Application of magnetic field over-modulation for improved EPR linewidth measurements using probes with Lorentzian lineshape

Magnetic field modulation in CW electron paramagnetic resonance (EPR) is used for signal detection. However, it can also distort signal lineshape. In experiments where the linewidth information is of particular importance, small modulation amplitude is usually used to limit the lineshape distortion. The use of small modulation amplitude, however, results in low signal-to-noise ratio and therefore affects the precision of linewidth measurements. Recently, a new spectral simulation model has been developed enabling accurate fitting of modulation-broadened EPR spectra in liquids. Since the use of large modulation amplitude (over-modulation) can significantly enhance the EPR signal, the precision of linewidth measurements is therefore greatly improved. We investigated the over-modulation technique in EPR oximetry experiments using the oxygen-sensing probe lithium octa-n-butoxy-substitued naphthalocyanine (LiNc-BuO). Modulation amplitudes 2-18 times the intrinsic linewidth of the probe were applied to increase the spectral signal-to-noise ratio. The intrinsic linewidth of the probe at different oxygen concentrations was accurately extracted through curve fitting from the enhanced spectra. Thus, we demonstrated that the over-modulation model is also applicable to particulate oxygen-sensing probes such as LiNc-BuO and that the lineshape broadening induced by oxygen is separable from that induced by over-modulation. Therefore, the over-modulation technique can be used to enhance sensitivity and improve linewidth measurements for EPR oximetry with particulate oxygen-sensing probes with Lorentzian lineshape. It should be particularly useful for in vivo oxygen measurements, in which direct linewidth measurements may not be feasible due to inadequate signal-to-noise ratio.     

The continuous wave electron paramagnetic resonance experiment revisited

When the modulation frequency used in continuous wave electron paramagnetic resonance (cw EPR) spectroscopy exceeds the linewidth, modulation sidebands appear in the spectrum. It is shown theoretically and experimentally that these sidebands are actually multiple photon transitions, sigma(+)+kxpi, where one microwave (mw) sigma(+) photon is absorbed from the mw radiation field and an arbitrary number k of radio frequency (rf) pi photons are absorbed from or emitted to the modulation rf field. Furthermore, it is demonstrated that both the derivative shape of the lines in standard cw EPR spectra and the distortions due to overmodulation are caused by the unresolved sideband pattern of these lines. The single-photon transition does not even give a contribution to the first-harmonic cw EPR signal. Multiple photon transitions are described semiclassically in a toggling frame and their existence is proven using second quantization. With the toggling frame approach and perturbation theory an effective Hamiltonian for an arbitrary sideband transition is derived. Based on the effective Hamiltonians an expression for the steady-state density operator in the singly rotating frame is derived, completely describing all sidebands in all modulation frequency harmonics of the cw EPR signal. The relative intensities of the sidebands are found to depend in a very sensitive way on the actual rf amplitude and the saturation of single sidebands is shown to depend strongly on the effective field amplitude of the multiple photon transitions. By comparison with the analogous solutions for frequency-modulation EPR it is shown that the field-modulation and the frequency-modulation technique are not equivalent. The experimental data fully verify the theoretical predictions with respect to intensities and lineshapes.     

Practical conditions and limitations for high-spatial-resolution multisite EPR oximetry

We have previously reported a high-spatial-resolution multisite electron paramagnetic resonance (EPR) oximetry method that is based on consecutive applications of magnetic field gradients with the same direction but different magnitudes. This method that could be called also two-gradient convolution EPR oximetry has no restrictions for the shape of solid paramagnetic materials implanted in tissue and is applicable for any particulate EPR oxygen-sensitive matieral with a Lorentzian line shape. To enhance the utilization of this method, a previously described algorithm was used to develop user-friendly Windows-based software. Practical conditions of application of the method were established using several different model systems. It has been shown that the spectral overlap from the adjacent sites can be neglected if the splitting between the corresponding lines exceeds the largest line width by at least a factor of 1.3. An additional requirement of the method is that the second field gradient should exceed by at least 30% the value of the first gradient. It was confirmed that the error in line width determination at L-band is proportional to the noise-to-signal ratio, and does not exceed 1% a noise-to-signal ratio of 0.1 in a typical in vivo experiment. We demonstrate that the line widths of up to 10 different sites can be determined.     

