Gordon Coupler with Inductive or Capacitive Iris for Small EPR Resonators for Aqueous Samples.

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.

Dispersion EPR: Considerations for Low-Frequency Experiments.

The hypothesis is made that the dispersion electron paramagnetic resonance (EPR) spectrum can yield a higher signal-to-noise ratio than the absorption spectrum in diagnostic examinations if phase noise in the bridge is under control. The rationale for this hypothesis is based on the observation that the dispersion spectrum becomes more intense than the absorption spectrum at high incident powers. The rationale is dependent on optimization of high microwave efficiency (Λ; mT/W1/2) and low quality factor (Q-value) resonators as well as the use of microwave sources with reduced phase noise. Microwave frequencies from 1.2 to 94 GHz are considered. Although the dispersion display appears to be observable with an adequate signal-to-noise ratio for most EPR research initiatives, a weakness of microwave bridges for studies at high incident microwave power was identified. Spurious leakage of incident microwave power through the circulator, thereby bypassing the probe leading to the resonator, can result in a decreased signal-to-noise ratio in both absorption and dispersion because of phase noise. For dispersion EPR with low Q-value sample resonators, this leakage is the primary contributor to phase noise at the receiver. In this work, we focus on the design of microwave reflection bridges and discuss possible methods to ameliorate this source of noise.

Characterization of the distribution of spin-lattice relaxation rates of lipid spin labels in fiber cell plasma membranes of eye lenses with a stretched-exponential function.

The stretched exponential function (SEF) was used to analyze and interpret saturation recovery (SR) electron paramagnetic resonance (EPR) data obtained from spin-labeled porcine eye-lens membranes. This function has two fitting parameters: the characteristic spin-lattice relaxation rate (T 1str -1) and the stretching parameter (ß), which ranges between zero and one. When ß = 1, the function is a single exponential. It is assumed that the SEF arises from a distribution of single exponential functions, each described by a T 1 value. Because T 1 -1s are determined primarily by the rotational diffusion of spin labels, they are a measure of membrane fluidity. Since ß describes the distribution of T 1 -1s, it can be interpreted as a measure of membrane heterogeneity. The SEF was used to analyze SR data obtained from intact cortical and nuclear fiber cell plasma membranes extracted from the eye lenses of two-year old animals and spinlabeled with phospholipid- and cholesterol-analogs. The lipid environment sensed by these probe molecules was found to be less fluid and more heterogeneous in nuclear membranes than in cortical membranes. Parameters T 1str -1 and ß were also used for a multivariate K-means cluster analysis of stretched-exponential data. This analysis indicates that SEF data can be assigned accurately to clusters in nuclear or cortical membranes. In future work, the SEF will be applied to analyze data from human eye lenses of donors with differing health histories.

Autobiography of James S. Hyde.

The papers, book chapters, reviews, and patents by James S. Hyde in the bibliography of this document have been separated into EPR and MRI sections, and within each section by topics. Within each topic, publications are listed chronologically. A brief summary is provided for each patent listed. A few publications and patents that do not fit this schema have been omitted. This list of publications is preceded by a scientific autobiography that focuses on selected topics that are judged to have been of most scientific importance. References to many of the publications and patents in the bibliography are made in the autobiography.

EPR UNIFORM FIELD SIGNAL ENHANCEMENT BY DIELECTRIC TUBES IN CAVITIES.

The dielectric tube resonator (DTR) for EPR spectroscopy is introduced. It is defined as a metallic cylindrical TE011 microwave cavity that contains a dielectric tube centered on the axis of the cylinder. Contour plots of dimensions of the metallic cylinder to achieve resonance at 9.5 GHz are shown for quartz, sapphire, and rutile tubes as a function of wall thickness and average radius. These contour plots were developed using analytical equations and confirmed by finite element modeling. They can be used in two ways: design of the metallic cylinder for use at 9.5 GHz that incorporates a readily available tube such as a sapphire tube intended for NMR, or design of a custom procured tube for optimized performance for specific sample-size constraints. The charts extend to the limiting condition where the dielectric fills the tube. However, the structure at this limit is not a dielectric resonator due to the metal wall and does not radiate. In addition, the uniform field (UF) DTR is introduced. Development of the UF resonator starting with a dielectric tube resonator is shown. The diameter of the tube remains constant along the cavity axis, and the diameter of the cylindrical metallic enclosure increases at the ends of the cavity to satisfy the uniform field condition. This structure has advantages over the previously developed UF TE011 resonators: higher resonator efficiency parameter Λ, convenient overall size when using sapphire tubes, and higher quality data for small samples. The DTR and UF DTR structures fill the gap between free space and dielectric resonator limits in a continuous manner.

