RIDME distance measurements using Gd(iii) tags with a narrow central transition.

Methods based on pulse electron paramagnetic resonance allow measurement of the electron-electron dipolar coupling between two spin labels. Here we compare the most popular technique, Double Electron-Electron Resonance (DEER or PELDOR), with the dead-time free 5-pulse Relaxation-Induced Dipolar Modulation Enhancement (RIDME) method for Gd(iii)-Gd(iii) distance measurements at W-band (94.9 GHz, ≈3.5 T) using Gd(iii) tags with a small zero field splitting (ZFS). Such tags are important because of their high EPR sensitivity arising from their narrow central transition. Two systems were investigated: (i) a rigid model compound with an inter-spin distance of 2.35 nm, and (ii) two mutants of a homodimeric protein, both labeled with a DOTA-based Gd(iii) chelate and characterized by an inter-spin distance of around 6 nm, one having a narrow distance distribution and the other a broad distribution. Measurements on the model compound show that RIDME is less sensitive to the complications arising from the failure of the weak coupling approximation which affect DEER measurements on systems characterized by short inter-spin distances between Gd(iii) tags having a narrow central transition. Measurements on the protein samples, which are characterized by a long inter-spin distance, emphasize the complications due to the appearance of harmonics of the dipolar interaction frequency in the RIDME traces for S > 1/2 spin systems, as well as enhanced uncertainties in the background subtraction. In both cases the sensitivity of RIDME was found to be significantly better than DEER. The effects of the experimental parameters on the RIDME trace are discussed.

Heteronuclear DNP of protons and deuterons with TEMPOL.

Dynamic nuclear polarization (DNP) experiments on samples with several types of magnetic nuclei sometimes exhibit "cross-talk" between the nuclei, such as different nuclei having DNP spectra with similar shapes and enhancements. In this work we demonstrate that while at 20 K the DNP spectra of (1)H and (2)H nuclei, in a sample composed of 50% v/v (1)H2O/DMSO-d6 and containing 40 mM TEMPOL, are different and can be analyzed using the indirect cross effect (iCE) model, at 6 K the DNP spectra of both (1)H and (2)H nuclei become identical. In addition we experimentally demonstrate that there exists an efficient polarization exchange between the two nuclear pools at this temperature. Both of these results are hallmark predictions of the thermal mixing (TM) formalism. However, the origin of these observations cannot, in our case, be explained using the standard TM formalism, as in our sample the electron reservoir cannot be described by a single non-Zeeman spin temperature, which is a prerequisite of TM. This conclusion follows from the analysis of the electron electron double resonance (ELDOR) experiments on our sample and is similar to the previously published results. Consequently, another mechanism must be used in order to explain these "cross-talk" effects. The heteronuclear cross effect (hnCE) DNP mechanism, previously introduced based on the simulations of the spin evolution in small model systems, results in "cross-talk" effects between two types of nuclei that are similar to the experimental ones seen in this work. In particular we show that the hnCE mechanism exhibits polarization transfer between the nuclei and that there exists a clear relationship between the steady state polarizations of the two types of nuclei which may, in the future, be correlated with the phenomenon observed in the two types of bulk nuclear signals in samples during DNP experiments. It is suggested that the hnCE electrons are a possible source for the process that equalizes the bulk enhancements of the (1)H and (2)H nuclei and are responsible for the observed cross-talk effects.

Gd(III) complexes as paramagnetic tags: Evaluation of the spin delocalization over the nuclei of the ligand.

