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A multi-arm W-band (94 GHz) electron paramagnetic resonance spectrometer that incorporates a loop-gap resonator with high bandwidth is described. A goal of the instrumental development is detection of free induction decay following rapid sweep of the microwave frequency across the spectrum of a nitroxide radical at physiological temperature, which is expected to lead to a capability for Fourier transform electron paramagnetic resonance. Progress toward this goal is a theme of the paper. Because of the low Q-value of the loop-gap resonator, it was found necessary to develop a new type of automatic frequency control, which is described in an appendix. Path-length equalization, which is accomplished at the intermediate frequency of 59 GHz, is analyzed. A directional coupler is favored for separation of incident and reflected power between the bridge and the loop-gap resonator. Microwave leakage of this coupler is analyzed. An oversize waveguide with hyperbolic-cosine tapers couples the bridge to the loop-gap resonator, which results in reduced microwave power and signal loss. Benchmark sensitivity data are provided. The most extensive application of the instrument to date has been the measurement of T values using pulse saturation recovery. An overview of that work is provided. [ABSTRACT FROM AUTHOR]

This paper builds on the work of Mett and Hyde (2003) and Sidabras et al. (2005) where multiple flat aqueous sample cells placed perpendicular to electric fields in microwave cavities were used to reduce the RF losses and increase the EPR signal. In this work, we present three novel sample holders for loop-gap resonators (LGRs) and cylindrical cavity geometries. Two sample holders have been commissioned and built by polytetrafluoroethylene (PTFE) extrusion techniques: a 1 mm O.D. capillary with a septum down the middle, named DoubleDee, and a 3.5 mm O.D. star shaped sample holder, named AquaStar. Simulations and experimental results at X-band show that the EPR signal intensity increases by factors of 1.43 and 3.87 in the DoubleDee and AquaStar respectively, over the current TPX 0.9 mm O.D. sample tube in a two-loop–one-gap LGR. Finally, combining the insight gained from the constructed sample holders and finite-element solutions, a third multi-lumen sample holder for a cylindrical TE 011 cavity is optimized, named AquaSun, where simulations show an EPR signal intensity increase by a factor of 8.2 over a standard 1 mm capillary. [ABSTRACT FROM AUTHOR]

The Gordon coupler was introduced for use in EPR experiments at liquid helium temperatures. It provides an evanescent wave incident on the iris of a microwave resonator. Match of power incident on the coupler to the resonator is obtained by variation of the amplitude of an evanescent wave that arises from displacement of a dielectric wedge in a tapered waveguide. Reduced microphonics from helium bubbling was reported. The Gordon coupler was subsequently extended from cavity resonators to loop-gap resonators, initially at helium temperatures but later for aqueous samples. Plastics with low dielectric constants, usually Teflon, were used. Here, we extend the Gordon coupler for application in X-band five-loop-four-gap resonators using fused quartz, sapphire, or rutile dielectrics, noting that the size of the coupler can then be commensurate with dimensions of dielectric loop-gap resonators as well as dielectric tube resonators. Finite element modeling of electromagnetic fields has been carried out, and use of a capacitive iris that interfaces with the Gordon coupler reduces pulling of the resonant frequency when matching the resonator.

Abstract: Direct irradiation of a sample using a quartz oscillator operating at 250MHz 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. [Copyright &y& Elsevier]