Saturation recovery EPR spin-labeling method for quantification of lipids in biological membrane domains.

The presence of integral membrane proteins induces the formation of distinct domains in the lipid bilayer portion of biological membranes. Qualitative application of both continuous wave (CW) and saturation recovery (SR) electron paramagnetic resonance (EPR) spin-labeling methods allowed discrimination of the bulk, boundary, and trapped lipid domains. A recently developed method, which is based on the CW EPR spectra of phospholipid (PL) and cholesterol (Chol) analog spin labels, allows evaluation of the relative amount of PLs (% of total PLs) in the boundary plus trapped lipid domain and the relative amount of Chol (% of total Chol) in the trapped lipid domain [M. Raguz, L. Mainali, W. J. O'Brien, and W. K. Subczynski (2015), Exp. Eye Res., 140:179-186]. Here, a new method is presented that, based on SR EPR spin-labeling, allows quantitative evaluation of the relative amounts of PLs and Chol in the trapped lipid domain of intact membranes. This new method complements the existing one, allowing acquisition of more detailed information about the distribution of lipids between domains in intact membranes. The methodological transition of the SR EPR spin-labeling approach from qualitative to quantitative is demonstrated. The abilities of this method are illustrated for intact cortical and nuclear fiber cell plasma membranes from porcine eye lenses. Statistical analysis (Student's t-test) of the data allowed determination of the separations of mean values above which differences can be treated as statistically significant (P ≤ 0.05) and can be attributed to sources other than preparation/technique.

Spin-labeled small unilamellar vesicles with the T1-sensitive saturation-recovery EPR display as an oxygen sensitive analyte for measurement of cellular respiration.

This study validated the use of small unilamellar vesicles (SUVs) made of 1-palmitoyl-2-oleoylphosphatidylcholine with 1 mol% spin label of 1-palmitoyl-2-(16-doxylstearoyl)phosphatidylcholine (16-PC) as an oxygen sensitive analyte to study cellular respiration. In the analyte the hydrocarbon environment surrounds the nitroxide moiety of 16-PC. This ensures high oxygen concentration and oxygen diffusion at the location of the nitroxide as well as isolation of the nitroxide moiety from cellular reductants and paramagnetic ions that might interfere with spin-label oximetry measurements. The saturation-recovery EPR approach was applied in the analysis since this approach is the most direct method to carry out oximetric studies. It was shown that this display (spin-lattice relaxation rate) is linear in oxygen partial pressure up to 100% air (159 mmHg). Experiments using a neuronal cell line in suspension were carried out at X-band for closed chamber geometry. Oxygen consumption rates showed a linear dependence on the number of cells. Other significant benefits of the analyte are: the fast effective rotational diffusion and slow translational diffusion of the spin-probe is favorable for the measurements, and there is no cross reactivity between oxygen and paramagnetic ions in the lipid bilayer.

Direct radiofrequency phase control in MRI by digital waveform playback at the Larmor frequency.

PURPOSE: A scalable multiband and multichannel digital magnetic resonance imaging system has been developed with the goal of reducing the time needed for acquisition of a single volume of gradient-recalled echo-planar images of the brain. METHODS: Transmit pulses are created by an offline computer equipped with a Pentek excitation card (PCIe model 78621) that was built around the Texas Instruments D/A converter (DAC5688). RESULTS: The spectral purity of pulses made in this way surpasses the quality of pulses made by the standard modulators of the scanner, even when using the same pulse-creation algorithm. There is no need to mix reference waveforms with the magnetic resonance imaging signal to obtain inter-k-space coherency for different repetitions. The key was the use of a system clock to create the Larmor frequency used for pulse formation. The 3- and 4-fold slice accelerations were tested using phantoms as well as functional and resting-state magnetic resonance imaging of the human brain. CONCLUSION: Synthesizers with limited modulation-time steps should be replaced not only because of the improved spectral quality of radiofrequency pulses but also for the exceptional coherence of pulses at different slice-selection frequencies.

