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Electron spin relaxation times for perdeuterated Finland trityl 99% enriched in 13 C at the central carbon ( 13 C 1 -dFT) were measured in phosphate buffered saline (pH = 7.2) (PBS) solution at X-band. The anisotropic 13 C 1 hyperfine (A x  = A y  = 18 ± 2, A z  = 162 ± 1 MHz) and g values (2.0033, 2.0032, 2.00275) in a 9:1 trehalose:sucrose glass at 293 K and in 1:1 PBS:glycerol at 160 K were determined by simulation of spectra at X-band and Q-band. In PBS at room temperature the tumbling correlation time, τ R , is 0.29 ± 0.02 ns. The linewidths are broadened by incomplete motional averaging of the hyperfine anisotropy and T 2 is 0.13 ± 0.02 µs, which is shorter than the T 2  ~ 3.8 µs for natural abundance dFT at low concentration in PBS. T 1 for 13 C 1 -dFT in deoxygenated PBS is 5.9 ± 0.5 µs, which is shorter than for natural abundance dFT in PBS (16 µs) but much longer than in air-saturated solution (0.48 ± 0.04 µs). The tumbling dependence of T 1 in PBS, 3:1 PBS:glycerol (τ R  = 0.80 ± 0.05 ns, T 1  = 9.7 ± 0.7 µs) and 1:1 PBS:glycerol (τ R  = 3.4 ± 0.3 ns, T 1  = 12.0 ± 1.0 µs) was modeled with contributions to the relaxation predominantly from modulation of hyperfine anisotropy and a local mode. The 1/T 1 rate for the 1% 12 C 1 -dFT in the predominantly 13 C labeled sample is about a factor of 6 more strongly concentration dependent than for natural abundance 12 C 1 -trityl, which reflects the importance of Heisenberg exchange with molecules with different resonance frequencies and faster relaxation rates. In glassy matrices at 160 K, T 1 and T m for 13 C 1 -dFT are in good agreement with previously reported values for 12 C 1 -dFT consistent with the expectation that modulation of nuclear hyperfine does not contribute to electron spin relaxation in a rigid lattice., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Inc. All rights reserved.)

As a demonstration of the application of rapid-scan EPR to imaging at low frequency and magnetic field, a multi-compartment phantom containing six different samples was imaged. The samples were nitroxide radicals, trityl (substituted triarylmethyl) radicals, and the oxygen-sensitive solid lithium phthalocyanine (LiPc), all of which are useful for in vivo imaging. The 2D spectral-spatial image demonstration was performed at 250 MHz, with samples in sealed tubes of various sizes arranged in a 3D-printed plastic holder. Maximum gradients of 10 G/cm gave a spatial resolution of about 0.1 mm for the narrow trityl and LiPc signals and about 1 mm for the nitroxide. The importance of proper selection of resonator bandwidth and scan rate for obtaining accurate linewidth information is demonstrated for a case in which the phantom is composed of species with signal linewidths and relaxation times that differ by more than a factor of 10., (Copyright © 2019 Elsevier Inc. All rights reserved.)

In rapid scan EPR the rapidly-changing magnetic field induces a background signal that may be larger than the EPR signal. A method has been developed to correct for that background signal by acquiring two sets of data, denoted as scan 1 and scan 2. In scan 2 the external field B 0 is reversed and the data acquisition trigger is offset by one half cycle of the scan field relative to the settings used in scan 1. For data acquired with a cross-loop resonator subtraction of scan 2 from scan 1 cancels the background and enhances the EPR signal. Experiments were performed at an EPR frequency of about 258 MHz, which is in the range that is commonly used for in vivo imaging. Samples include nitroxide radicals, a trityl radical, a dinitroxide, and a nitroxide in the presence of a magnetic field gradient. This method has the advantage that no assumption is made about the shape of the background signal, and it provides an approach to automating the background correction., (Copyright © 2018 Elsevier Inc. All rights reserved.)

An iodoacetamide azaadamantyl spin label was studied in fluid solution and in 9:1 trehalose:sucrose glass. In 9:1 toluene:CH 2 Cl 2 solution at 293 K, the isotropic nitrogen hyperfine coupling is 19.2 G, T 1 is 0.37 µs and T 2 is 0.30-0.35 µs. Between about 80 and 150 K 1/T m in 9:1 trehalose:sucrose is approximately independent of temperature demonstrating that the absence of methyl groups decreases 1/T m relative to that which is observed in spin labels with methyl groups on the alpha carbons. Spin lattice relaxation rates between about 80 and 293 K in 9:1 trehalose:sucrose are similar to those observed for other nitroxide spin labels, consistent with the expectation that relaxation is dominated by Raman and local mode processes. Although complexation of the azaadamantyl spin label with β-cyclodextrin slows tumbling in aqueous solution by about a factor of 10, it has little impact on 1/T 1 or 1/T m in 9:1 trehalose:sucrose between 80 and 293 K.