Advantages of digital phase-sensitive detection for upgrading an obsolete CW EPR spectrometer

The replacement of the commonly used analog phase-sensitive detection (PSD) by digital PSD for demodulation of electron paramagnetic resonance (EPR) signals is suggested for upgrading of an out-of-date EPR spectrometer. Connection of the microwave bridge output to a fast analog-digital converter (ADC) eliminates some of the spectrometer’s components: the electronics responsible for analog PSD, ADC for sampling of demodulated signals, and a computer, as well as the usage of some of the spectrometer’s settings. The spectrometer is reduced to a magnet, microwave bridge, and personal computer containing an ADC board. EPR signals digitized for a set of magnetic field positions form a two-dimensional array which is stored in a personal computer. Demodulation and filtering are done numerically to produce a conventional EPR spectrum. In comparison with analog PSD, this numerical approach does not eliminate the out-of-phase component of the signal and the signals at the higher harmonics of the modulation frequency. The details of modernizing the Bruker ER200E SRC EPR spectrometer are discussed to demonstrate these and other advantages of digital demodulation.     

EPR spectra of a GdMnO3 thin film on a SrTiO3 substrate

Electron paramagnetic resonance (EPR) spectra of a GdMnO₃/SrTiO₃ thin film in the X band have been measured in the temperature interval from 200 to 450 K. Signals from two types of paramagnetic centers have been observed in the spectra. The first paramagnetic center is a subsystem of Gd³⁺ ions, in the EPR spectrum of which the fine structure lines are resolved below 350 K. The second paramagnetic center is a system of manganese and gadolinium ions, in the EPR spectrum of which an exchange-narrowed line is observed with the width ΔH several times less than the width ΔH of an exchange-narrowed line observed in the GdMnO₃ single crystal. Unusual magnetic properties are due to the mismatch of the lattice parameters of the GdMnO₃ thin film and the SrTiO₃ substrate.     

Reconstruction of the first-derivative EPR spectrum from multiple harmonics of the field-modulated continuous wave signal

Selection of the amplitude of magnetic field modulation for continuous wave electron paramagnetic resonance (EPR) often is a trade-off between sensitivity and resolution. Increasing the modulation amplitude improves the signal-to-noise ratio, S/N, at the expense of broadening the signal. Combining information from multiple harmonics of the field-modulated signal is proposed as a method to obtain the first derivative spectrum with minimal broadening and improved signal-to-noise. The harmonics are obtained by digital phase-sensitive detection of the signal at the modulation frequency and its integer multiples. Reconstruction of the first-derivative EPR line is done in the Fourier conjugate domain where each harmonic can be represented as the product of the Fourier transform of the 1st derivative signal with an analytical function. The analytical function for each harmonic can be viewed as a filter. The Fourier transform of the 1st derivative spectrum can be calculated from all available harmonics by solving an optimization problem with the goal of maximizing the S/N. Inverse Fourier transformation of the result produces the 1st derivative EPR line in the magnetic field domain. The use of modulation amplitude greater than linewidth improves the S/N, but does not broaden the reconstructed spectrum. The method works for an arbitrary EPR line shape, but is limited to the case when magnetization instantaneously follows the modulation field, which is known as the adiabatic approximation.     

EPR Spectra of Nitrogen in Ultra-Dispersed Diamonds

The analysis of the electron paramagnetic resonance (EPR) line shape of ultra-dispersed diamond (UDD) obtained by conversion of trotyl and hexogen mixture and purified from other phases and metal compounds is carried out. The observed wide line with g = 2.0028 and a line width of 8.7 G is shown to be formed by superposition of three lines with line widths of 15.3, 8.5 and 3 G and with a ratio of integral intensities of 70:30:1. The procedure of decomposition and subtraction of wide lines has revealed the resolved hyperfine structure (HFS) from donor nitrogen with parameters A = 40.8 G and B = 29.2 G. Experimentally obtained dependence of the line width of the exchange line on the concentration of donor nitrogen in synthetic diamonds assumed that variations in line widths of the EPR spectrum components are caused by different local concentration of donor nitrogen due to distribution of nitrogen impurity during crystallization of diamond nanoparticles. EPR spectra of UDD after annealing in vacuum and at high pressures in the range of diamond phase stability are also discussed. At high-pressure annealing above 973 K, the areas with high concentration of defects are graphitized and a narrow Dyson-shape line from conductivity electrons and a resolved HFS from donor nitrogen can be observed without additional treatment of the EPR spectrum.     