C7 nerve root sensory distribution in peripheral nerves: a bold functional magnetic resonance imaging investigation at 9.4 T.

INTRODUCTION: In this study we used a rat model to elucidate the linear make-up of each major nerve of the upper limb by the C7 root through sensory stimulation and functional magnetic resonance imaging (fMRI). METHODS: The C7 nerve root and major nerves of the right forelimb were stimulated electrically. Blood oxygen level-dependent functional magnetic resonance imaging (BOLD fMRI) was performed concurrently. Voxel overlap within the primary sensory cortex was calculated. RESULTS: C7 comprised sensation in <1% in the musculocutaneous nerve, 6% in the ulnar nerve, 16% in the radial nerve, and 19% in the median nerve (P<0.005 for each). The overlap was always <25% for each major nerve. CONCLUSIONS: This study helps explain why C7 is a suitable donor for brachial plexus injury treatment and why there is only a transient sensory deficit after transfer.

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) 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.

A pressure-jump EPR system to monitor millisecond conformational exchange rates of spin-labeled proteins.

Site-directed spin labeling electron paramagnetic resonance (SDSL-EPR) using nitroxide spin labels is a well-established technology for mapping site-specific secondary and tertiary structure and for monitoring conformational changes in proteins of any degree of complexity, including membrane proteins, with high sensitivity. SDSL-EPR also provides information on protein dynamics in the time scale of ps-µs using continuous wave lineshape analysis and spin lattice relaxation time methods. However, the functionally important time domain of µs-ms, corresponding to large-scale protein motions, is inaccessible to those methods. To extend SDSL-EPR to the longer time domain, the perturbation method of pressure-jump relaxation is implemented. Here, we describe a complete high-pressure EPR system at Q-band for both static pressure and millisecond-timescale pressure-jump measurements on spin-labeled proteins. The instrument enables pressure jumps both up and down from any holding pressure, ranging from atmospheric pressure to the maximum pressure capacity of the system components (~3500 bar). To demonstrate the utility of the system, we characterize a local folding-unfolding equilibrium of T4 lysozyme. The results illustrate the ability of the system to measure thermodynamic and kinetic parameters of protein conformational exchange on the millisecond timescale.

Toward discovery science of human brain function.

Although it is being successfully implemented for exploration of the genome, discovery science has eluded the functional neuroimaging community. The core challenge remains the development of common paradigms for interrogating the myriad functional systems in the brain without the constraints of a priori hypotheses. Resting-state functional MRI (R-fMRI) constitutes a candidate approach capable of addressing this challenge. Imaging the brain during rest reveals large-amplitude spontaneous low-frequency (<0.1 Hz) fluctuations in the fMRI signal that are temporally correlated across functionally related areas. Referred to as functional connectivity, these correlations yield detailed maps of complex neural systems, collectively constituting an individual's "functional connectome." Reproducibility across datasets and individuals suggests the functional connectome has a common architecture, yet each individual's functional connectome exhibits unique features, with stable, meaningful interindividual differences in connectivity patterns and strengths. Comprehensive mapping of the functional connectome, and its subsequent exploitation to discern genetic influences and brain-behavior relationships, will require multicenter collaborative datasets. Here we initiate this endeavor by gathering R-fMRI data from 1,414 volunteers collected independently at 35 international centers. We demonstrate a universal architecture of positive and negative functional connections, as well as consistent loci of inter-individual variability. Age and sex emerged as significant determinants. These results demonstrate that independent R-fMRI datasets can be aggregated and shared. High-throughput R-fMRI can provide quantitative phenotypes for molecular genetic studies and biomarkers of developmental and pathological processes in the brain. To initiate discovery science of brain function, the 1000 Functional Connectomes Project dataset is freely accessible at www.nitrc.org/projects/fcon_1000/.