Complexes of the Gd(III) ion are currently being established as spin labels for distance determination in biomolecules by pulse dipolar spectroscopy. Because Gd(III) is an f ion, one expects electron spin density to be localized on the Gd(III) ion - an important feature for the mentioned application. Most of the complex ligands have nitrogens as Gd(III) coordinating atoms. Therefore, measurement of the (14)N hyperfine coupling gives access to information on the localization of the electron spin on the Gd(III) ion. We carried out W-band, 1D and 2D (14)N and (1)H ENDOR measurements on the Gd(III) complexes Gd-DOTA, Gd-538, Gd-595, and Gd-PyMTA that serve as spin labels for Gd-Gd distance measurements. The obtained (14)N spectra are particularly well resolved, revealing both the hyperfine and nuclear quadrupole splittings, which were assigned using 2D Mims ENDOR experiments. Additionally, the spectral contributions of the two different types of nitrogen atoms of Gd-PyMTA, the aliphatic N atom and the pyridine N atom, were distinguishable. The (14)N hyperfine interaction was found to have a very small isotropic hyperfine component of -0.25 to -0.37MHz. Furthermore, the anisotropic hyperfine interactions with the (14)N nuclei and with the non-exchangeable protons of the ligands are well described by the point-dipole approximation using distances derived from the crystal structures. We therefore conclude that the spin density is fully localized on the Gd(III) ion and that the spin density distribution over the nuclei of the ligands is rightfully ignored when analyzing distance measurements.

Protein conformation by EPR spectroscopy using gadolinium tags clicked to genetically encoded p-azido-L-phenylalanine.

Quantitative cysteine-independent ligation of a Gd(3+) tag to genetically encoded p-azido-L-phenylalanine via Cu(I)-catalyzed click chemistry is shown to deliver an exceptionally powerful tool for Gd(3+)-Gd(3+) distance measurements by double electron-electron resonance (DEER) experiments, as the position of the Gd(3+) ion relative to the protein can be predicted with high accuracy.

Effects of the electron polarization on dynamic nuclear polarization in solids.

Dynamic Nuclear Polarization (DNP) experiments on solid dielectrics can be described in terms of the Solid Effect (SE) and Cross Effect (CE) mechanisms. These mechanisms are best understood by following the spin dynamics in electron-nuclear and electron-electron-nuclear model systems, respectively. Recently it was shown that the frequency swept DNP enhancement profiles can be reconstructed by combining basic SE and CE DNP spectra. However, this analysis did not take into account the role of the electron spectral diffusion (eSD), which can result in a dramatic loss of electron polarization along the EPR line. In this paper we extend the analysis of DNP spectra by including the influence of the eSD process on the enhancement profiles. We show for an electron-electron-nuclear model system that the change in nuclear polarization can be caused by direct MW irradiation on the CE electron transitions, resulting in a direct CE (dCE) enhancement, or by the influence of the eSD process on the spin system, resulting in nuclear enhancements via a process we term the indirect CE (iCE). We next derive the dependence of the basic SE, dCE, and iCE DNP spectra on the electron polarization distribution along the EPR line and on the MW irradiation frequency. The electron polarization can be obtained from ELDOR experiments, using a recent model which describes its temporal evolution in real samples. Finally, DNP and ELDOR spectra, recorded for a 40 mM TEMPOL sample at 10-40 K, are analyzed. It is shown that the iCE is the major mechanism responsible for the bulk nuclear enhancement at all temperatures.

The electron depolarization during dynamic nuclear polarization: measurements and simulations.

Dynamic nuclear polarization is typically explained either using microscopic systems, such as in the solid effect and cross effect mechanisms, or using the macroscopic formalism of spin temperature which assumes that the state of the electrons can be described using temperature coefficients, giving rise to the thermal mixing mechanism. The distinction between these mechanisms is typically made by measuring the DNP spectrum - i.e. the nuclear enhancement profile as a function of irradiation frequency. In particular, we have previously used the solid effect and cross effect mechanisms to explain temperature dependent DNP spectra. Our past analysis has however neglected the effect of depolarization of the electrons resulting from the microwave (MW) irradiation. In this work we concentrate on this electron depolarization process and perform electron-electron double resonance (ELDOR) experiments on TEMPOL and trityl frozen solutions, using a 3.34 Tesla magnet and at 2.7-30 K, in order to measure the state of the electron polarization during DNP. The experiments indicate that a significant part of the EPR line is affected by the irradiation due to spectral diffusion. Using a theoretical framework based on rate equations for the polarizations of the different electron spin packets and for those of the nuclei we simulated the various ELDOR line-shapes and reproduced the MW frequency and irradiation time dependence. The obtained electron polarization distribution cannot be described using temperature coefficients as required by the classical thermal mixing mechanism, and therefore the DNP mechanism cannot be described by thermal mixing. Instead, the theoretical framework presented here for the analysis of the ELDOR data forms a basis for future interpretation of DNP spectra in combination with EPR measurements.

