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.

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.

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.

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.

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.

Uniform field loop-gap resonator and rectangular TEU02 for aqueous sample EPR at 94GHz.

In this work we present the design and implementation of two uniform-field resonators: a seven-loop-six-gap loop-gap resonator (LGR) and a rectangular TEU02 cavity resonator. Each resonator has uniform-field-producing end-sections. These resonators have been designed for electron paramagnetic resonance (EPR) of aqueous samples at 94GHz. The LGR geometry employs low-loss Rexolite end-sections to improve the field homogeneity over a 3mm sample region-of-interest from near-cosine distribution to 90% uniform. The LGR was designed to accommodate large degassable Polytetrafluorethylen (PTFE) tubes (0.81mm O.D.; 0.25mm I.D.) for aqueous samples. Additionally, field modulation slots are designed for uniform 100kHz field modulation incident at the sample. Experiments using a point sample of lithium phthalocyanine (LiPC) were performed to measure both the uniformity of the microwave magnetic field and 100kHz field modulation, and confirm simulations. The rectangular TEU02 cavity resonator employs over-sized end-sections with sample shielding to provide an 87% uniform field for a 0.1×2×6mm3 sample geometry. An evanescent slotted window was designed for light access to irradiate 90% of the sample volume. A novel dual-slot iris was used to minimize microwave magnetic field perturbations and maintain cross-sectional uniformity. Practical EPR experiments using the application of light irradiated rose bengal (4,5,6,7-tetrachloro-2',4',5',7'-tetraiodofluorescein) were performed in the TEU02 cavity. The implementation of these geometries providing a practical designs for uniform field resonators that continue resonator advancements towards quantitative EPR spectroscopy.

Broadband W-band Rapid Frequency Sweep Considerations for Fourier Transform EPR.

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 T1 values using pulse saturation recovery. An overview of that work is provided.

Extruded dielectric sample tubes of complex cross section for EPR signal enhancement of aqueous samples.

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 1mm O.D. capillary with a septum down the middle, named DoubleDee, and a 3.5mm 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 0.9mm 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 TE011 cavity is optimized, named AquaSun, where simulations show an EPR signal intensity increase by a factor of 8.2 over a standard 1mm capillary.

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.

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.

Axially uniform magnetic field-modulation excitation for electron paramagnetic resonance in rectangular and cylindrical cavities by slot cutting.

In continuous-wave (CW) Electron Paramagnetic Resonance (EPR) a low-frequency time-harmonic magnetic field, called field modulation, is applied parallel to the static magnetic field and incident on the sample. Varying amplitude of the field modulation incident on the sample has consequences on spectral line-shape and line-height over the axis of the sample. Here we present a method of coupling magnetic field into the cavity using slots perpendicular to the sample axis where the slot depths are designed in such a way to produce an axially uniform magnetic field along the sample. Previous literature typically assumes a uniform cross-section and axial excitation due to the wavelength of the field modulation being much larger than the cavity. Through numerical analysis and insights obtained from the eigenfunction expansion of dyadic Green's functions, it is shown that evanescent standing-wave modes with complex cross-sections are formed within the cavity. From this analysis, a W-band (94GHz) cylindrical cavity is designed where modulation slots are optimized to present a uniform 100kHz field modulation over the length of the sample.

Hyperbolic-cosine waveguide tapers and oversize rectangular waveguide for reduced broadband insertion loss in W-band electron paramagnetic resonance spectroscopy. II. Broadband characterization.

Experimental results have been reported on an oversize rectangular waveguide assembly operating nominally at 94 GHz. It was formed using commercially available WR28 waveguide as well as a pair of specially designed tapers with a hyperbolic-cosine shape from WR28 to WR10 waveguide [R. R. Mett et al., Rev. Sci. Instrum. 82, 074704 (2011)]. The oversize section reduces broadband insertion loss for an Electron Paramagnetic Resonance (EPR) probe placed in a 3.36 T magnet. Hyperbolic-cosine tapers minimize reflection of the main mode and the excitation of unwanted propagating waveguide modes. Oversize waveguide is distinguished from corrugated waveguide, overmoded waveguide, or quasi-optic techniques by minimal coupling to higher-order modes. Only the TE10 mode of the parent WR10 waveguide is propagated. In the present work, a new oversize assembly with a gradual 90° twist was implemented. Microwave power measurements show that the twisted oversize waveguide assembly reduces the power loss in the observe and pump arms of a W-band bridge by an average of 2.35 dB and 2.41 dB, respectively, over a measured 1.25 GHz bandwidth relative to a straight length of WR10 waveguide. Network analyzer measurements confirm a decrease in insertion loss of 2.37 dB over a 4 GHz bandwidth and show minimal amplitude distortion of approximately 0.15 dB. Continuous wave EPR experiments confirm these results. The measured phase variations of the twisted oversize waveguide assembly, relative to an ideal distortionless transmission line, are reduced by a factor of two compared to a straight length of WR10 waveguide. Oversize waveguide with proper transitions is demonstrated as an effective way to increase incident power and the return signal for broadband EPR experiments. Detailed performance characteristics, including continuous wave experiment using 1 µM 2,2,6,6-tetramethylpiperidine-1-oxyl in aqueous solution, provided here serve as a benchmark for other broadband low-loss probes in millimeter-wave EPR bridges.

MRI surface-coil pair with strong inductive coupling.