Abstract: Spin–lattice relaxation times (T 1s) of small water-soluble spin-labels in the aqueous phase as well as lipid-type spin-labels in membranes increase when the microwave frequency increases from 2 to 35GHz (Hyde, et al., J. Phys. Chem. B 108 (2004) 9524–9529). The T 1s measured at W-band (94GHz) for the water-soluble spin-labels CTPO and TEMPONE (Froncisz, et al., J. Magn. Reson. 193 (2008) 297–304) are, however, shorter than when measured at Q-band (35GHz). In this paper, the decreasing trends at W-band have been confirmed for commonly used lipid-type spin-labels in model membranes. It is concluded that the longest values of T 1 will generally be found at Q-band, noting that long values are advantageous for measurement of bimolecular collisions with oxygen. The contribution of dissolved molecular oxygen to the relaxation rate was found to be independent of microwave frequency up to 94GHz for lipid-type spin-labels in membranes. This contribution is expressed in terms of the oxygen transport parameter , which is a function of both concentration and translational diffusion of oxygen in the local environment of a spin-label. The new capabilities in measurement of the oxygen transport parameter using saturation-recovery (SR) EPR at Q- and W-band have been demonstrated in saturated (DMPC) and unsaturated (POPC) lipid bilayer membranes with the use of stearic acid (n-SASL) and phosphatidylcholine (n-PC) spin-labels, and compared with results obtained earlier at X-band. SR EPR spin-label oximetry at Q- and W-band has the potential to be a powerful tool for studying samples of small volume, ∼30nL. These benefits, together with other factors such as a higher resonator efficiency parameter and a new technique for canceling free induction decay signals, are discussed. [Copyright &y& Elsevier]

Abstract: Region-selected intensity determination (RSID) is a method for obtaining the temporal changes in electron paramagnetic resonance (EPR) signal intensity from a target region, without the use of complicated procedures employed in the conventional imaging methods. An in vivo 700-MHz radio frequency EPR spectrometer equipped with a bridged loop-gap resonator was used with the RSID method to estimate intracerebral reducing ability in the rat following acute administration of olanzapine (OZP) or haloperidol (HPD). To this end, temporal changes in EPR signal intensity of target regions (the striatum and the prefrontal cortex) of rats which had received a blood-brain-barrier-permeable nitroxide radical (3-hydroxymethyl-2,2,5,5-tetramethylpyrrolidine-1-oxyl) via an intravenous route were observed. The half-lives of EPR signal intensity in both regions of OZP- or HPD-treated rats were significantly longer than in control animals. This indicated that reducing abilities of the striatum and cerebral cortex decreased in the rats to which either OZP or HPD had been acutely administered. [Copyright &y& Elsevier]

[Display omitted] • A 2.0-GHz ESR spectrometer was developed to detect lubrication oil degradation. • The prototype spectrometer is compact for use in the automobile industry. • A marginal oscillator can help detect ESR absorption at 2.0 GHz. • The oscillator circuit involves a loop-gap resonator to realize oscillation. This article reports a simple, compact, and cost-effective electron spin resonance (ESR) spectrometer for monitoring automobile lubrication oil degradation. Lubrication oil degradation strongly correlates with the concentration of stable free radicals caused by hydrocarbon chain decomposition due to heating. For the prototype spectrometer, the amplitude shift in a marginal oscillator output detects ESR absorption in a sample. The spectrometer's spin sensitivity of 2.3 × 1014 spins for a used oil sample was achieved using the marginal oscillator with a loop-gap resonator. For the prototype spectrometer, the oscillation frequency was 2.09 GHz. The volume of the prototype spectrometer was 1.3 L, including a permanent magnet, microwave circuits, and digital communication circuitry on printed circuit boards. The weight of the spectrometer setup was 1.45 kg. This prototype spectrometer successfully detected the ESR signal from a 50 μL oil sample (spin concentration 8.3 x1019 spins/L) with a signal-to-noise ratio of 37 and an acquisition time of 30 s. [ABSTRACT FROM AUTHOR]