EPR and optical absorption studies of X-irradiated cobalt doped sodium chloride crystals

Sodium chloride crystals containing small concentrations of cobalt (< 10 ppm) do not show any EPR line. A thick block of crystal containing ～25 ppm of Co showed two partially resolved lines, with approximate g-values 2.036 and 2.011. These g-values are not close to those of Co⁺⁺ (4.0 to 4.5) in other crystals. On X-irradiation, pure NaCl crystals show a complex EPR spectrum. X-irradiated Co doped NaCl crystals showed an EPR line superimposed on the complex EPR spectrum. Cobalt doped highly pure crystals, on X-irradiation, showed an EPR line superimposed on the F center EPR line. The g-value of the former is 2.049±0.002 and half width is 62±3 gauss. These results combined with those of dielectric loss and optical studies show that X-irradiation of Co doped crystals produces new centers, labelled as S centers, which produce a dielectric loss peak, a decrease in electrical conductivity, an optical band at 210 nm and the EPR line. Possible models of the S centers are discussed.     

Importance of volume limitation for tissue redox status measurements using nitroxyl contrast agents: a comparison of X-band EPR bile flow monitoring (BFM) method and 300 MHz in vivo EPR measurement

Methods proposed for in vivo redox status estimation, X-band (9.4 GHz) electron paramagnetic resonance (EPR) bile flow monitoring (BFM) and 300 MHz in vivo EPR measurement, were compared. The spin probe 3-carbamoyl-2,2,5,5-tetramethylpyrrolidine-1-oxyl (carbamoyl-PROXYL) was utilized for both methods, due to its suitable lipophilicity. EPR signal decay of a nitroxyl spin probe in the bile flow and in the liver region (upper abdomen) of several rat groups with different selenium status were measured by both the BFM and the in vivo EPR method, respectively. The nitroxyl radical clearance measured with in vivo EPR method may be affected not only by the redox status in the liver but also by information from other tissues in the measured region of the rat. On the other hand, the time course of nitroxyl radical level in the bile flow of rats was found to be a reliable index of redox status. Measurement site and/or volume limitation, which was achieved by the BFM method in this paper, is quite important in estimating reasonable EPR signal decay information as an index of tissue/organ redox status.     

EPR study of Mn2+ ions adsorption on silica gel

The paper deals with the study of the spectra of the electron paramagnetic resonance (EPR) of the Mn²⁺ ions adsorbed on the silica gel. Further on there have been studied the changes in the EPR spectrum of the Mn²⁺ ions adsorbed on the silica gel that are occuring as a result of dehydratation of the introduced samples, as well as the spectra changes of these samples that have arisen owing to the natural adsorption of the water vapour from the air under normal physical conditions.At low concentrations of the Mn²⁺ ions adsorbed on the silica gel, the EPR resonance spectrum contains six hyperfine structure (HFS) components the widths of which enlarge towards higher magnetic fields and do not change with the increase of the Mn²⁺ ions concentration.The spectrum further contains a system of lines that have been identified as the so-called forbidden transitions, when simultaneously with the orientation of the electron spin the orientation of the nuclear spin changes as well. This spectrum corresponds to the Mn²⁺ ions adsorbed in the individual positions considerably far from each other, without mutual dipole-dipole interaction.During further increase of the adsorbed Mn²⁺ concentration, there appears in the EPR spectrum, besides the above-described HFS, a broad symetric line the width of which does not change with increasing concentration, except for high concentrations when it becomes narrower probably due to the exchange interaction. This signal corresponds to the Mn²⁺ ions adsorbed in clusters, probably in the silca gel pores, when the HFS gets smudged as a result of dipole-dipole interaction.It appears that the width of the EPR spectrum broad line is dependent upon the temperature at which the dehydratation process was passing. At the adsorption of water vapour from the air under normal conditions on the dehydrated sample, there arise expressive changes in the EPR spectrum shape. The broad line decomposes in 6 HFS components, the widths of which change with duration time of the water vapour adsorption. Since the HFS of the individually adsorbed Mn²⁺ ions does not change after the dehydratation and the ensuing water vapour adsorption, the EPR spectrum of these samples becomes a superposition of two hyperfine structures coresponding to the two inequivalently bound groups of Mn²⁺ ions.     