In rapid-scan EPR the magnetic field or frequency is repeatedly scanned through the spectrum at rates that are much faster than in conventional continuous wave EPR. The signal is directly-detected with a mixer at the source frequency. Rapid-scan EPR is particularly advantageous when the scan rate through resonance is fast relative to electron spin relaxation rates. In such scans, there may be oscillations on the trailing edge of the spectrum. These oscillations can be removed by mathematical deconvolution to recover the slow-scan absorption spectrum. In cases of inhomogeneous broadening, the oscillations may interfere destructively to the extent that they are not visible. The deconvolution can be used even when it is not required, so spectra can be obtained in which some portions of the spectrum are in the rapid-scan regime and some are not. The technology developed for rapid-scan EPR can be applied generally so long as spectra are obtained in the linear response region. The detection of the full spectrum in each scan, the ability to use higher microwave power without saturation, and the noise filtering inherent in coherent averaging results in substantial improvement in signal-to-noise relative to conventional continuous wave spectroscopy, which is particularly advantageous for low-frequency EPR imaging. This overview describes the principles of rapid-scan EPR and the hardware used to generate the spectra. Examples are provided of its application to imaging of nitroxide radicals, diradicals, and spin-trapped radicals at a Larmor frequency of ca. 250MHz., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Thiol redox status is an important physiologic parameter that affects the success or failure of cancer treatment. Rapid scan electron paramagnetic resonance (RS EPR) is a novel technique that has shown higher signal-to-noise ratio than conventional continuous-wave EPR in in vitro studies. Here we used RS EPR to acquire rapid three-dimensional images of the thiol redox status of tumors in living mice. This work presents, for the first time, in vivo RS EPR images of the kinetics of the reaction of 2 H, 15 N-substituted disulfide-linked dinitroxide (PxSSPx) spin probe with intracellular glutathione. The cleavage rate is proportional to the intracellular glutathione concentration. Feasibility was demonstrated in a FSa fibrosarcoma tumor model in C3H mice. Similar to other in vivo and cell model studies, decreasing intracellular glutathione concentration by treating mice with l-buthionine sulfoximine (BSO) markedly altered the kinetic images., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Targeted delivery of molecular probes into cells enables cellular imaging through optical and magnetic modalities. Probe molecules that are well retained by cells can accumulate to higher intracellular concentrations, and thus increase the signal-to-noise ratio of, and widen the temporal window for, imaging. Here we synthesize a paramagnetic spin probe bearing six ionic functional groups and show that it has long intracellular half-life (>12 h) and exceptional biostability in living cells. We demonstrate that judicious incorporation of ionic substituents on probe molecules systematically increases intracellular retention time, and should therefore be beneficial to imaging experiments.

X-band rapid scan electron paramagnetic resonance (EPR) measures the free radicals in irradiated clipped fingernails with higher signal-to-noise (S/N) and lower standard deviation of the signal amplitude for replicate measurements than does conventional continuous wave (CW) EPR in the same measurement time. For a clipped fingernail sample irradiated to 10 Gy and data acquisition time of 30 s with B 1  = 8.5 μT, the S/N for rapid scan is >2000 for the absorption spectrum and 1200 for the corresponding first derivative. The standard deviation for replicate measurements of signal amplitude is ~1%. For CW spectra, the S/N depends on the modulation amplitude. The CW signal has a first derivative peak-to-peak linewidth of 0.95 mT. For 30 s of CW acquisition time, the S/N was 30 for a conservative modulation amplitude of 0.2 mT and B 1 of 2.3 μT or 90 for a modulation amplitude of 0.4 mT and B 1 of 3.2 μT, which resulted in standard deviations of replicate measurements of 5% or 2%, respectively., (© The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

We demonstrate a superior method of 2D spectral-spatial imaging of stable radical reporter molecules at 250 MHz using rapid-scan electron-paramagnetic-resonance (RS-EPR), which can provide quantitative information under in vivo conditions on oxygen concentration, pH, redox status and concentration of signaling molecules (i.e., OH • , NO • ). The RS-EPR technique has a higher sensitivity, improved spatial resolution (1 mm), and shorter acquisition time in comparison to the standard continuous wave (CW) technique. A variety of phantom configurations have been tested, with spatial resolution varying from 1 to 6 mm, and spectral width of the reporter molecules ranging from 16 µT (160 mG) to 5 mT (50 G). A cross-loop bimodal resonator decouples excitation and detection, reducing the noise, while the rapid scan effect allows more power to be input to the spin system before saturation, increasing the EPR signal. This leads to a substantially higher signal-to-noise ratio than in conventional CW EPR experiments.