Zero field splitting fluctuations induced phase relaxation of Gd3+ in frozen solutions at cryogenic temperatures.

Distance measurements using double electron-electron resonance (DEER) and Gd(3+) chelates for spin labels (GdSL) have been shown to be an attractive alternative to nitroxide spin labels at W-band (95GHz). The maximal distance that can be accessed by DEER measurements and the sensitivity of such measurements strongly depends on the phase relaxation of Gd(3+) chelates in frozen, glassy solutions. In this work, we explore the phase relaxation of Gd(3+)-DOTA as a representative of GdSL in temperature and concentration ranges typically used for W-band DEER measurements. We observed that in addition to the usual mechanisms of phase relaxation known for nitroxide based spin labels, GdSL are subjected to an additional phase relaxation mechanism that features an increase in the relaxation rate from the center to the periphery of the EPR spectrum. Since the EPR spectrum of GdSL is the sum of subspectra of the individual EPR transitions, we attribute this field dependence to transition dependent phase relaxation. Using simulations of the EPR spectra and its decomposition into the individual transition subspectra, we isolated the phase relaxation of each transition and found that its rate increases with |ms|. We suggest that this mechanism is due to transient zero field splitting (tZFS), where its magnitude and correlation time are scaled down and distributed as compared with similar situations in liquids. This tZFS induced phase relaxation mechanism becomes dominant (or at least significant) when all other well-known phase relaxation mechanisms, such as spectral diffusion caused by nuclear spin diffusion, instantaneous and electron spin spectral diffusion, are significantly suppressed by matrix deuteration and low concentration, and when the temperature is sufficiently low to disable spin lattice interaction as a source of phase relaxation.

The (13)C solid DNP mechanisms with perchlorotriphenylmethyl radicals--the role of (35,37)Cl.

The microwave frequency swept DNP enhancement, referred to as the DNP spectrum, is strongly dependent on the EPR spectrum of the polarizing radical and it reveals the underlying DNP mechanisms. Here we focus on two chlorinated trityl radicals that feature axially symmetric powder patterns at 95 GHz, the width of which are narrower than those of TEMPOL or TOTAPOL but broader than that of the trityl derivative OX63. The static DNP lineshapes of these commonly used radicals in DNP, have been recently analyzed in terms of a superposition of basic Solid Effect (SE) and Cross Effect (CE)-DNP lineshapes, with their relative contributions as a fit parameter. To substantiate the generality of this approach and further investigate an earlier suggestion that a (35,37)Cl-(13)C polarization transfer pathway, termed "hetero-nuclear assisted DNP", may be in effect in the chlorinated radicals (C. Gabellieri et al., Angew. Chem., Int. Ed., 2010, 49, 3360-3362), we measured the static (13)C-glycerol DNP spectra of solutions of ca. ∼10 mM of the two chlorinated trityl radicals as a function of temperature (10-50 K) and microwave power. Analysis of the DNP lineshapes was first done in terms of the SE/CE superposition model calculated assuming a direct e-(13)C polarization transfer. The CE was found to prevail at the high temperature range (40-50 K), whereas at the low temperature end (10-20 K) the SE dominates, as was observed earlier for (13)C DNP with OX63 and (1)H DNP with TEMPOL and TOTAPOL, thus indicating that this is rather general behavior. Furthermore, it was found that at low temperatures it is possible to suppress the SE, and increase the CE by merely lowering the microwave power. While this analysis gave a good agreement between experimental and calculated lineshapes when the CE dominates, some significant discrepancies were observed at low temperatures, where the SE dominates. We show that by explicitly taking into account the presence of (35/37)Cl nuclei through a e-(35,37)Cl-(13)C polarization pathway in the SE-DNP lineshape calculations, as proposed earlier, we can improve the fit significantly, thus supporting the existence of the "hetero-nuclear assisted DNP" pathway.

Dynamic nuclear polarization using frequency modulation at 3.34 T.