A novel inductively coupled coil pair was used to obtain magnetic resonance phantom images. Rationale for using such a structure is described in R. R. Mett et al. [Rev. Sci. Instrum. 87, 084703 (2016)]. The original rationale was to increase the Q-value of a small diameter surface coil in order to achieve dominant loading by the sample. A significant improvement in the vector reception field (VRF) is also seen. The coil assembly consists of a 3-turn 10 mm tall meta-metallic self-resonant spiral (SRS) of inner diameter 10.4 mm and outer diameter 15.1 mm and a single-loop equalization coil of 25 mm diameter and 2 mm tall. The low-frequency parallel mode was used in which the rf currents on each coil produce magnetic fields that add constructively. The SRS coil assembly was fabricated and data were collected using a tissue-equivalent 30% polyacrylamide phantom. The large inductive coupling of the coils produces phase-coherency of the rf currents and magnetic fields. Finite-element simulations indicate that the VRF of the coil pair is about 4.4 times larger than for a single-loop coil of 15 mm diameter. The mutual coupling between coils influences the current ratio between the coils, which in turn influences the VRF and the signal-to-noise ratio (SNR). Data on a tissue-equivalent phantom at 9.4 T show a total SNR increase of 8.8 over the 15 mm loop averaged over a 25 mm depth and diameter. The experimental results are shown to be consistent with the magnetic resonance theory of the emf induced by spins in a coil, the theory of inductively coupled resonant circuits, and the superposition principle. The methods are general for magnetic resonance and other types of signal detection and can be used over a wide range of operating frequencies.

Separation of parallel encoded complex-valued slices (SPECS) from a single complex-valued aliased coil image.

PURPOSE: Achieving a reduction in scan time with minimal inter-slice signal leakage is one of the significant obstacles in parallel MR imaging. In fMRI, multiband-imaging techniques accelerate data acquisition by simultaneously magnetizing the spatial frequency spectrum of multiple slices. The SPECS model eliminates the consequential inter-slice signal leakage from the slice unaliasing, while maintaining an optimal reduction in scan time and activation statistics in fMRI studies. MATERIALS AND METHODS: When the combined k-space array is inverse Fourier reconstructed, the resulting aliased image is separated into the un-aliased slices through a least squares estimator. Without the additional spatial information from a phased array of receiver coils, slice separation in SPECS is accomplished with acquired aliased images in shifted FOV aliasing pattern, and a bootstrapping approach of incorporating reference calibration images in an orthogonal Hadamard pattern. RESULT: The aliased slices are effectively separated with minimal expense to the spatial and temporal resolution. Functional activation is observed in the motor cortex, as the number of aliased slices is increased, in a bilateral finger tapping fMRI experiment. CONCLUSION: The SPECS model incorporates calibration reference images together with coefficients of orthogonal polynomials into an un-aliasing estimator to achieve separated images, with virtually no residual artifacts and functional activation detection in separated images.

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.

Restoring susceptibility induced MRI signal loss in rat brain at 9.4 T: A step towards whole brain functional connectivity imaging.

The aural cavity magnetic susceptibility artifact leads to significant echo planar imaging (EPI) signal dropout in rat deep brain that limits acquisition of functional connectivity fcMRI data. In this study, we provide a method that recovers much of the EPI signal in deep brain. Needle puncture introduction of a liquid-phase fluorocarbon into the middle ear allows acquisition of rat fcMRI data without signal dropout. We demonstrate that with seeds chosen from previously unavailable areas, including the amygdala and the insular cortex, we are able to acquire large scale networks, including the limbic system. This tool allows EPI-based neuroscience and pharmaceutical research in rat brain using fcMRI that was previously not feasible.

Spin-label CW microwave power saturation and rapid passage with triangular non-adiabatic rapid sweep (NARS) and adiabatic rapid passage (ARP) EPR spectroscopy.

Non-adiabatic rapid passage (NARS) electron paramagnetic resonance (EPR) spectroscopy was introduced by Kittell et al. (2011) as a general purpose technique to collect the pure absorption response. The technique has been used to improve sensitivity relative to sinusoidal magnetic field modulation, increase the range of inter-spin distances that can be measured under near physiological conditions (Kittell et al., 2012), and enhance spectral resolution in copper (II) spectra (Hyde et al., 2013). In the present work, the method is extended to CW microwave power saturation of spin-labeled T4 Lysozyme (T4L). As in the cited papers, rapid triangular sweep of the polarizing magnetic field was superimposed on slow sweep across the spectrum. Adiabatic rapid passage (ARP) effects were encountered in samples undergoing very slow rotational diffusion as the triangular magnetic field sweep rate was increased. The paper reports results of variation of experimental parameters at the interface of adiabatic and non-adiabatic rapid sweep conditions. Comparison of the forward (up) and reverse (down) triangular sweeps is shown to be a good indicator of the presence of rapid passage effects. Spectral turning points can be distinguished from spectral regions between turning points in two ways: differential microwave power saturation and differential passage effects. Oxygen accessibility data are shown under NARS conditions that appear similar to conventional field modulation data. However, the sensitivity is much higher, permitting, in principle, experiments at substantially lower protein concentrations. Spectral displays were obtained that appear sensitive to rotational diffusion in the range of rotational correlation times of 10(-3) to 10(-7) s in a manner that is analogous to saturation transfer spectroscopy.

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.