Deconvolution of sinusoidal rapid EPR scans

In rapid scan EPR the magnetic field is scanned through the signal in a time that is short relative to electron spin relaxation times. Previously it was shown that the slow-scan lineshape could be recovered from triangular rapid scans by Fourier deconvolution. In this paper a general Fourier deconvolution method is described and demonstrated to recover the slow-scan lineshape from sinusoidal rapid scans. Since an analytical expression for the Fourier transform of the driving function for a sinusoidal scan was not readily apparent, a numerical method was developed to do the deconvolution. The slow scan EPR lineshapes recovered from rapid triangular and sinusoidal scans are in excellent agreement for lithium phthalocyanine, a trityl radical, and the nitroxyl radical, tempone. The availability of a method to deconvolute sinusoidal rapid scans makes it possible to scan faster than is feasible for triangular scans because of hardware limitations on triangular scans.     

Application of in vivo ESR spectroscopy to pharmaceutical sciences: Evaluation of in vivo inhibitory mechanism of antigastric lesion drugs

In order to analyze free radical reactions in living stomach, we developed a noninvasive measurement by an in vivo electron spin resonance (ESR) and spin probe technique and applied it to mucosal injury. NH₄OH-induced gastric lesions were prepared in rats. A nitroxyl probe was administered intragastrically or intravenously, and then in vivo ESR spectra of the gastric region were obtained by 300 MHz ESR spectroscopy. The signal of the intragastrically administered spin probe decreased gradually and the decay significantly enhanced 30 min after NH₄OH administration. The enhanced signal decay was attributed to the OH radical generation, since it was completely suppressed by mannitol, catalase, and desferrioxamine. Two commercially available antigastric lesion drugs, rebamipide and taurine, were tested with a NH₄OH-induced gastric lesion model. Both intraperitoneal administration of rebamipide and intravenous administration of taurine suppressed gastric lesion formation induced by NH₄OH in a dose-dependent manner. Intraperitoneal preadministration of rebamipide also suppressed the enhanced signal decay, but neither pre- nor coadministration of taurine showed any effect on the enhanced signal decay. The results strongly indicate that the inhibitory mechanism on gastric lesion formation in NH₄OH-treated rats is quite different for the two antigastric lesion drugs rebamipide and taurine.     

Comparisons of EPR imaging and T1-weighted MRI for efficient imaging of nitroxyl contrast agents

The resolution and signal to noise ratio of EPR imaging and T(1)-weighted MRI were compared using an identical phantom. Several solutions of nitroxyl contrast agents with different EPR spectral shapes were tested. The feasibility of T(1)-weighted MRI to detect nitroxyl contrast agents was described. T(1)-weighted MRI can detect nitroxyl contrast agents with a complicated EPR spectrum easier and quicker; however, T(1)-weighted MRI has less quantitative ability especially for lipophilic nitroxyl contrast agents, because T(1)-relaxivity, i.e. accessibility to water, is affected by the hydrophilic/hydrophobic micro-environment of a nitroxyl contrast agent. The less quantitative ability of T(1)-weighted MRI may not be a disadvantage of redox imaging, which obtains reduction rate of a nitroxyl contrast. Therefore, T(1)-weighted MRI has a great advantage to check the pharmacokinetics of newly modified and/or designed nitroxyl contrast agents.     

Using of Digital Demodulation of Multiharmonic Overmodulated EPR Signals to Improve EPR Oximetry Reliability

Overmodulation of electron paramagnetic resonance (EPR) lines is routinely used in EPR oximetry in order to increase the signal-to-noise ratio and thus to improve the accuracy with which the line width of a spin probe can be measured. For a known probe type, the line width is easily translated into the oxygen partial pressure. A standard EPR spectrometer uses the analog phase-sensitive detection (PSD) to demodulate the EPR signal. PSD imposes the restriction that only one spectrum is measured at a time, which is normally the first-harmonic EPR line. Information about EPR signals centered at the other harmonics of the modulation frequency is irreversibly destroyed by PSD. The question is raised whether this information can be utilized for EPR oximetry, for overmodulation enhances the second- and the other harmonic spectra, so that they approach the first-harmonic spectrum in intensity. To find an answer, numerical simulation and experimental measurements have been conducted. The experiment required modification of the detection scheme, so that all EPR-related information in the overmodulated signal is preserved. This permits measuring of the multiharmonic EPR spectrum, which when fitted to a set of the corresponding theoretical lines produces more accurate results in comparison with the standard overmodulation method.     