The widest scan that had been demonstrated previously for rapid scan EPR was a 155G sinusoidal scan. As the scan width increases, the voltage requirement across the resonating capacitor and scan coils increases dramatically and the background signal induced by the rapidly changing field increases. An alternate approach is needed to achieve wider scans. A field-stepped direct detection EPR method that is based on rapid-scan technology is now reported, and scan widths up to 6200G have been demonstrated. A linear scan frequency of 5.12kHz was generated with the scan driver described previously. The field was stepped at intervals of 0.01 to 1G, depending on the linewidths in the spectra. At each field data for triangular scans with widths up to 11.5G were acquired. Data from the triangular scans were combined by matching DC offsets for overlapping regions of successive scans. This approach has the following advantages relative to CW, several of which are similar to the advantages of rapid scan. (i) In CW if the modulation amplitude is too large, the signal is broadened. In direct detection field modulation is not used. (ii) In CW the small modulation amplitude detects only a small fraction of the signal amplitude. In direct detection each scan detects a larger fraction of the signal, which improves the signal-to-noise ratio. (iii) If the scan rate is fast enough to cause rapid scan oscillations, the slow scan spectrum can be recovered by deconvolution after the combination of segments. (iv) The data are acquired with quadrature detection, which permits phase correction in the post processing. (v) In the direct detection method the signal typically is oversampled in the field direction. The number of points to be averaged, thereby improving the signal-to-noise ratio, is determined in post processing based on the desired field resolution. A degased lithium phthalocyanine sample was used to demonstrate that the linear deconvolution procedure can be employed with field-stepped direct detection EPR signals. Field-stepped direct detection EPR spectra were obtained for Cu(2+) doped in Ni(diethyldithiocarbamate)2, Cu(2+) doped in Zn tetratolylporphyrin, perdeuterated tempone in sucrose octaacetate, vanadyl ion doped in a parasubstituted Zn tetratolylporphyrin, Mn(2+) impurity in CaO, and an oriented crystal of Mn(2+) doped in Mg(acetylacetonate)2(H2O)2., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Multiharmonic electron paramagnetic resonance spectroscopy was demonstrated for two samples with both narrow and broad lines: (i) α,γ-Bisdiphenylene-β-phenylallyl (BDPA) with ΔBpp of 0.85 G plus ultramarine blue with ΔBpp of 17 G, and (ii) a nitroxide radical immobilized in sucrose octaacetate. Modulation amplitudes up to 17 G at 41 kHz were generated with a rapid scan coil driver and Litz wire coils that provide uniform magnetic field sweeps over samples with heights of 5mm. Data were acquired with a 2-D experiment in the Xepr software through the transient signal path of a Bruker E500T and digitized in quadrature with a Bruker SpecJet II. Signals at the modulation frequency and its harmonics were calculated by digital phase-sensitive detection. The number of harmonics with signal intensity greater than noise increases as the ratio of the modulation amplitude to the narrowest peak increases. Spectra reconstructed by the multiharmonic method from data obtained with modulation amplitudes up to five times the peak-to-peak linewidths of the narrowest features have linewidths that are broadened by up to only about 10% relative to linewidths in spectra obtained at low modulation amplitudes. The signal-to-noise improves with increasing modulation amplitude up to the point where the modulation amplitude is slightly larger than the linewidth of the narrowest features. If this high a modulation amplitude had been used in conventional methodology the linewidth of the narrowest features would have been severely broadened. The multiharmonic reconstruction methodology means that the selection of the modulation amplitude that can be used without spectral distortion is no longer tightly tied to the linewidth of the narrowest line., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Nitroxide spin labels are used for double electron-electron resonance (DEER) measurements of distances between sites in biomolecules. Rotation of gem-dimethyls in commonly used nitroxides causes spin echo dephasing times (Tm) to be too short to perform DEER measurements at temperatures between ∼80 and 295 K, even in immobilized samples. A spirocyclohexyl spin label has been prepared that has longer Tm between 80 and 295 K in immobilized samples than conventional labels. Two of the spirocyclohexyl labels were attached to sites on T4 lysozyme introduced by site-directed spin labeling. Interspin distances up to ∼4 nm were measured by DEER at temperatures up to 160 K in water/glycerol glasses. In a glassy trehalose matrix the Tm for the doubly labeled T4 lysozyme was long enough to measure an interspin distance of 3.2 nm at 295 K, which could not be measured for the same protein labeled with the conventional 1-oxyl-2,2,5,5-tetramethyl-3-pyrroline-3-(methyl)methanethio-sulfonate label., (Copyright © 2015 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Rapid-scan electron paramagnetic resonance is based on continuous direct detection of the spin response as the magnetic field is scanned upfield and downfield through resonance thousands of times per second. The method provides improved signal-to-noise for a wide range of samples, including rapidly tumbling and immobilized radicals. This chapter provides an introduction to the method and practical examples of implementation for organic radicals., (© 2015 Elsevier Inc. All rights reserved.)

In fluid solution, motion-dependent processes dominate electron spin-lattice relaxation for nitroxides and semiquinones at frequencies between 250 MHz and 34 GHz. For triarylmethyl radicals, motion-dependent processes dominate spin-lattice relaxation at frequencies below about 3 GHz. The frequency dependence of relaxation provides invaluable information about dynamic processes occurring with characteristic times on the order of the electron Zeeman frequency. Relaxation mechanisms, methods of measuring spin-lattice relaxation, and motional processes for nitroxide, semiquinone, and triarylmethyl radicals are discussed., (© 2015 Elsevier Inc. All rights reserved.)

The spin-lattice relaxation rates at 293 K for three anionic semiquinones (2,5-di-t-butyl-1,4-benzosemiquinone, 2,6-di-t-butyl-1,4-benzosemiquinone, and 2,3,5,6-tetramethoxy-1,4-benzosemiquinone) were studied at up to 8 frequencies between 250 MHz and 34 GHz in ethanol or methanol solution containing high concentrations of OH(-). The relaxation rates are about a factor of 2 faster at lower frequencies than at 9 or 34 GHz. However, in perdeuterated alcohols the relaxation rates exhibit little frequency dependence, which demonstrates that the dominant frequency-dependent contribution to relaxation is modulation of dipolar interactions with solvent nuclei. The relaxation rates were modeled as the sum of two frequency-independent contributions (spin rotation and a local mode) and two frequency-dependent contributions (modulation of dipolar interaction with solvent nuclei and a much smaller contribution from modulation of g anisotropy). The correlation time for modulation of the interaction with solvent nuclei is longer than the tumbling correlation time of the semiquinone and is consistent with hydrogen bonding of the alcohol to the oxygen atoms of the semiquinones., (Copyright © 2014 Elsevier Inc. All rights reserved.)

X-band electron paramagnetic resonance spectra of immobilized nitroxides were obtained by rapid scan at 293 K. Scan widths were 155 G with 13.4 kHz scan frequency for (14)N-perdeuterated tempone and for T4 lysozyme doubly spin labeled with an iodoacetamide spirocyclohexyl nitroxide and 100 G with 20.9 kHz scan frequency for (15)N-perdeuterated tempone. These wide scans were made possible by modifications to our rapid-scan driver, scan coils made of Litz wire, and the placement of highly conducting aluminum plates on the poles of a Bruker 10″ magnet to reduce resistive losses in the magnet pole faces. For the same data acquisition time, the signal-to-noise for the rapid-scan absorption spectra was about an order of magnitude higher than for continuous wave first-derivative spectra recorded with modulation amplitudes that do not broaden the lineshapes., (Copyright © 2014 Elsevier Inc. All rights reserved.)