Cross-correlation: an fMRI signal-processing strategy.

The discovery of functional MRI (fMRI), with the first papers appearing in 1992, gave rise to new categories of data that drove the development of new signal-processing strategies. Workers in the field were confronted with image time courses, which could be reshuffled to form pixel time courses. The waveform in an active pixel time-course was determined not only by the task sequence but also by the hemodynamic response function. Reference waveforms could be cross-correlated with pixel time courses to form an array of cross-correlation coefficients. From this array of numbers, colorized images could be created and overlaid on anatomical images. An early paper from the authors' laboratory is extensively reviewed here (Bandettini et al., 1993. Magn. Reson. Med. 30:161-173). That work was carried out using the vocabulary of vector algebra. Cross-correlation methodology was central to the discovery of functional connectivity MRI (fcMRI) by Biswal et al. (1995. Magn. Reson. Med. 34:537-541). In this method, a whole volume time course of images is collected while the brain is nominally at rest and connectivity is studied by cross-correlation of pixel time courses.

Interhemispheric neuroplasticity following limb deafferentation detected by resting-state functional connectivity magnetic resonance imaging (fcMRI) and functional magnetic resonance imaging (fMRI).

Functional connectivity magnetic resonance imaging (fcMRI) studies in rat brain show brain reorganization following peripheral nerve injury. Subacute neuroplasticity was observed 2 weeks following transection of the four major nerves of the brachial plexus. Direct stimulation of the intact radial nerve reveals a functional magnetic resonance imaging (fMRI) activation pattern in the forelimb regions of the sensory and motor cortices that is significantly different from that observed in normal rats. Results of this fMRI experiment were used to determine seed voxel regions for fcMRI analysis. Intrahemispheric connectivities in the sensorimotor forelimb representations in both hemispheres are largely unaffected by deafferentation, whereas substantial disruption of interhemispheric sensorimotor cortical connectivity occurs. In addition, significant intra- and interhemispheric changes in connectivities of thalamic nuclei were found. These are the central findings of the study. They could not have been obtained from fMRI studies alone-both fMRI and fcMRI are needed. The combination provides a general marker for brain plasticity. The rat visual system was studied in the same animals as a control. No neuroplastic changes in connectivities were found in the primary visual cortex upon forelimb deafferentation. Differences were noted in regions responsible for processing multisensory visual-motor information. This incidental discovery is considered to be significant. It may provide insight into phantom limb epiphenomena.

EPR of Cu2+ prion protein constructs at 2 GHz using the g(perpendicular) region to characterize nitrogen ligation.

A double octarepeat prion protein construct, which has two histidines, mixed with copper sulfate in a 3:2 molar ratio provides at most three imidazole ligands to each copper ion to form a square-planar Cu(2+) complex. This work is concerned with identification of the fourth ligand. A new (to our knowledge) electron paramagnetic resonance method based on analysis of the intense features of the electron paramagnetic resonance spectrum in the g( perpendicular) region at 2 GHz is introduced to distinguish between three and four nitrogen ligands. The methodology was established by studies of a model system consisting of histidine imidazole ligation to Cu(2+). In this spectral region at 2 GHz (S-band), g-strain and broadening from the possible rhombic character of the Zeeman interaction are small. The most intense line is identified with the M(I) = +1/2 extra absorption peak. Spectral simulation demonstrated that this peak is insensitive to cupric A(x) and A(y) hyperfine interaction. The spectral region to the high-field side of this peak is uncluttered and suitable for analysis of nitrogen superhyperfine couplings to determine the number of nitrogens. The spectral region to the low-field side of the intense extra absorption peak in the g( perpendicular) part of the spectrum is sensitive to the rhombic distortion parameters A(x) and A(y). Application of the method to the prion protein system indicates that two species are present and that the dominant species contains four nitrogen ligands. A new loop-gap microwave resonator is described that contains approximately 1 mL of frozen sample.

A protocol for use of medetomidine anesthesia in rats for extended studies using task-induced BOLD contrast and resting-state functional connectivity.