Free radical EPR spectroscopy analysis using blind source separation

In this paper, we propose a novel approach for electron paramagnetic resonance (EPR) mixture spectra analysis based on blind source separation (BSS) technique. EPR spectrum of a free radical is often superimposed by overlapping spectra of other species. It is important and challenging to accurately identify and quantify the 'pure' spectra from such mixtures. In this study, an automated BSS method implementing independent component analysis is used to extract the components from mixed EPR spectra that contain overlapping components of different paramagnetic centers. To apply this method, there is no requirement to know the component spectra or the number of components in advance. The method is applied to analyze free radical EPR spectra which are collected from standard chemical system, cultured cell suspense, and ex vivo rat kidneys by spin trapping EPR technique. Results show that the BSS method proposed here is capable of identifying the component EPR spectra from mixtures with unknown compositions. The BSS technique can offer powerful aids in resolving spectral overlapping problems in general EPR spectroscopy analysis.     

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

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

Noninvasive mapping of the redox status in septic mouse by in vivo electron paramagnetic resonance imaging

Increased reactive oxygen species (ROS) contribute to numerous brain disorders, and ROS generation has been examined in diverse experimental models of lipopolysaccharide (LPS)-induced inflammation. The in vivo electron paramagnetic resonance (EPR)/nitroxide spin probe method has been used to analyze the redox status in animal models modulated by ROS generation. In this study, a blood–brain barrier (BBB)-permeable nitroxide spin probe, 3-hydroxymethyl-2,2,5,5-tetramethylpyrrolidine-1-oxyl (HMP), was used as a redox-sensitive nitroxide probe. Magnetic resonance images of mouse head after the injection of HMP showed that HMP was distributed throughout all regions of the mouse head including the brain, suggesting that HMP can reveal redox information in all regions of the mouse head. After the injection of HMP through the mouse tail vein 6 h after the injection of LPS, three-dimensional (3D) EPR images were obtained each minute under a field scanning of 0.3 s and with 81 projections. The reduction reaction of HMP in septic mouse heads was remarkably accelerated compared to that in control mice, and this accelerated reaction was inhibited by aminoguanidine and allopurinol, which inhibit enzymatic activities of induced nitric oxide synthase and xanthine oxidase, respectively. Based on the pharmacokinetics of HMP in mouse heads, the half-life mapping of HMP was performed in LPS-treated mouse head. Half-life maps clearly show a difference in the redox status induced by ROS generation in the presence or absence of inhibitors of ROS-generating enzymes. The present results suggest that a 3D in vivo EPR imaging system combined with BBB-permeable HMP is a useful noninvasive tool for assessing changes in the redox status in rodent models of brain disease under oxidative stress.     

Development and testing of a CW-EPR apparatus for imaging of short-lifetime nitroxyl radicals in mouse head

This article describes a method for reducing the acquisition time in three-dimensional (3D) continuous-wave electron paramagnetic resonance (CW-EPR) imaging. To visualize nitroxyl spin probes, which have a short lifetime in living organisms, the acquisition time for a data set of spectral projections should be shorter than the lifetime of the spin probes. To decrease the total time required for data acquisition, the duration of magnetic field scanning was reduced to 0.5 s. Moreover, the number of projections was decreased by using the concept of a uniform distribution. To demonstrate this faster data acquisition, two kinds of nitroxyl radicals with different decay rates were measured in mice. 3D EPR imaging of 4-hydroxy-2,2,6,6-tetramethylpiperidine-d₁₇-1-¹⁵N-1-oxyl in mouse head was successfully carried out. 3D EPR imaging of nitroxyl spin probes with a half-life of a few minutes was achieved for the first time in live animals.     