An algorithm is derived and demonstrated that reconstructs an EPR spectral-spatial image from projections with arbitrarily selected gradients. This approach permits imaging wide spectra without the use of the very large sweep widths and gradients that would be required for spectral-spatial imaging with filtered back projection reconstruction. Each projection is defined as the sum of contributions at the set of locations in the object. At each location gradients shift the spectra in the magnetic field domain, which is equivalent to a phase change in the Fourier-conjugate frequency domain. This permits solution of the problem in the frequency domain. The method was demonstrated for 2D images of phantoms consisting of (i) two tubes containing (14)N and (15)N nitroxide and (ii) two tubes containing a pH sensitive trityl radical at pH 7.0 and 7.2. In each case spectral slices through the image agree well with the full spectra obtained in the absence of gradient., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Projections for 2D spectral-spatial images were obtained by continuous wave and rapid-scan electron paramagnetic resonance using a bimodal cross-loop resonator at 251MHz. The phantom consisted of three 4mm tubes containing different (15)N,(2)H-substituted nitroxides. Rapid-scan and continuous wave images were obtained with 5min total acquisition times. For comparison, images also were obtained with 29s acquisition time for rapid scan and 15min for continuous wave. Relative to continuous wave projections obtained for the same data acquisition time, rapid-scan projections had significantly less low-frequency noise and substantially higher signal-to-noise at higher gradients. Because of the improved image quality for the same data acquisition time, linewidths could be determined more accurately from the rapid-scan images than from the continuous wave images., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Recent advances in electron paramagnetic resonance (EPR) include capabilities for applications to areas as diverse as archeology, beer shelf life, biological structure, dosimetry, in vivo imaging, molecular magnets, and quantum computing. Enabling technologies include multifrequency continuous wave, pulsed, and rapid scan EPR. Interpretation is enhanced by increasingly powerful computational models., (Copyright © 2012 Elsevier Inc. All rights reserved.)

X-band rapid-scan EPR spectra were obtained for dilute aqueous solutions of nitroxyl radicals (15)N-mHCTPO (4-hydro-3-carbamoyl-2,2,5,5-tetra-perdeuteromethyl-pyrrolin-1-(15)N-oxyl-d(12)) and (15)N-PDT (4-oxo-2,2,6,6-tetra-perdeuteromethyl-piperidinyl-(15)N-oxyl-d(16)). Simulations of spectra for (15)N-mHCTPO and (15)N-PDT agreed well with the experimental spectra. As the scan rate is increased in the rapid scan regime, the region in which signal amplitude increases linearly with B(1) extends to higher power and the maximum signal amplitude increases. In the rapid scan regime, the signal-to-noise for rapid-scan spectra was about a factor of 2 higher than for unbroadened CW EPR, even when the rapid scan spectra were obtained in a mode that had only 4% duty cycle for data acquisition. Further improvement in signal-to-noise per unit time is expected for higher duty cycles. Rapid scan spectra have higher bandwidth than CW spectra and therefore require higher detection bandwidths at faster scan rates. However, when the scan rate is increased by increasing the scan frequency, the increase in noise from the detection bandwidth is compensated by the decrease in noise due to increased number of averages per unit time. Because of the higher signal bandwidth, lower resonator Q is needed for rapid scan than for CW, so the rapid scan method is advantageous for lossy samples that inherently lower resonator Q., (Copyright © 2011 Elsevier Inc. All rights reserved.)

A digital EPR spectrometer was constructed by replacing the traditional bridge with an arbitrary waveform generator (AWG) to produce excitation patterns and a high-speed digitizer for direct detection of the spin system response at the carrier frequency. Digital down-conversion produced baseband signals in quadrature with very precise orthogonality. Real-time resonator tuning was performed by monitoring the Fourier transforms of signals reflected from the resonator during frequency sweeps generated by the AWG. The capabilities of the system were demonstrated by rapid magnetic field scans at 256 MHz carrier frequency, and FID and spin echo experiments at 1 and 10 GHz carrier frequencies. For the rapid scan experiments the leakage through a cross-loop resonator was compensated by adjusting the amplitude and phase of a sinusoid at the carrier frequency that was generated with another AWG channel., (Copyright © 2011 Elsevier Inc. All rights reserved.)

In rapid frequency scan EPR with triangular scans, sufficient time must be allowed to insure that the magnetization in the x, y plane decays to baseline at the end of the scan, which typically is about 5T(2) after the spins are excited. To permit relaxation of signals excited toward the extremes of the scan the total scan time required may be much longer than 5T(2). However, with periodic, saw-tooth excitation, the slow-scan EPR spectrum can be recovered by Fourier deconvolution of data recorded with a total scan period of 5T(2), even if some spins are excited later in the scan. This scan time is similar to polyphase excitation methods. The peak power required for either polyphase excitation or rapid frequency scans is substantially smaller than for pulsed EPR. The use of an arbitrary waveform generator (AWG) and cross loop resonator facilitated implementation of the rapid frequency scan experiments reported here. The use of constant continuous low B(1), periodic excitation waveform, and constant external magnetic field is similar to polyphase excitation, but could be implemented without the AWG that is required for polyphase excitation., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Polyphase continuous excitation based on the Frank sequence is suggested as an alternative to single pulse excitation in EPR. The method allows reduction of the source power, while preserving the excitation bandwidth of a single pulse. For practical EPR implementation the use of a cross-loop resonator is essential to provide isolation between the spin system and the resonator responses to the excitation. Provided that a line broadening of about 5% is acceptable, the cumulative turning angle of the magnetization vector generated by the excitation sequence can be quite large and can produce signal amplitudes that are comparable to that achieved with a higher power 90° pulse., (Copyright © 2011 Elsevier Inc. All rights reserved.)