Abstract: A reference arm W-band (94GHz) microwave bridge with two sample-irradiation arms for saturation recovery (SR) EPR and ELDOR experiments is described. Frequencies in each arm are derived from 2GHz synthesizers that have a common time-base and are translated to 94GHz in steps of 33 and 59GHz. Intended applications are to nitroxide radical spin labels and spin probes in the liquid phase. An enabling technology is the use of a W-band loop-gap resonator (LGR) [J.W. Sidabras, R.R. Mett, W. Froncisz, T.G. Camenisch, J.R. Anderson, J.S. Hyde, Multipurpose EPR loop-gap resonator and cylindrical TE011 cavity for aqueous samples at 94GHz, Rev. Sci. Instrum. 78 (2007) 034701]. The high efficiency parameter (8.2GW−1/2 with sample) permits the saturating pump pulse level to be just 5mW or less. Applications of SR EPR and ELDOR to the hydrophilic spin labels 3-carbamoyl-2,2,5,5-tetra-methyl-3-pyrroline-1-yloxyl (CTPO) and 2,2,6,6,-tetramethyl-4-piperidone-1-oxyl (TEMPONE) are described in detail. In the SR ELDOR experiment, nitrogen nuclear relaxation as well as Heisenberg exchange transfer saturation from pumped to observed hyperfine transitions. SR ELDOR was found to be an essential method for measurements of saturation transfer rates for small molecules such as TEMPONE. Free induction decay (FID) signals for small nitroxides at W-band are also reported. Results are compared with multifrequency measurements of T 1e previously reported for these molecules in the range of 2–35GHz [J.S. Hyde, J.-J. Yin, W.K. Subczynski, T.G. Camenisch, J.J. Ratke, W. Froncisz, Spin label EPR T1 values using saturation recovery from 2 to 35GHz. J. Phys. Chem. B 108 (2004) 9524–9529]. The values of T 1e decrease at 94GHz relative to values at 35GHz. [Copyright &y& Elsevier]

Abstract: Sample heating induced by radio frequency (RF) irradiation presents a significant challenge to solid state NMR experiments in proteins and other biological systems, causing the sample to dehydrate which may result in distorted spectra and a damaged sample. In this work we describe a large volume, low-E 19F–1H solid state NMR probe, which we developed for the 2D 19F CPMG studies of dilute membrane proteins in a static and electrically lossy environment at 600MHz field. In 19FCPMG and related multi-pulse 19F–1H experiments the sample is heated by the conservative electric fields E produced in the sample coil at both 19F and 1H frequencies. Instead of using a traditional sample solenoid, our low-E 19F–1H probe utilizes two orthogonal loop-gap resonators in order to minimize the conservative electric fields responsible for sample heating. Absence of the wavelength effects in loop-gap resonators results in homogeneous RF fields and enables the study of large sample volumes, an important feature for the dilute protein preparations. The orthogonal resonators also provide intrinsic isolation between the 19F and 1H channels, which is another major challenge for the 19F–1H circuits where Larmor frequencies are only 6% apart. We detail steps to reduce 19F background signals from the probe, which included careful choice of capacitor lubricants and manufacture of custom non-fluorinated coaxial cables. Application of the probe for two-dimensional 19F CPMG spectroscopy in oriented lipid membranes is demonstrated with Flufenamic acid (FFA), a non-steroidal anti-inflammatory drug. [Copyright &y& Elsevier]

Abstract: 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 59GHz; similarly, the EPR signal was translated back to Q-band for detection. A cavity resonant in the cylindrical TE011 mode suitable for use with 100kHz field modulation has also been developed. Results using microwave frequency modulation (FM) at 50kHz as an alternative to magnetic field modulation are described. FM was accomplished by modulating a varactor coupled to the 59GHz oscillator. A spin-label study of sensitivity was performed under conditions of overmodulation and γ 2 H 1 2 T 1 T 2 <1. EPR spectra were obtained, both absorption and dispersion, by lock-in detection at the fundamental modulation frequency (50kHz), and also at the second and third harmonics (100 and 150kHz). 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 3dB points of about 1GHz), the resonator efficiency parameter was calculated to be 9.3G at one W incident power, and the frequency deviation was 11.3MHz p-p, which is equivalent to a field modulation amplitude of 4G. W-band EPR using an LGR is a favorable configuration for microwave FM experiments. [Copyright &y& Elsevier]