Estimation of the in vivo decay rate of EPR signals for a nitroxide radical in rat brains by a region-selected intensity determination method

The region-selected intensity determination (RSID) method was proposed to obtain the temporal changes in electron paramagnetic resonance (EPR) signal intensity from a selected region by a stationary magnetic field gradient. To select the region, the subtraction field that was derived from the distance between the center and the projection of the selected region to the direction of the field gradient was applied to the main field. The directions of the stationary magnetic field gradient at a constant strength were systematically changed in a three-dimensional space after each acquisition of the spectrum. All spectra under the field gradient were accumulated and the resultant spectrum was deconvoluted by a spectrum without the field gradient. The center height of the deconvoluted spectrum indicates the signal intensity of the selected region. To verify this method, a phantom or in vivo study was conducted on a 700 MHz radio-frequency EPR spectrometer equipped with a bridged loop-gap resonator. In the temporal EPR measurements of phantoms including a nitroxide radical aqueous solution with and without ascorbic acid, the selected regions were alternatively changed at the position of the two phantoms. The signal intensity derived from the one phantom showed an exponential decay, and for the other phantom, no temporal changes. The spatial resolution of this method was estimated to be 2.7 mm by using a pinpoint phantom that included diphenylpicrylhydrazyl powder. In the in vivo temporal EPR measurements, the selected regions were alternatively changed at the cerebral cortex and the striatum of rats that had received a blood-brain barrier-permeative nitroxide radical. The decay rate of the signal intensity at each region obtained by this method was consistent with those previously reported.     

Spin relaxation measurements using first-harmonic out-of-phase absorption EPR signals: rotational motion effects

A recent survey of nonlinear continuous-wave (CW) EPR methods revealed that the first-harmonic absorption EPR signal, detected 90 degrees out of phase with respect to the Zeeman modulation (V(1)(')-EPR), is the most appropriate for determining spin-lattice relaxation enhancements of spin labels (V. A. Livshits, T. Páli, and D. Marsh, 1998, J. Magn. Reson. 134, 113-123). The sensitivity of such V(1)(')-EPR spectra to molecular rotational motion is investigated here by spectral simulations for nitroxyl spin labels, over the entire range of rotational correlation times. Determination of the effective spin-lattice relaxation times is less dependent on rotational mobility than for other nonlinear CW EPR methods, especially at a Zeeman modulation frequency of 25 kHz which is particularly appropriate for spin labels. This relative insensitivity to molecular motion further enhances the usefulness of the V(1)(')-EPR method. Calibrations of the out-of-phase to in-phase spectral intensity (and amplitude) ratios are given as a function of spin-lattice relaxation time, for the full range of spin-label rotational correlation times. Experimental measurements on spin labels in the slow, intermediate, and fast motional regimes of molecular rotation are used to test and validate the method.     

In vivo temporal EPR imaging for estimating the kinetics of a nitroxide radical in the renal parenchyma and pelvis in rats

The kinetics of a nitroxide radical in the renal parenchyma and pelvis in rats were investigated by employing an in vivo EPR imaging system equipped with a surface-coil-type resonator (SCR). The exposed kidney of a living rat was inserted into the single-turn coil of the SCR, with the renal major axis aligned with the direction of alternative magnetic field (B(1)). After the injection of nitroxide radical via the tail vein, EPR measurements were repeated. From the temporal EPR images of the kidney on the 2-D projection to the plane which is perpendicular to the direction of B(1,) the decay rate of nitroxide radical in the renal parenchyma and pelvis was estimated. The parenchymal decay rate was found to be significantly shorter than that for the pelvis.     

EPR imaging to estimate the in vivo intracerebral reducing ability of mature rats after neonatal hypoxic-ischemic brain injury

A rat model of neonatal hypoxic-ischemic encephalopathy (Rice's model) was obtained by unilateral ligation of the common carotid artery of 7-day-old rats with hypoxia (exposure to 8% oxygen). To estimate the in vivo intracerebral reducing ability of the mature rats (8 weeks old) of Rice's model, temporal electron paramagnetic resonance (EPR) imaging of the brain of a rat receiving a blood-brain barrier-permeable nitroxide radical, 3-hydroxymethyl-2,2,5,5-tetramethylpyrrolidine-1-oxyl, was performed. In this imaging technique, the decay rate of the EPR signal intensity in a selected region of the brain is indicative of region-specific reducing ability. The effect of neonatal treatment of an antioxidant agent, 3-methyl-1-phenyl-2-pyrazolin-5-one (MCI-186), after a hypoxic-ischemic insult was also tested. It was found that the reducing ability had been depleted in the contralateral hemisphere of Rice's model rats; this depletion was suppressed by administering MCI-186.     