Simultaneous acquisition of pulse EPR orientation selective spectra

High resolution pulse EPR methods are usually applied to resolve weak magnetic electron-nuclear or electron-electron interactions that are otherwise unresolved in the EPR spectrum. Complete information regarding different magnetic interactions, namely, principal components and orientation of principal axis system with respect to the molecular frame, can be derived from orientation selective pulsed EPR measurements that are performed at different magnetic field positions within the inhomogeneously broadened EPR spectrum. These experiments are usually carried out consecutively, namely a particular field position is chosen, data are accumulated until the signal to noise ratio is satisfactory, and then the next field position is chosen and data are accumulated. Here we present a new approach for data acquisition of pulsed EPR experiments referred to as parallel acquisition. It is applicable when the spectral width is much broader than the excitation bandwidth of the applied pulse sequence and it is particularly useful for orientation selective pulse EPR experiments. In this approach several pulse EPR measurements are performed within the waiting (repetition) time between consecutive pulse sequences during which spin lattice relaxation takes place. This is achieved by rapidly changing the main magnetic field, B(0), to different values within the EPR spectrum, performing the same experiment on the otherwise idle spins. This scheme represents an efficient utilization of the spectrometer and provides the same spectral information in a shorter time. This approach is demonstrated on W-band orientation selective electron-nuclear double resonance (ENDOR), electron spin echo envelope modulation (ESEEM), electron-electron double resonance (ELDOR)--detected NMR and double electron-electron resonance (DEER) measurements on frozen solutions of nitroxides. We show that a factors of 3-6 reduction in total acquisition time can be obtained, depending on the experiment applied.     

Temperature-induced changes in the EPR spectrum of the magnetic center in kaolin

Studies of kaolin have revealed an effect characterized by an unusual temperature-induced change of the EPR spectrum of the Fe3+ ion, which is the magnetic probe in kaolin-clay. At low temperature (T=4.2 K) a resonance line with an effective g-value g1=4.13 +/- 0.16 is observed. At high temperature (T=288 K) one observes a resonance line with the effective g-value g2=2.15 +/- 0.1. The transition from the low- to high-temperature spectrum is gradual and it is accompanied by a redistribution of the absorption intensity. The observed properties of the temperature dependence of the EPR spectrum are characteristic of systems with a multiminimum potential.     

Application of the Raman heterodyne technique for the detection of EPR and ENDOR

Raman heterodyne detection is a coherent optical-RF double resonance technique where the optical and RF fields induce coherence within a three level system and a resultant Raman field is measured using heterodyne detection. This approach has been used previously to detect NMR and more recently EPR. In this paper the parameters that affect the amplitude and signal to noise ratio of the Raman heterodyne signals are considered. The power levels in relation to the oscillator strength and dephasing times, the amplitude and spectrum of the laser frequency jitter in relation to the optical homogeneous linewidths and holeburning rates, and the sample properties such as absorption strength and optical quality, are all factors that affect the Raman signal. The presentation is focused on the Raman heterodyne detected EPR of the nitrogen-vacancy pair centre in diamond making comparisons with Raman heterodyne detected NMR signals obtained for rare earth ion systems. RF-RF double resonance studies, RF holeburning and ENDOR, which give information about the hyperfine levels are also reported for the nitrogen-vacancy centre. The resonance frequencies are in agreement with those predicted from the spin Hamiltonian. The factors affecting the lineshapes and relative intensities of the double resonance signals are discussed.     