The Frank polyphase sequence has been applied to pulsed EPR of triarylmethyl radicals at 25 6 MHz (9.1 mT magnetic field), using 256 phase pulses. In EPR, as in NMR, use of a Frank sequence of phase steps permits pulsed FID signal acquisition with very low power microwave/RF pulses (ca. 1.5 mW in the application reported here) relative to standard pulsed EPR. A 0.2 mM aqueous solution of a triarylmethyl radical was studied using a 16 mm diameter cross-loop resonator to isolate the EPR signal detection system from the incident pulses., (Copyright © 2011 Elsevier Inc. All rights reserved.)

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., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Human electron transfer flavoprotein (ETF) is a soluble mitochondrial heterodimeric flavoprotein that links fatty acid β-oxidation to the main respiratory chain. The crystal structure of human ETF bound to medium chain acyl-CoA dehydrogenase indicates that the flavin adenine dinucleotide (FAD) domain (αII) is mobile, which permits more rapid electron transfer with donors and acceptors by providing closer access to the flavin and allows ETF to accept electrons from at least 10 different flavoprotein dehydrogenases. Sequence homology is high and low-angle X-ray scattering is identical for Paracoccus denitrificans (P. denitrificans) and human ETF. To characterize the orientations of the αII domain of P. denitrificans ETF, distances between enzymatically reduced FAD and spin labels in the three structural domains were measured by double electron-electron resonance (DEER) at X- and Q-bands. An FAD to spin label distance of 2.8 ± 0.15 nm for the label in the FAD-containing αII domain (A210C) agreed with estimates from the crystal structure (3.0 nm), molecular dynamics simulations (2.7 nm), and rotamer library analysis (2.8 nm). Distances between the reduced FAD and labels in αI (A43C) were between 4.0 and 4.5 ± 0.35 nm and for βIII (A111C) the distance was 4.3 ± 0.15 nm. These values were intermediate between estimates from the crystal structure of P. denitrificans ETF and a homology model based on substrate-bound human ETF. These distances suggest that the αII domain adopts orientations in solution that are intermediate between those which are observed in the crystal structures of free ETF (closed) and ETF bound to a dehydrogenase (open)., (Copyright © 2011 The Protein Society.)

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., (Copyright © 2010 Elsevier Inc. All rights reserved.)

Experimental data obtained with an electron paramagnetic resonance (EPR) rapid scan spectrometer were translated through the reverse transfer functions of the spectrometer hardware to the sample position. Separately, theoretical calculations were performed to predict signal and noise amplitudes at the sample position for specified experimental conditions. A comparison was then made between the translated experimental values and the calculated values. Excellent agreement was obtained., (Copyright (c) 2010. Published by Elsevier Inc.)

A 2 mm diameter by 10mm long cylinder of fused SiO2 (quartz) gamma-irradiated to 1 kGy with 60Co contains about 2x10(16) spins/cm3. It is proposed as a standard for monitoring signal-to-noise (S/N) performance of X-band pulsed EPR spectrometers. This sample yields S/N of about 25 on modern spin echo spectrometers, which permits measurement of both signal and noise under the same conditions with an 8-bit digitizer., (Copyright (c) 2010 Elsevier Inc. All rights reserved.)

Direct detection of the rapid-scan EPR signal with quadrature detection and without automatic frequency control provides both the absorption and dispersion components of the signal. The use of a cross-loop resonator results in similar signal-to-noise in the two channels. The dispersion signal can be converted to an equivalent absorption signal by means of Kramers-Kronig relations. The converted signal is added to the directly measured absorption signal. Since the noise in the two channels is not correlated, this procedure increases the signal-to-noise ratio of the resultant absorption signal by up to a factor of square root 2. The utility of this method was demonstrated for 2D spectral-spatial imaging of a phantom containing three tubes of LiPc with different oxygen concentrations and therefore different linewidths., (2010 Elsevier Inc. All rights reserved.)

Electron spin lattice relaxation rates for five semiquinones (2,5-di-t-butyl-1,4-benzosemiquinone, 2,5-di-t-amyl-1,4-benzosemiquinone, 2,5-di-phenyl-1,4-benzosemiquinone, 2,6-di-t-butyl-1,4-benzosemiquinone, tetrahydroxy-1,4-benzosemiquione) were studied by long-pulse saturation recovery EPR in 1:4 glycerol:ethanol, 1:1 glycerol:ethanol, and triethanolamine between 25 and 295K. Although the dominant process changes with temperature, relaxation rates vary smoothly with temperature, even near the glass transition temperatures, and could be modeled as the sum of contributions that have the temperature dependence that is predicted for the direct, Raman, local mode and tumbling-dependent processes. At 85K, which is in a temperature range where the Raman process dominates, relaxation rates along the g(xx) (g approximately 2.006) and g(yy) (g approximately 2.005) axes are about 2.7-1.5 times faster than along the g(zz) axis (g=2.0023). In highly viscous triethanolamine, contributions from tumbling-dependent processes are negligible. At temperatures above 100K relaxation rates in triethanolamine are unchanged between X-band (9.5GHz) and Q-band (34GHz), so the process that dominates in this temperature interval was assigned as a local mode rather than a thermally activated process. Because the largest proton hyperfine couplings are only 2.2G, spin rotation makes a larger contribution than tumbling-dependent modulation of hyperfine anisotropy. Since g anisotropy is small, tumbling-dependent modulation of g anisotropy makes a smaller contribution than spin rotation at X-band. Although there was negligible impact of methyl rotation on T(1), rotation of t-butyl or t-amyl methyl groups enhances spin echo dephasing between 85 and 150K.

