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Rapid Scan Electron Paramagnetic Resonance Opens New Avenues for Imaging Physiologically Important Parameters <em>In Vivo</em>

We demonstrate a superior method of 2D spectral-spatial imaging of stable radical reporter molecules at 250 MHz using rapid-scan ...

University of Denver

Optimization of nitroxides as probes for EPR imaging requires detailed understanding of spectral properties. Spin lattice relaxation times, spin packet line widths, nuclear hyperfine splitting, and overall lineshapes were characterized for six low molecular weight nitroxides in dilute deoxygenated aqueous solution at X-band. The nitroxides included 6-member, unsaturated 5-member, or saturated 5-member rings, most of which were isotopically labeled. The spectra are near the fast tumbling limit with T(1)∼T(2) in the range of 0.50-1.1 μs at ambient temperature. Both spin-lattice relaxation T(1) and spin-spin relaxation T(2) are longer for (15)N- than for (14)N-nitroxides. The dominant contributions to T(1) are modulation of nitrogen hyperfine anisotropy and spin rotation. Dependence of T(1) on nitrogen nuclear spin state m(I) was observed for both (14)N and (15)N. Unresolved hydrogen/deuterium hyperfine couplings dominate overall line widths. Lineshapes were simulated by including all nuclear hyperfine couplings and spin packet line widths that agreed with values obtained by electron spin echo. Line widths and relaxation times are predicted to be about the same at 250 MHz as at X-band.

Relaxation times and line widths of isotopically-substituted nitroxides in aqueous solution at X-band.

Electron spin relaxation times of perdeuterated tempone (PDT) 1 and of a nitronyl nitroxide (2-(4-carboxy-phenyl)-4,4,5,5-tetramethylimidazoline-3-oxide-1-oxyl) 2 in aqueous solution at room temperature were measured by 2-pulse electron spin echo (T(2)) or 3-pulse inversion recovery (T(1)) in the frequency range of 250 MHz to 34 GHz. At 9 GHz values of T(1) measured by long-pulse saturation recovery were in good agreement with values determined by inversion recovery. Below 9 GHz for 1 and below 1.5 GHz for 2,T(1)~T(2), as expected in the fast tumbling regime. At higher frequencies T(2) was shorter than T(1) due to incomplete motional averaging of g and A anisotropy. The frequency dependence of 1/T(1) is modeled as the sum of spin rotation, modulation of g and A-anisotropy, and a thermally-activated process that has maximum contribution at about 1.5 GHz. The spin lattice relaxation times for the nitronyl nitroxide were longer than for PDT by a factor of about 2 at 34 GHz, decreasing to about a factor of 1.5 at 250 MHz. The rotational correlation times, τ(R) are calculated to be 9 ps for 1 and about 25 ps for 2. The longer spin lattice relaxation times for 2 than for 1 at 9 and 34 GHz are due predominantly to smaller contributions from spin rotation that arise from slower tumbling. The smaller nitrogen hyperfine couplings for the nitronyl 2 than for 1 decrease the contribution to relaxation due to modulation of A anisotropy. However, at lower frequencies the slower tumbling of 2 results in a larger value of ωτ(R) (ω is the resonance frequency) and larger values of the spectral density function, which enhances the contribution from modulation of anisotropic interactions for 2 to a greater extent than for 1.

Frequency dependence of electron spin relaxation times in aqueous solution for a nitronyl nitroxide radical and perdeuterated-tempone between 250 MHz and 34 GHz.