Anisotropic motion effects in CW non-linear EPR spectra: relaxation enhancement of lipid spin labels

Continuous-wave (CW) EPR measurements of enhancements in spin-lattice (T(1)-) relaxation rate find wide application for determining spin-label locations in biological systems. Often, especially in membranes, the spin-label rotational motion is anisotropic and subject to an orientational potential. We investigate here the effects of anisotropic diffusion and ordering on non-linear CW-EPR methods for determining T(1) of nitroxyl spin labels. Spectral simulations are performed for progressive saturation of the conventional in-phase, first-harmonic EPR signal, and for the first-harmonic absorption EPR signals detected 90 degrees -out-of-phase with respect to the Zeeman field modulation. Motional models used are either rapid rotational diffusion, or strong-jump diffusion of unrestricted frequency, within a cone of fixed maximum amplitude. Calculations of the T(1)-sensitive parameters are made for both classes of CW-experiment by using motional parameters (i.e., order parameters and correlation times), intrinsic homogeneous and inhomogeneous linewidth parameters, and spin-Hamiltonian hyperfine- and g-tensors, that are established from simulation of the linear CW-EPR spectra. Experimental examples are given for spin-labelled lipids in membranes.     

EPR of radiation defects in LiBaF 3 crystals

EPR spectra of LiBaF 3 crystals have been investigated after X-irradiation at RT. A spectrum consisting of approximately 35 nearly equidistant EPR lines has a strong angular dependence on the line intensities. The spectrum is caused by a hyperfine interaction (hfs) of a spin S =1/2 with neighbouring groups of nuclei. The observed large number of hfs lines required Li nuclei being in the first shell and fluorine nuclei in the more distant second shell. We analysed the spectrum in the F -centre model, taking reduced hfs values of the F -centre in LiF and found qualitative explanation of the number of hfs lines. The angular dependence of the line intensities could be explained by an anisotropy of the g -tensor with its main axis along the [1 v 0 v 0] axis of the crystal.     

A new method for the determination of oxygen solubilities by EPR

Free radicals dissolved in oxygen-containing solutions give rise to EPR spectra characterized by very broad lines due to Heisenberg spin exchange. In the method herein proposed oxygen is consumed at a constant rate, within the cavity of an EPR spectrometer, by alkyl radicals generatedin situ by thermal decomposition of an aliphatic azo compound in the presence of a nitroxide probe. The effect of decreasing the oxygen concentration is to reduce the width, and therefore to increase the height of the spectral lines of the nitroxide, which reach a maximum when oxygen has been completely consumed. From the knowledge of the rate of generation of the alkyl radicals, the oxygen solubility in a given solvent can be easily determined.     

Separation and Enrichment of the Active Component of Carbon Based Paramagnetic Materials for Use in EPR Oximetry

Carbon based paramagnetic materials are frequently used for EPR oximetry, especiallyin vivo,but the EPR spectra of these materials often have more than one paramagnetic center and/or relatively low signal intensity. To determine whether the multi-components of carbon based materials could be separated and enriched in the active component, we used density gradient centrifugation to separate the materials into several fractions. We studied two types of coals, gloxy and Pocahontas, and found these materials to have large density distribution. The separated density fractions had very different EPR spectra and intensities. The active component from the coal material had a more homogeneous EPR signal and significantly increased EPR signal intensity, whereas for India ink, only slight changes were observed. This result can be very useful in the development of better probes for EPR oximetry.     

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.     

Angular variation of electron paramagnetic resonance spectrum: simulation of a polycrystalline EPR spectrum

A procedure based on homotopy, involving a quick calculation of EPR line positions for various orientations of the external magnetic field by the method of least-squares fitting and Taylor-series expansion, using a known line position at an infinitesimally close orientation of the external magnetic field as the initial value, by using the eigenvectors and eigenvalues of the spin-Hamiltonian matrix in single crystals, has been exploited to simulate a polycrystalline EPR spectrum. This requires rigorous calculations of intensities of resonant lines, along with their positions. Specifically, details are given of the numerical techniques involving time-efficient matrix diagonalization to obtain the eigenvalues and eigenvectors required to calculate positions and intensities of EPR lines by the method of least-squares fitting. Finally, the procedure of how to simulate a polycrystalline EPR spectrum is outlined, the required steps are listed, and illustrative examples are given.     

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.