Abstract: RF heating of solid-state biological samples is known to be a destabilizing factor in high-field NMR experiments that shortens the sample lifetime by continuous dehydration during the high-power cross-polarization and decoupling pulses. In this work, we describe specially designed, large volume, low-E 15N–1H solid-state NMR probes developed for 600 and 900MHz PISEMA studies of dilute membrane proteins oriented in hydrated and dielectrically lossy lipid bilayers. The probes use an orthogonal coil design in which separate resonators pursue their own aims at the respective frequencies, resulting in a simplified and more efficient matching network. Sample heating at the 1H frequency is minimized by a loop-gap resonator which produces a homogeneous magnetic field B 1 with low electric field E. Within the loop-gap resonator, a multi-turn solenoid closely matching the shape of the sample serves as an efficient observe coil. We compare power dissipation in a typical lossy bilayer sample in the new low-E probe and in a previously reported 15N–1H probe which uses a double-tuned 4-turn solenoid. RF loss in the sample is measured in each probe by observing changes in the 1H 360° pulse lengths. For the same values of 1H B 1 field, sample heating in the new probe was found to be smaller by an order of magnitude. Applications of the low-E design to the PISEMA study of membrane proteins in their native hydrated bilayer environment are demonstrated at 600 and 900MHz. [Copyright &y& Elsevier]

Abstract: Measurement of the bimolecular collision rate between a spin label and oxygen is conveniently carried out using a gas permeable plastic sample tube of small diameter that fits a loop–gap resonator. It is often desirable to concentrate the sample by centrifugation in order to improve the signal-to-noise ratio (SNR), but the deformable nature of small plastic sample tubes presents technical problems. Solutions to these problems are described. Two geometries were considered: (i) a methylpentene polymer, TPX, from Mitsui Chemicals, at X-band and (ii) Teflon tubing with 0.075mm wall thickness at Q-band. Sample holders were fabricated from Delrin that fit the Eppendorf microcentrifuge tubes and support the sample capillaries. For TPX, pressure of the sealant at the end of the sample tube against the Delrin sample holder provided an adequate seal. For Teflon, the holder permitted introduction of water around the tube in order to equalize pressures across the sealant during centrifugation. Typically, the SNR was improved by a factor of five to eight. Oxygen accessibility applications in site-directed spin labeling studies are discussed. [Copyright &y& Elsevier]

• Single crystal rutile can give 6 times the X-band EPR resonator efficiency of an LGR. • A factor of two higher saturable EPR signal for aqueous sample sizes near 400 nL. • Dielectric in the inner loop of an LGR can perform similarly to a DR. • Experimental results are compared with analytic theory and finite element simulations. • Polycrystalline rutile has background signals that make it unsuitable for EPR use. The performance of a metallic microwave resonator that contains a dielectric depends on the separation between metallic and dielectric surfaces, which affects radio frequency currents, evanescent waves, and polarization charges. The problem has previously been discussed for an X-band TE 011 cylindrical cavity resonator that contains an axial dielectric tube (Hyde and Mett, 2017). Here, a short rutile dielectric tube inserted into a loop-gap resonator (LGR) at X-band, which is called a dielectric LGR (dLGR), is considered. The theory is developed and experimental results are presented. It was found that a central sample loop surrounded by four "flux-return" loops (i.e., 5-loop–4-gap) is preferable to a 3-loop–2-gap configuration. For sufficiently small samples (less than 1 µL), a rutile dLGR is preferred relative to an LGR both at constant Λ (B 1 / P l ) and at constant incident power. Introduction of LGR technology to X-band EPR was a significant advance for site-directed spin labeling because of small sample size and high Λ. The rutile dLGR introduced in this work offers further extension to samples that can be as small as 50 nL when using typical EPR acquisition times. [ABSTRACT FROM AUTHOR]

Spin-label W-band (94 GHz) EPR with a five-loop-four-gap resonator (LGR) was successfully applied to study membrane properties (L. Mainali, J.S. Hyde, W.K. Subczynski, Using spin-label W-band EPR to study membrane fluidity in samples of small volume, J. Magn. Reson. 226 (2013) 35-44). 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.