The alpha-2-adrenoreceptor agonist, medetomidine, which exhibits dose-dependent sedative effects and is gaining acceptance in small-animal functional magnetic resonance imaging (fMRI), has been studied. Rats were examined on the bench using the classic tail-pinch method with three infusion sequences: 100 microg/kg/h, 300 microg/kg/h, or 100 microg/kg/h followed by 300 microg/kg/h. Stepping the infusion rate from 100 to 300 microg/kg/h after 2.5 h resulted in a prolonged period of approximately level sedation that cannot be achieved by a constant infusion of either 100 or 300 microg/kg/h. By stepping the infusion dosage, experiments as long as 6 h are possible. Functional MRI experiments were carried out on rats using a frequency dependent electrical stimulation protocol-namely, forepaw stimulation at 3, 5, 7, and 10 Hz. Each rat was studied for a four-hour period, divided into two equal portions. During the first portion, rats were started at a 100 microg/kg/h constant infusion. During the second portion, four secondary levels of infusion were used: 100, 150, 200, and 300 microg/kg/h. The fMRI response to stimulation frequency was used as an indirect measure of modulation of neuronal activity through pharmacological manipulation. The frequency response to stimulus was attenuated at the lower secondary infusion dosages 100 or 150 microg/kg/h but not at the higher secondary infusion dosages 200 or 300 microg/kg/h. Parallel experiments with the animal at rest were carried out using both electroencephalogram (EEG) and functional connectivity MRI (fcMRI) methods with consistent results. In the secondary infusion period using 300 microg/kg/h, resting-state functional connectivity is enhanced.

Uniform Field Resonators for EPR Spectroscopy: A Review.

Cavity resonators are often used for electron paramagnetic resonance (EPR). Rectangular TE102 and cylindrical TE011 are common modes at X-band even though the field varies cosinusoidally along the Z-axis. The authors found a way to create a uniform field (UF) in these modes. A length of waveguide at cut-off was introduced for the sample region, and tailored end sections were developed that supported the microwave resonant mode. This work is reviewed here. The radio frequency (RF) magnetic field in loop-gap resonators (LGR) at X-band is uniform along the Z-axis of the sample, which is a benefit of LGR technology. The LGR is a preferred structure for EPR of small samples. At Q-band and W-band, the LGR often exhibits nonuniformity along the Z-axis. Methods to trim out this nonuniformity, which are closely related to the methods used for UF cavity resonators, are reviewed. In addition, two transmission lines that are new to EPR, dielectric tube waveguide and circular ridge waveguide, were recently used in UF cavity designs that are reviewed. A further benefit of UF resonators is that cuvettes for aqueous samples can be optimum in cross section along the full sample axis, which improves quantification in EPR spectroscopy of biological samples.

Rutile dielectric loop-gap resonator for X-band EPR spectroscopy of small aqueous samples.

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 TE011 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 (B1/Pl) 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.

Three-dimensional dynamic structure of the liquid-ordered domain in lipid membranes as examined by pulse-EPR oxygen probing.

Membranes made of dimyristoylphosphatidylcholine and cholesterol, one of the simplest paradigms for the study of liquid ordered-disordered phase separation, were investigated using a pulse-EPR spin-labeling method in which bimolecular collision of molecular oxygen with the nitroxide spin label is measured. This method allowed discrimination of liquid-ordered, liquid-disordered, and solid-ordered domains because the collision rates (OTP) differ in these domains. Furthermore, the oxygen transport parameter (OTP) profile across the bilayer provides unique information about the three-dimensional dynamic organization of the membrane domains. First, the OTP in the bilayer center in the liquid-ordered domain was comparable to that in the liquid-disordered domain without cholesterol, but the OTP near the membrane surface (up to carbon 9) was substantially smaller in the ordered domain, i.e., the cholesterol-based liquid-ordered domain is ordered only near the membrane surface, still retaining high levels of disorder in the bilayer center. This property may facilitate lateral mobility in ordered domains. Second, in the liquid-disordered domain, the domains with approximately 5 mol % cholesterol exhibited higher OTP than those without cholesterol, everywhere across the membrane. Third, the transmembrane OTP profile in the liquid-ordered domain that contained 50 mol % cholesterol dramatically differed from that which contained 27 mol % cholesterol.

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.