Between 65 and 175 K, nitroxyl radicals with spirocyclohexyl groups at the 2- and 6-positions of the piperidine ring exhibit spin echo dephasing rates that are slower than for nitroxyl radicals with 2,5-gem-dimethyl or 2,6-gem-dimethyl substituents that are currently used as spin labels, and are slow enough to permit DEER measurements at temperatures up to about 125 K for spin labels with analogous ring structures.

In rapid scan EPR the changing magnetic field creates a background signal with components at the scan frequency and its harmonics. The amplitude of the background signal increases with scan width and is more significant for weak EPR signals such as are obtained in the presence of magnetic field gradients. A procedure for distinguishing this background from the EPR signal is proposed, mathematically described, and tested for various experimental conditions.

A new algorithm for EPR imaging oximetry is described and tested with experimental data for the case of one spatial and one spectral dimension. A single species with variable linewidth is assumed. Instead of creating a 2D image, two one-dimensional profiles are reconstructed: the concentration of the radical and the corresponding oxygen concentration, which reduces the dimensionality of the problem. The algorithm (i) seeks to minimize the discrepancy between experimental data and projections calculated from the profiles and (ii) uses Tikhonov regularization to constrain the smoothness of the results. This approach controllably smoothes profiles rather than the data, while preserving sharp features.

Electron transfer flavoprotein-ubiquinone oxidoreductase (ETF-QO) is a membrane-bound electron transfer protein that links primary flavoprotein dehydrogenases with the main respiratory chain. Human, porcine, and Rhodobacter sphaeroides ETF-QO each contain a single [4Fe-4S](2+,1+) cluster and one equivalent of FAD, which are diamagnetic in the isolated enzyme and become paramagnetic on reduction with the enzymatic electron donor or with dithionite. The anionic flavin semiquinone can be reduced further to diamagnetic hydroquinone. The redox potentials for the three redox couples are so similar that it is not possible to poise the proteins in a state where both the [4Fe-4S](+) cluster and the flavoquinone are fully in the paramagnetic form. Inversion recovery was used to measure the electron spin-lattice relaxation rates for the [4Fe-4S](+) between 8 and 18K and for semiquinone between 25 and 65K. At higher temperatures the spin-lattice relaxation rates for the [4Fe-4S](+) were calculated from the temperature-dependent contributions to the continuous wave linewidths. Although mixtures of the redox states are present, it was possible to analyze the enhancement of the electron spin relaxation of the FAD semiquinone signal due to dipolar interaction with the more rapidly relaxing [4Fe-4S](+) and obtain point-dipole interspin distances of 18.6+/-1A for the three proteins. The point-dipole distances are within experimental uncertainty of the value calculated based on the crystal structure of porcine ETF-QO when spin delocalization is taken into account. The results demonstrate that electron spin relaxation enhancement can be used to measure distances in redox poised proteins even when several redox states are present.

Reconstruction of two-dimensional images by filtered back-projection (FBP) and by the maximum entropy method (MEM) was compared for spectral-spatial EPR images with differing signal-to-noise ratios. Experimental projections were recorded using direct-detected rapid scans in the presence of a series of magnetic field gradients. The slow-scan absorption lineshapes were calculated by Fourier deconvolution. A Hamming filter was used in conjunction with FBP, but not for MEM. Imperfections in real experimental data, as well as random noise, contribute to discrepancies between the reconstructed image and experimental projections, which may make it impossible to achieve the customary MEM criterion for convergence. The Cambridge MEM algorithm, with allowance for imperfections in experimental data, produced images with more linear intensity scales and more accurate linewidths for weak signals than was obtained with another MEM method. The more effective elimination of noise in baseline regions by MEM made it possible to detect weak trityl (13)C trityl hyperfine lines that could not be distinguished from noise in images reconstructed by FBP. Another advantage of MEM is that projections do not need to be equally spaced. FBP has the advantages that computational time is less, the amplitude scale is linear, and there is less noise superimposed on peaks in images. It is useful to reconstruct images by both methods and compare results. Our observations indicate that FBP works well when the number of projections is large enough that the star effect is negligible. When there is a smaller number of projections, projections are unequally spaced, and/or signal-to-noise is lower MEM is advantageous.

