-gap resonator with inner diameter of 42 mm and 48 mm length was designed and constructed for whole body EPR imaging of small animals. The resonator has good field homogeneity and stability to animal-induced motional noise. Resonator stability was achieved with electrical and mechanical design utilizing a fixed position double coupling loop of novel geometry, thus minimizing the number of moving parts. Using this resonator, high quality EPR images of lossy phantoms and living mice were obtained. This design provides good sensitivity, ease of sample access, excellent stability and uniform B(1) field homogeneity for in vivo whole body EPR imaging of mice at 1.2 GHz.     

Impact of the Characteristic Impedance of Coaxial Lines on the Sensitivity of a 750-MHz Electronically Tunable EPR Resonator

A 750-MHz electronically tunable resonator was investigated in terms of the sensitivity of electron paramagnetic resonance (EPR) signal detection. The conversion efficiency of the radio-frequency magnetic field was calculated for resonators with 50- and 100-Ω coaxial coupling lines using three-dimensional (3D) microwave field and microwave circuit simulators. Based on the simulation results, two tunable resonators were physically constructed and compared in terms of EPR signal sensitivity using a nitroxyl radical solution. While the resonator with 100-Ω coaxial lines provided 14% greater signal intensity, its signal-to-noise ratio was lower than that of the resonator with 50-Ω lines. To demonstrate the capability of the constructed tunable resonator for EPR imaging experiments, a solution of nitroxyl radical and the leg of a tumor-bearing mouse were visualized.     

Spin-label W-band EPR with Seven-Loop–Six-Gap Resonator: Application to Lens Membranes Derived from Eyes of a Single Donor

Spin-label W-band (94 GHz) electron paramagnetic resonance (EPR) with a five-loop–four-gap resonator (LGR) was successfully applied to study membrane properties (Mainali et al. J Magn Reson 226:35–44, 2013). In that study, samples were equilibrated with the selected gas mixture outside the resonator in a sample volume ~100 times larger than the sensitive volume of the LGR and transferred to the resonator in a quartz capillary. A seven-loop–six-gap W-band resonator has been developed. This resonator permits measurements on aqueous samples of 150 nL volume positioned in a polytetrafluoroethylene (PTFE) gas permeable sample tube. Samples can be promptly deoxygenated or equilibrated with an air/nitrogen mixture inside the resonator, which is significant in saturation-recovery measurements and in spin-label oximetry. This approach was tested for lens lipid membranes derived from lipids extracted from two porcine lenses (single donor). Profiles of membrane fluidity and the oxygen transport parameter were obtained from saturation-recovery EPR using phospholipid analog spin-labels. Cholesterol analog spin-labels allowed discrimination of the cholesterol bilayer domain and acquisition of oxygen transport parameter profiles across this domain. Results were compared with those obtained previously for membranes derived from a pool of 100 lenses. Results demonstrate that EPR at W-band can be successfully used to study aqueous biological samples of small volume under controlled oxygen concentration.     

Raising the sensitivity of the electron-paramagnetic-resonance spectrometer using a ferroelectric resonator

In order to raise the sensitivity of microwave electron-paramagnetic-resonance (EPR) spectrometers, it is proposed to use a piece of ferroelectric material as an additional resonator. The method has been tested using the RE-1307 microwave EPR spectrometer and a pulsed microwave spectrometer. The possibility of raising the signal-to-noise ratio when using ferroelectric resonators of rectangular-parallelepiped and spherical shape has been considered. For a potassium-tantalate ferroelectric resonator of rectangular-parallelepiped shape, the signal-to-noise ratio has been raised by a factor of 16 at 331 K and by a factor of 10 at 292 K. In the pulse experiment, the presence of the ferroelectric resonator permits a reduction in microwave power, required for sample saturation, by a factor of 50 at 50 K.     

Transverse oriented electric field re-entrant resonator (TERR) with automatic tuning and coupling control for EPR spectroscopy and imaging of the beating heart

Sample motion, particularly that of a beating heart, induces baseline noise and spectral distortion on an EPR spectrum. In order to quench motional noise and restore the EPR signal amplitude and line-width, an L-band transverse oriented electric field re-entrant resonator (TERR) was designed and constructed with provisions for automatic tuning control (ATC) and automatic coupling control (ACC) suited for studies of isolated beating rat hearts. Two sets of electronic circuits providing DC biased voltage to two varactor diodes were implemented to electronically adjust coupling and tuning. The resonator has a rectangular cross-sectional sample arm of 25 mm diameter with a Q value of 1100 without sample. Once inserted with lossy aqueous samples of 0.45% NaCl, Q value drops to 400 with a volume of 0.5 ml and 150 with 5 ml. The ATC/ACC functions were tested with a moving phantom and isolated beating rat hearts with the improvement of signal to noise ratio (S/N, peak amplitude of signal over peak amplitude of baseline noise) of 6.7-, and 4 to 6-fold, respectively. With these improvements, EPR imaging could be performed on an isolated beating rat heart. Thus, this TERR resonator with ATC/ACC enables application of EPR spectroscopy and imaging for the measurement and imaging of radical metabolism, redox state, and oxygenation in the isolated beating rat heart.     