Electron spin relaxation times at 295 K were measured at frequencies between 250 MHz and 34 GHz for perdeuterated 2,2,6,6-tetramethyl-4-piperidone-1-oxyl (PDT) in five solvents with viscosities that result in tumbling correlation times, τR, between 4 and 50 ps and for three (14)N/(15)N pairs of nitroxides in water with τR between 9 and 19 ps. To test the impact of structure on relaxation three additional nitroxides with τR between 10 and 26 ps were studied. In this fast tumbling regime T2(-1)~T1(-1) at frequencies up to about 9 GHz. At 34 GHz T2(-1)>T1(-1) due to increased contributions to T2(-1) from incomplete motional averaging of g-anisotropy, and T2(-1)-T1(-1) is proportional to τR. The contribution to T1(-1) from spin rotation is independent of frequency and decreases as τR increases. Spin rotation dominates T1(-1) at 34 GHz for all τR studied, and at all frequencies studied for τR=4 ps. The contribution to T1(-1) from modulation of nitrogen hyperfine anisotropy increases as frequency decreases and as τR increases; it dominates at low frequencies for τR>~15 ps. The contribution from modulation of g anisotropy is significant only at 34 GHz. Inclusion of a thermally-activated process was required to account for the observation that for most of the radicals, T1(-1) was smaller at 250 MHz than at 1-2 GHz. The significant (15)N/(14)N isotope effect, the small H/D isotope effect, and the viscosity dependence of the magnitude of the contribution from the thermally-activated process suggest that it arises from intramolecular motions of the nitroxide ring that modulate the isotropic A values.

Electron spin-lattice relaxation mechanisms of rapidly-tumbling nitroxide radicals.

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.

Imaging of nitroxides at 250MHz using rapid-scan electron paramagnetic resonance.

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.

New spectral-spatial imaging algorithm for full EPR spectra of multiline nitroxides and pH sensitive trityl radicals.

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.

Rapid-scan EPR of immobilized nitroxides.

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.

Frequency dependence of electron spin-lattice relaxation for semiquinones in alcohol solutions.

Radicals, including hydroxyl, superoxide, and nitric oxide, play key signaling roles in vivo. Reaction of these free radicals with a spin trap affords more stable paramagnetic nitroxides, but concentrations in vivo still are so low that detection by electron paramagnetic resonance (EPR) is challenging. Three innovative enabling technologies have been combined to substantially improve sensitivity for imaging spin-trapped radicals at 250 MHz. 1) Spin-trapped adducts of BMPO have lifetimes that are long enough to make imaging by EPR at 250 MHz feasible. 2) The signal-to-noise ratio of rapid-scan EPR is substantially higher than for conventional continuous-wave EPR. 3) An improved algorithm permits image reconstruction with a spectral dimension that encompasses the full 50 G spectrum of the BMPO-OH spin adduct without requiring the wide sweeps that would be needed for filtered backprojection. A 2D spectral-spatial image is shown for a phantom containing ca. 5 μM BMPO-OH.

Improved sensitivity for imaging spin trapped hydroxyl radical at 250 MHz.

Carboxy-substituted trityl (triarylmethyl) radicals are valuable in vivo probes because of their stability, narrow lines, and sensitivity of their spectroscopic properties to oxygen. Amino-substituted trityl radicals have the potential to monitor pH in vivo, and the suitability for this application depends on spectral properties. Electron spin relaxation times T1 and T2 were measured at X-band for the protonated and deprotonated forms of two amino-substituted triarylmethyl radicals. Comparison with relaxation times for carboxy-substituted triarylmethyl radicals shows that T1 exhibits little dependence on protonation or the nature of the substituent, which makes it useful for measuring O2 concentration, independent of pH. Insensitivity of T1 to changes in substituents is consistent with the assignment of the dominant contribution to spin lattice relaxation as a local mode that involves primarily atoms in the carbon and sulfur core. Values of T2 vary substantially with pH and the nature of the aryl group substituent, reflecting a range of dynamic processes. The narrow spectral widths for the amino-substituted triarylmethyl radicals facilitate spectral-spatial rapid scan electron paramagnetic resonance imaging, which was demonstrated with a phantom. The dependence of hyperfine splittings patterns on pH is revealed in spectral slices through the image.

Electron spin relaxation times and rapid scan EPR imaging of pH-sensitive amino-substituted trityl radicals.

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.

Field-stepped direct detection electron paramagnetic resonance.