The temperature dependence, between 10 and 120 K, of electron spin-lattice relaxation at X-band was analyzed for a series of eight pyrrolate-imine complexes and for ten other copper(II) complexes with varying ligands and geometry including copper-containing prion octarepeat domain and S100 type proteins. The geometry of the CuN4 coordination sphere for pyrrolate-imine complexes with R=H, methyl, n-butyl, diphenylmethyl, benzyl, 2-adamantyl, 1-adamantyl, and tert-butyl has been shown to range from planar to pseudo-tetrahedral. The fit to the recovery curves was better for a distribution of values of T1 than for a single time constant. Distributions of relaxation times may be characteristic of Cu(II) in glassy solution. Long-pulse saturation recovery and inversion recovery measurements were performed. The temperature dependence of spin-lattice relaxation rates was analyzed in terms of contributions from the direct process, the Raman process, and local modes. It was necessary to include more than one process to fit the experimental data. There was a small contribution from the direct process at low temperature. The Raman process was the dominant contribution to relaxation between about 20 and 60 K. Debye temperatures were between 80 and 120 K. For samples with similar Debye temperatures the coefficient of the Raman process tended to increase as gz increased, as expected if modulation of spin-orbit coupling is a major factor in relaxation rates. Above about 60 K local modes with energies in the range of 260-360 K (180-250 cm-1) dominated the relaxation. For molecules with similar geometry, relaxation rates were faster for more flexible molecules than for more rigid ones. Relaxation rates for the copper protein samples were similar to rates for small molecules with comparable coordination spheres. At each temperature studied the range of relaxation rates was less than an order of magnitude. The spread was smaller between 20 and 60 K where the Raman process dominates, than at higher temperatures where local modes dominate the relaxation. Spin echo dephasing time constants, Tm, were calculated from two-pulse spin echo decays. Near 10 K Tm was dominated by proton spins in the surroundings. As temperature was increased motion and spin-lattice relaxation made increasing contributions to Tm. Near 100 K spin-lattice relaxation dominated Tm.

Direct-detected rapid-scan EPR signals were recorded using triangular field scan rates between 1.7 and 150 kG/s for deoxygenated samples of lithium phthalocyanine (LiPc) and Nycomed trityl-CD3. These scan rates are rapid relative to the reciprocals of the electron spin relaxation times and cause characteristic oscillations in the signals. Fourier deconvolution with an analytical function permitted recovery of lineshapes that are in good agreement with experimental slow-scan spectra. Unlike slow-scan EPR, direct detection rapid-scan EPR does not use phase sensitive detection and records the absorption signal directly instead of the first derivative of the absorption signal. The amplitude of the signal decreases approximately linearly with applied magnetic field gradient. Images of phantoms constructed from samples of LiPc and trityl-CD3 were reconstructed by filtered back-projection from data sets with a missing angle. The lineshapes in spectral slices from the image are in good agreement with slow-scan spectra and the spacing between sample tubes matches well with the known sample geometry.

The spin-lattice relaxation times for the alanine radical in dosimeters were measured by long-pulse saturation recovery and by inversion recovery. The strong dependence of the recovery time constants on experimental conditions show that spectral diffusion processes contribute to enhanced relaxation rates, analogous to what was observed previously for 60Co gamma-irradiated polycrystalline l-alanine. The spectral diffusion processes make it possible to run quantitative CW spectra of the alanine radical at substantially higher microwave powers than could be used in the absence of spectral diffusion processes.

Electron spin relaxation times for four triarylmethyl (trityl) radicals at room temperature were measured by long-pulse saturation recovery, inversion recovery, and electron spin echo at 250 MHz, 1.5, 3.1, and 9.2 GHz in mixtures of water and glycerol. At 250 MHz T(1) is shorter than at X-band and more strongly dependent on viscosity. The enhanced relaxation at 250 MHz is attributed to modulation of electron-proton dipolar coupling by tumbling of the trityl radicals at rates that are comparable to the reciprocal of the resonance frequency. Deuteration of the solvent was used to distinguish relaxation due to solvent protons from the relaxation due to intra-molecular electron-proton interactions at 250 MHz. For trityl-CD(3), which contains no protons, modulation of dipolar interaction with solvent protons dominates T(1). For proton-containing radicals the relative importance of modulation of intra- and inter-molecular proton interactions varies with solution viscosity. The viscosity and frequency dependence of T(1) was modeled based on dipolar interaction with a defined number of protons at specified distances from the unpaired electron. At each of the frequencies examined T(2) decreases with increasing viscosity consistent with contributions from T(1) and from incomplete motional averaging of anisotropic hyperfine interaction.

EPR spectra at 250 MHz for a single crystal of lithium phthalocyanine (LiPc) in the absence of oxygen and for a deoxygenated aqueous solution of a Nycomed triarylmethyl (trityl-CD3) radical were obtained at scan rates between 1.3 x 10(3) and 3.4 x 10(5)G/s. These scan rates are rapid relative to the reciprocals of the electron spin relaxation times (LiPc: T1 = 3.5 micros and T2 = 2.5 micros; trityl: T1 = 12 micros and T2 = 11.5 micros) and cause characteristic oscillations in the direct-detected absorption spectra. For a given scan rate, shorter values of T2 and increased inhomogeneous broadening cause less deep oscillations that damp out more quickly than for longer T2. There is excellent agreement between experimental and calculated lineshapes and signal amplitudes as a function of radiofrequency magnetic field (B1) and scan rate. When B1 is adjusted for maximum signal amplitude as a function of scan rate, signal intensity for constant number of scans is enhanced by up to a factor of three relative to slow scans. The number of scans that can be averaged in a defined period of time is proportional to the scan rate, which further enhances signal amplitude per unit time. Longer relaxation times cause the maximum signal intensity to occur at slower scan rates. These experiments provide the first systematic characterization of direct-detected rapid-scan EPR signals.