A study of the molecular structure of bacterial lysodektose by quantum-chemical calculations and EPR spectroscopy

The standard redox potentials of the sequential oxidation of lysodektose to the corresponding nitrone were estimated by quantum chemistry methods. It follows from these estimates that the experimentally observed accumulation of the intermediate nitroxyl radical in substantial amounts during the oxidation of lysodektose can be explained by high medium reorganization energy in the oxidation of the nitrosyl radical with simultaneous proton abstraction. The EPR spectra of the radical lysodektose form were modeled. Arguments in favor of the suggestion that one nonequivalent proton appeared in the formation of an intramolecular H-bond were presented. Quantum-chemical calculations of the hyperfine structure constants were in satisfactory agreement with experiment.     

Dielectric Resonator-Based Flow and Stopped-Flow EPR with Rapid Field Scanning: A Methodology for Increasing Kinetic Information

We report methodology which combines recently developed dielectric resonator-based, rapid-mix, stopped-flow EPR (appropriate for small, aqueous, lossy samples) with rapid scanning of the external (Zeeman) magnetic field where the scanning is preprogrammed to occur at selected times after the start of flow. This methodology gave spectroscopic information complementary to that obtained by stopped-flow EPR at single fields, and with low reactant usage, it yielded more graphic insight into the time evolution of radical and spin-labeled species. We first used the ascorbyl radical as a test system where rapid scans triggered after flow was stopped provided “snapshots” of simultaneously evolving and interacting radical species. We monitored ascorbyl radical populations either as brought on by biologically damaging peroxynitrite oxidant or as chemically and kinetically interacting with a spectroscopically overlapping nitroxide radical. In a different biophysical application, where a spin-label lineshape reflected rapidly changing molecular dynamics of folding spin-labeled protein, rapid scan spectra were taken during flow with different flow rates and correspondingly different times after the mixing-induced inception of protein folding. This flow/rapid scan method is a means for monitoring early immobilization of the spin probe in the course of the folding process.     

EPR studies of15N- and2H-substituted pH-sensitive spin probes of imidazoline and imidazolidine types

The isotopically substituted analogs of pH-sensitive imidazoline and imidazolidine radicals have been synthesized and investigated with electron paramagnetic resonance (EPR) spectroscopy. The introduction of²H and¹⁵N into the structure of the radical is a useful approach to enhance the information obtained from spin-labeling experiments. The spectra of the radicals have been analyzed with 9.8 (X-band) and 130 GHz (D-band) EPR spectroscopy. The substitution of¹H for²H leads to significant narrowing of Gaussian line width, while the substitution of¹⁴N for¹⁵N in the nitroxyl fragment decreases both the number of spectral lines and Lorentzian line width. These effects result in a significant increase in the peak intensities up to 5–7 times for X-band EPR spectra of one of the imidazoline radicals (R4). The increase in spectral resolution allowed us to reveal the hyperfine interaction splitting with the attached proton (0.36 G) in the protonated form of the radical R4. The influence of proton exchange of the radicals with phosphate and acetate buffers on their EPR spectra has been studied in H₂O and D₂O. The corresponding rate constants of the proton exchange have been calculated from fitting of the simulated EPR spectra line shapes to experimental spectra. The data obtained demonstrated the advantages of the isotopically substituted spin pH probes in spectral resolution and sensitivity which can be an important factor particularly for applications in vivo where the fundamental sensitivity is much lower. The sensitivity of EPR spectra of these spin probes to the buffer capacity could be of practical importance taking into account the biological relevance of monitoring this parameter in some pathological states.     