Rapid-scan EPR signals for semiquinones with very-small well-resolved hyperfine splittings exhibit coherence signals at a time after passing through the EPR line that is proportional to the reciprocal of the hyperfine splitting. Such coherences are a general phenomenon due to constructive interference of the responses to transient excitation of spins by rapid scan of the magnetic field across equally spaced spin packets. Examples are shown for 2,3,5,6-tetramethoxy-1,4-benzosemiquinone with aH=46 mG for 12 protons and for 2,5-di-t-butyl-1,4-benzosemiquinone with aH=59 mG for 18 protons.

Rapid-scan coherence signals in X-band EPR spectra of semiquinones with small hyperfine splittings.

Measurement of thiol-disulfide redox status is crucial for characterization of tumor physiology. The electron paramagnetic resonance (EPR) spectra of disulfide-linked dinitroxides are readily distinguished from those of the corresponding monoradicals that are formed by cleavage of the disulfide linkage by free thiols. EPR spectra can thus be used to monitor the rate of cleavage and the thiol redox status. EPR spectra of (1)H,(14)N- and (2)H,(15)N-disulfide dinitroxides and the corresponding monoradicals resulting from cleavage by glutathione have been characterized at 250 MHz, 1.04 GHz, and 9 GHz and imaged by rapid-scan EPR at 250 MHz.

Imaging disulfide dinitroxides at 250 MHz to monitor thiol redox status.

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 B1 = 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 B1 of 2.3 μT or 90 for a modulation amplitude of 0.4 mT and B1 of 3.2 μT, which resulted in standard deviations of replicate measurements of 5% or 2%, respectively.

Article	Year
Relaxation times and line widths of isotopically-substituted nitroxides in aqueous solution at X-band. Journal of magnetic resonance (San Diego, Calif. : 1997)\| PubMed ID: 21843961	2011
Frequency dependence of electron spin relaxation times in aqueous solution for a nitronyl nitroxide radical and perdeuterated-tempone between 250 MHz and 34 GHz. Journal of magnetic resonance (San Diego, Calif. : 1997)\| PubMed ID: 23123770	2012
Electron spin-lattice relaxation mechanisms of rapidly-tumbling nitroxide radicals. Journal of magnetic resonance (San Diego, Calif. : 1997)\| PubMed ID: 24056272	2013
Imaging of nitroxides at 250MHz using rapid-scan electron paramagnetic resonance. Journal of magnetic resonance (San Diego, Calif. : 1997)\| PubMed ID: 24650729	2014
New spectral-spatial imaging algorithm for full EPR spectra of multiline nitroxides and pH sensitive trityl radicals. Journal of magnetic resonance (San Diego, Calif. : 1997)\| PubMed ID: 25058914	2014
Rapid-scan EPR of immobilized nitroxides. Journal of magnetic resonance (San Diego, Calif. : 1997)\| PubMed ID: 25240151	2014
Frequency dependence of electron spin-lattice relaxation for semiquinones in alcohol solutions. Journal of magnetic resonance (San Diego, Calif. : 1997)\| PubMed ID: 25261741	2014
Improved sensitivity for imaging spin trapped hydroxyl radical at 250 MHz. Chemphyschem : a European journal of chemical physics and physical chemistry\| PubMed ID: 25488257	2015
Electron spin relaxation times and rapid scan EPR imaging of pH-sensitive amino-substituted trityl radicals. Magnetic resonance in chemistry : MRC\| PubMed ID: 25504559	2015
Field-stepped direct detection electron paramagnetic resonance. Journal of magnetic resonance (San Diego, Calif. : 1997)\| PubMed ID: 26232363	2015
Rapid-scan coherence signals in X-band EPR spectra of semiquinones with small hyperfine splittings. Journal of magnetic resonance (San Diego, Calif. : 1997)\| PubMed ID: 26277376	2015
Imaging disulfide dinitroxides at 250 MHz to monitor thiol redox status. Journal of magnetic resonance (San Diego, Calif. : 1997)\| PubMed ID: 26415686	2015
Comparison of Continuous Wave and Rapid Scan X-band Electron Paramagnetic Resonance of Irradiated Clipped Fingernails. Radiation protection dosimetry\| PubMed ID: 27590467	2016

Hanan Elajaili

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