Mitogen-activated protein kinases are regulated by occupancy at two phosphorylation sites near the active site cleft. Previous studies using hydrogen exchange to investigate the canonical mitogen-activated protein kinase, extracellular signal-regulated protein kinase-2, have shown that phosphorylation alters backbone conformational mobility >10 A distal to the site of phosphorylation, including decreased mobility within amino acids 102-105 and increased mobility within 108-109. To further describe changes after enzyme activation, site-directed spin labeling at amino acids 101, 105-109, 111, 112 and electron paramagnetic resonance spectroscopy were used to investigate this region. The anisotropic hyperfine splitting of the spin labels in glassy samples was unchanged by phosphorylation, consistent with previous crystallographic studies that indicate no structural change in this region. At positions 101, 111, and 112, the mobility of the spin label was unchanged by diphosphorylation, consistent with little or no conformational change. However, diphosphorylation caused small but significant changes in rotational diffusion rates at positions 105-108 and altered proportions of probe in a motionally constrained state at positions 105, 107, and 109. Thus, electron paramagnetic resonance indicates reproducible changes in nanosecond side-chain mobilities at specific residues within the interdomain region, far from the site of phosphorylation and conformational change.

The effects of methyl rotation on electron spin-lattice relaxation times were examined by pulsed electron paramagnetic resonance for the major radicals in gamma-irradiated polycrystalline alpha-amino isobutyric acid, dimethyl-malonic acid, and L-valine. The dominant radical is the same in irradiated dimethyl-malonic acid and alpha-amino isobutyric acid. Continuous wave saturation recovery was measured between 10 and 295 K at S-band and X-band. Inversion recovery, echo-detected saturation recovery, and pulsed electron-electron double resonance (ELDOR) data were obtained between 77 and 295 K. For the radicals in the three solids, recovery time constants measured by the various techniques were not the same, because spectral diffusion processes contribute differently for each measurement. Hyperfine splitting due to the protons of two methyl groups is resolved in the EPR spectra for each of the samples. Pulsed ELDOR data were obtained to characterize the spectral diffusion processes that transfer magnetization between hyperfine lines. Time constants were obtained for electron spin-lattice relaxation (T(1e)), nuclear spin relaxation (T(1n)), cross-relaxation (T(x1)), and spin diffusion (T(s)). Between 77 and 295 K rapid cross-relaxation (deltaM(s) = +/- 1, deltaM(I) = -/+ 1) was observed for each sample, which is attributed to methyl rotation at a rate that is approximately equal to the electron Larmor frequency. The large temperature range over which cross-relaxation was observed suggests that methyl groups in the radical and in the lattice, with different activation energies for rotation, contribute to the rapid cross-relaxation. Activation energies for methyl and amino group rotation between 160 and 1900 K (1.3-16 kJ/mol) were obtained by analysis of the temperature dependence of 1/T(1e) at S-band and X-band in the temperature intervals where the dynamic process dominates T(1e)., (Copyright 2002 Elsevier Science (USA))

Experimental EPR signal intensities at 250 MHz, 1.5 GHz, and 9.1 GHz agree within experimental error with predictions from first principles. When both the resonator size and the sample size are scaled with the inverse of RF/microwave frequency, omega, the EPR signal at constant B(1) scales as omega(-1/4). Comparisons were made for three different samples in two pairs of loop gap resonators. Each pair was geometrically scaled by a factor of 6. One pair of resonators was scaled from 250 MHz to 1.5 GHz, and the other pair was scaled from 1.5 GHz to 9 GHz. All terms in the comparison were measured directly, and their uncertainties estimated. The theory predicts that the signal at the lower frequency will be larger than the signal at the higher frequency by the ratio 1.57. For 250 MHz to 1.5 GHz, the experimental ratio was 1.52 and for the 1.5-GHz to 9-GHz comparison the ratio was 1.14.

To understand the signals that are observed under rapid-passage conditions for samples with long electron spin relaxation times, the E' defect in irradiated vitreous SiO(2) was studied. For these samples at room temperature, T(1) is 200 mciro s and T(2) ranged from 35 to 200 micro s, depending on spin concentration. At X band with 100-kHz modulation frequency and 1-G modulation amplitude there was minimal lineshape difference between the low-power, in-phase spectra and high-power spectra detected 90 degrees out-of-phase with respect to the magnetic field modulation. Signal enhancement, defined as the ratio of the intensities of the out-of-phase to the in-phase signals when B(1) for both observation modes is adjusted to give maximum signal, was 3.4 to 9.5 at room temperature. The origin of the out-of-phase signal was modeled by numerical integration of the Bloch equations including magnetic field modulation. The waveforms for the E' signal, prior to phase sensitive detection, were simulated by summing the contributions of many individual spin packets. Good agreement was obtained between experimental and calculated waveforms. At low B(1) the experimental values of T(1) and T(2) were used in the simulations. However, at higher B(1), T(2) was adjusted to match the experimental signal intensity and increased with increasing B(1). At high B(1), T(2)=T(1), consistent with Redfield's and Hyde's models. For the spin concentrations examined, the out-of-phase signals at very high power (B(1) approximately 0.33 G) displayed a linear relationship between peak-to-peak signal amplitude and spin concentration. Under the conditions used for spin quantitation the signal-to-noise for these spectra was up to 5 times higher than for the in-phase signal, which greatly facilitates quantitation for these types of samples. For samples in which T(2) is dominated by electron spin-spin interaction, lower spin concentration results in longer T(2) and the enhancement is increased.

The electron paramagnetic resonance pulsed free induction decay (FID) of a degassed solution of a triaryl methyl radical, methyl tris(8-carboxy-2,2,6,6-tetramethyl(-d3)-benzo[1,2-d:4,5-d']bis(1,3)dithiol-4-yl) tripotassium salt, 0.2 mM in H2O, was measured at VHF (247.5 MHz) and L-band (1.40 GHz). The calculated and observed FID signal amplitudes (in millivolts) agreed within 1 and 6%, and the ratio of the normalized FID signals at the two frequencies agreed within 5%. The FID decay time constant was 2.7 micros at both frequencies.