Influence of the lens effect in a sample with large dielectric constant in a loop-gap resonator on the EPR signal intensity at 700 MHz

The influence of the lens effect on the electron paramagnetic resonance (EPR) signal intensity was investigated in a loop-gap resonator (LGR) with an inner diameter of 41 mm. TheQ- value and EPR signal intensity were measured when the phantoms containing 3-carbamoyl-2,2,5,5-tetramethyl-pyrrolidin-l-yloxy dissolved in sodium chloride aqueous solutions were put in the LGR. TheQ- value and signal intensity reduced with increasing concentrations of sodium chloride in the phantom, indicating that the imaginary part of the dielectric constant is larger in the phantom with the higher concentration of sodium chloride. However, relationships betweenQ-values of the resonator and EPR signal intensities were not proportional and signal intensities were relatively higher compared with theQ-values. These findings suggest that the signal reduction due to lowQ is slightly compensated by the lens effect in the sample with the large real part of the dielectric constant. In the distribution of the signal intensities of a pinpoint sample made of diphenylpicrylhydrazyl in the agar medium containing sodium chloride in the LGR, it was found that the signal intensity decreased according to the distance from the center and the difference in the signal intensity within 10 mm from the center was about 20%, indicating the inhomogeneity of the alternating magnetic field at the center and marginal region in the sample with the large dielectric constant caused by the lens effect.     

Measurement of specific absorption rate for clinical EPR at 1200 MHz

The specific absorption rate (SAR) has been measured experimentally at 1200 MHz under the conditions and in the configuration used for in vivo electron paramagnetic resonance (EPR) spectroscopy of human subjects. The measurement of SAR was based on an analysis of the time dependence of the temperature of a model tissue (isolated bovine skeletal muscle) placed adjacent to the surface loop of the EPR resonator. The measured SAR in the tissue at the position that should have the greatest density of microwaves, directly under the wire of the loop, was 3.7±1.2 W/kg at 100 mW incident radio-frequency power to a surface loop resonator with an efficiency of about 0.1 mT/W^1/2. This is substantially below the recommended limit in the relevant regulation for extremities. The method of measurement of SAR used in this study can be adapted for other resonators and other types of clinical applications of EPR spectroscopy and imaging.     

Analysis of the tuning and operation of reflection resonator EPR spectrometers

This paper investigates basic characteristics of the electron paramagnetic resonance (EPR) signal obtained from spectrometers employing reflection resonators. General equations are presented which reveal the phase and amplitude dependence on instrumental parameters of both components of the continuous wave (CW) EPR signal (absorption and dispersion). New phase vector diagrams derived from these general equations are presented for the analysis of the EPR response. The dependence of the phase and absolute value of the CW EPR signal on the local oscillator (LO) phase and on resonator offset and coupling is presented and analyzed. The EPR spectrometer tuning procedures for both balanced and unbalanced heterodyne receivers are analyzed in detail using the new phase diagrams. Extraneous signals at the RF input of the microwave receiver (resulting from circulator leakage and reflections in the resonator transmission line) have been taken into account and analyzed. It is shown that a final tuning condition that corresponds to an extremum of the receiver output as a function of the resonator frequency is necessary and sufficient for the acquisition of pure absorption signal. This condition is universal: it applies to all spectrometer configurations in all frequency ranges. High Frequency EPR spectrometer (130 GHz) data are used to generate experimental phase diagrams that illustrate the theoretical concepts presented in the paper. Conditions are presented under which the absorption signal can be measured with complete suppression of the dispersion, independent of the mutual frequency offset between the microwave source and the EPR sample resonator. Equations describing the approximate relationship between changes of the resonator properties (Q-factor and frequency) and paramagnetic susceptibility are derived and analyzed.     

A novel loop-gap resonator probehead for EPR and ENDOR at X-band

An EPR and ENDOR probehead with a loop-gap resonator for X-band is described. The novel feature of the construction is that an iris-type coupling of the resonator is used instead of the conventional antenna coupling. The ENDOR coil combines the role of creating the radio frequency field and that of a shield for the microwave loop-gap structure. Hence, in order to accommodate the iris and waveguide, a pair of RF coils is used in conjunction with a reduced waveguide with dielectric filling. This arrangement simplifies matching the resonator to the microwave bridge, and standard EPR cryostats can be used making sample manipulation more convenient.     