NMR and EPR Study of the Nitroxide Radical Tempo Interaction with Phenols

The 2,2,6,6-tetramethyl-I-piperidinyloxy free radical (TEMPO) was used as a probe to study the changes in hydrogen bonding between the phenolic OH group and the ON group of the radical by means of NMR and EPR. ¹³C NMR contact shifts induced by TEMPO were measured for five phenols. Formation of intermolecular hydrogen bond between a phenol and TEMPO molecule causes noticeable increase of ¹⁴N hyperfme coupling constant in the radical and appearance of negative spin density on carbon nuclei of C-OH fragment in the phenol.     

Effects of resveratrol and its analogs on scavenging hydroxyl radicals: Evaluation by EPR spin trapping method

Resveratrol (3,4′,5-trihydroxy-trans-stilbene) and six analogs, polyhydroxystilbenes, were synthesized. Their effects on scavenging hydroxyl radicals were studied by electron paramagnetic resonance (EPR) spin trapping method. The EPR signal intensity of the spin adduct of hydroxyl radical to 5,5-dimethyl-1-pyrroline N-oxide was detected and used as a standard for the evaluation of the effect of the seven compounds on scavenging hydroxyl radicals. While all seven compounds exhibited hydroxyl radical-scavenging activity, some of them proved to be more effective than resveratrol in this model. Another stable but low-intensity spin adduct was also observed by EPR. A possible assignment is proposed.     

In vivo temporal EPR study using a region-selected intensity determination method to estimate cerebral reducing ability in rats treated with olanzapine

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.     

EPR of photo-excited triplet states: A personal account

A sketch is presented of the path that has led from Zavoisky’s pioneering experiments to modern investigations by electron paramagnetic resonance (EPR) of the phosphorescent (S = 1) triplet state of polyatomic molecules or ions. The group-theoretical method first introduced by Wigner in his analysis of the multiplets of atomic spectroscopy, likewise provides a key for understanding the zero-field splitting and selection rules for radiative decay of the phosphorescent triplet state. Examples to illustrate the progress made through EPR experiments are selected from three fields. (i) Conformational instability on excitation. Both the zero-field splitting and the electron spin density distribution provide unique fingerprints of a triplet state’s geometry — structural information of a kind that is nonexistent for singlet states! Illustrations are provided by benzene C₆H₆ and fullerene C₆₀. (ii) The optical pumping cycle. The spin selectivity of singlet-to-triplet intersystem crossing and radiative decay of the individual spin components of the triplet state is discussed. In practice this selectivity is put to advantage by performing EPR on triplet states in zero-field by means of optical detection. In turn, such experiments have led to a detailed insight into the spin-orbit coupling mechanisms responsible for the spin selectivity of the above processes. The high sensitivity attainable with optical detection has recently culminated in EPR experiments on single molecules. (iii) Quantum interference. In a triplet state of low symmetry two of the spin sublevels may decay to the ground state by the emission of photons of a common polarization (i.e., out of plane for an aromatic hydrocarbon). In such a situation quantum interference between the two decay channels can be induced by an appropriate preparation of the excited state. An example is shown where flash-excitation in the singlet manifold followed by rapid intersystem crossing causes theS = 1 spin angular momentum to be created in a spin state which is not an eigenstate of the zero-field splitting tensor. This nonstationary character of the initial triplet state, which reflects the spin-orbit coupling pathway, is observed through the detection of a spontaneous microwave signal following the 25 ps laser flash.     

Rate constant measurements for initial addition reactions of radicals at the propagation step of photo‐polymerization as studied by pulsed EPR spectroscopy

Pulsed EPR spectroscopy was employed to determine reaction rate constants at an early stage of addition reactions in radical polymerizations triggered by four initiator radicals, which were generated by photodissociation of four parent molecules. Two monomers (tert‐butylacrylate and tert‐butylmethacrylate) were examined as reactant. Stern–Volmer analysis on the measured decay time of electron spin echo intensity of reacting radicals provides rate constants for addition reactions. We focused on rate constants for the second step reaction between monomer and adduct radical that is produced by the first step addition reaction between initiator radical and monomer. The rate constant measured by pulsed EPR was evaluated by theoretical calculations in the light of (1) enthalpy difference between product radical and reactants and (2) charge transfer interaction between reacting radical and monomer. Copyright © 2016 John Wiley & Sons, Ltd.