Microwave frequency modulation in CW EPR at W-band using a loop-gap resonator

Loop-gap resonator (LGR) technology has been extended to W-band (94GHz). One output of a multiarm Q-band (35GHz) EPR bridge was translated to W-band for sample irradiation by mixing with 59 GHz; similarly, the EPR signal was translated back to Q-band for detection. A cavity resonant in the cylindrical TE011 mode suitable for use with 100 kHz field modulation has also been developed. Results using microwave frequency modulation (FM) at 50 kHz as an alternative to magnetic field modulation are described. FM was accomplished by modulating a varactor coupled to the 59 GHz oscillator. A spin-label study of sensitivity was performed under conditions of overmodulation and gamma2H1(2)T1T2<1. EPR spectra were obtained, both absorption and dispersion, by lock-in detection at the fundamental modulation frequency (50 kHz), and also at the second and third harmonics (100 and 150 kHz). Source noise was deleterious in first harmonic spectra, but was very low in second and third harmonic spectra. First harmonic microwave FM was transferred to microwave modulation at second and third harmonics by the spins, thus satisfying the "transfer of modulation" principle. The loaded Q-value of the LGR with sample was 90 (i.e., a bandwidth between 3 dB points of about 1 GHz), the resonator efficiency parameter was calculated to be 9.3 G at one W incident power, and the frequency deviation was 11.3 MHz p-p, which is equivalent to a field modulation amplitude of 4 G. W-band EPR using an LGR is a favorable configuration for microwave FM experiments.     

Electronically tunable surface-coil-type resonator for L-band EPR spectroscopy

The automatic frequency control (AFC) circuit in conventional electron paramagnetic resonance (EPR) spectrometers automatically tunes the microwave source to the resonance frequency of the resonator. The circuit works satisfactorily for samples stable enough that the geometric relations in the resonance structure do not change in a significant way. When EPR signals are measured during in vivo experiments with small rodents, however, the distance between the signal source and the surface-coil detector can change rapidly. When a conventional AFC circuit keeps the oscillator tuned to the resonator under those conditions, the resultant frequency change may exceed +/-5 MHz and markedly shift the position of the EPR signal. Such a shift results in unacceptable effects on the spectra, especially when the experimenter is dealing with narrow EPR lines. The animal movement also causes a mismatching of the resonator and the 50-ohm transmission line. Direct results of this mismatching are increased noise; shifts in the position of the baseline; and a high probability of overdriving the signal preamplifier with consequent loss of the EPR signal. We therefore designed, built, and tested a new surface-coil resonator using varactor diodes for tuning the resonance frequency to the fixed frequency oscillator and for capacitive matching of the resonator to the 50-ohm transmission line. The performance of the automatic matching system was tested in vivo by measuring EPR spectra of lithium phthalocyanine implanted in rats. Stability and sensitivity of the spectrometer were evaluated by measuring EPR spectra with and without the use of the automatic matching system. The overall experimental performance of the spectrometer was found to significantly improve during in vivo experiments using the automatic matching system. Excellent matching between the 50-ohm transmission line and the resonator was maintained under all experimental circumstances that were tested. This should allow us now to carry out experiments that previously were not possible.     

Impact of High-Dielectric-Loss Materials on the Microwave Field in EPR Experiments

A dielectric material distorts the microwave field inside an EPR resonator, which results in distortion of the EPR signal from spins inside the material. In this paper, the effects of a spherical bulb filled with a dielectric liquid such as water or a water–ethanol mixture were examined. EPR spectra were recorded for small samples inside and outside of the sphere. The studies include CW and ESE experiments at two microwave frequencies, X band (9.2 GHz) and L band (1.03 GHz). The double integral (area) of an EPR signal depends on[formula]at the position of the sample, causing a large difference in EPR signal intensities between samples in regions of different dielectrics. The phase of the EPR signal also is affected by the presence of the dielectric. These results were compared with three methods of calculating electromagnetic fields (quasi-static method, plane-wave-superposition method, and numerical analysis). Good agreement was found between experimental and calculated results.     

Transfer matrix method for optimizing quasioptical EPR cavities

We present a convenient method for characterizing and optimizing the performance of quasioptical electron paramagnetic resonance (EPR) sample cavities. The formalism is based on the transfer matrix method used in transmission line analysis. Transfer matrix representations are defined for each of the essential components of an open resonator, and the method is demonstrated by application to selected practical examples. Emphasis is given to optimization of quasioptical EPR for aqueous biological samples.