During dynamic nuclear polarization (DNP) experiments polarization is transferred from unpaired electrons to their neighboring nuclear spins, resulting in dramatic enhancement of the NMR signals. While in most cases this is achieved by continuous wave (cw) irradiation applied to samples in fixed external magnetic fields, here we show that DNP enhancement of static samples can improve by modulating the microwave (MW) frequency at a constant field of 3.34 T. The efficiency of triangular shaped modulation is explored by monitoring the (1)H signal enhancement in frozen solutions containing different TEMPOL radical concentrations at different temperatures. The optimal modulation parameters are examined experimentally and under the most favorable conditions a threefold enhancement is obtained with respect to constant frequency DNP in samples with low radical concentrations. The results are interpreted using numerical simulations on small spin systems. In particular, it is shown experimentally and explained theoretically that: (i) The optimal modulation frequency is higher than the electron spin-lattice relaxation rate. (ii) The optimal modulation amplitude must be smaller than the nuclear Larmor frequency and the EPR line-width, as expected. (iii) The MW frequencies corresponding to the enhancement maxima and minima are shifted away from one another when using frequency modulation, relative to the constant frequency experiments.

Increasing sensitivity of pulse EPR experiments using echo train detection schemes.

Modern pulse EPR experiments are routinely used to study the structural features of paramagnetic centers. They are usually performed at low temperatures, where relaxation times are long and polarization is high, to achieve a sufficient Signal/Noise Ratio (SNR). However, when working with samples whose amount and/or concentration are limited, sensitivity becomes an issue and therefore measurements may require a significant accumulation time, up to 12h or more. As the detection scheme of practically all pulse EPR sequences is based on the integration of a spin echo--either primary, stimulated or refocused--a considerable increase in SNR can be obtained by replacing the single echo detection scheme by a train of echoes. All these echoes, generated by Carr-Purcell type sequences, are integrated and summed together to improve the SNR. This scheme is commonly used in NMR and here we demonstrate its applicability to a number of frequently used pulse EPR experiments: Echo-Detected EPR, Davies and Mims ENDOR (Electron-Nuclear Double Resonance), DEER (Electron-Electron Double Resonance|) and EDNMR (Electron-Electron Double Resonance (ELDOR)-Detected NMR), which were combined with a Carr-Purcell-Meiboom-Gill (CPMG) type detection scheme at W-band. By collecting the transient signal and integrating a number of refocused echoes, this detection scheme yielded a 1.6-5 folds SNR improvement, depending on the paramagnetic center and the pulse sequence applied. This improvement is achieved while keeping the experimental time constant and it does not introduce signal distortion.

Information content of SNR/resolution trade-offs in three-dimensional magnetic resonance imaging.

In MRI, a trade-off exists between resolution and signal-to-noise ratio, since different fractions of the available scan time can be used to acquire data at higher spatial frequencies and to perform signal averaging. By comparing a wide variety of 3D isotropic MR scans with different combinations of SNR, resolution, and scan duration, the impact of this trade-off on the image information content was assessed. The information content of mouse brain, mouse whole-body, and human brain images was evaluated using a simple numerical approach, which sums the information contribution of each individual k-space data point. Results show that, with a fixed receiver bandwidth and field of view, the information content of trade-off images is always maximized when the SNR is equal to about 16. The optimal imaging resolution is dependent on the scan duration, as well as certain MR system properties, such as field strength and coil sensitivity. These properties are, however, easily accounted for with the acquisition of a single scout MR image, and the optimal imaging resolution can then be calculated using a simple mathematical relationship. If the imaging task is approached with a predetermined resolution requirement, the same scout scan can be used to calculate the scan duration that will provide the maximum possible information. Using these relationships to maximize the image information content is an excellent technique for guiding the initial selection of imaging parameters.

Diffusive diffraction of the local ESR pulse-gradient spin-echo signal in a restricted one-dimensional conductor.

The diffusive motion of the conducting electrons in the one-dimensional organic conductor FA2PF6 (FA, Fluoranthene) is studied with the ESR pulse-gradient spin-echo (PGSE) signal combined with spatial density ESR imaging. A local measurement of the short restricted regions reveals diffraction patterns in the local PGSE data. A model calculation adapted to the local measurements provides a highly accurate quantitative description of the results. The appearance of these patterns provides information about the diffusive motion of the charge at the crystal boundaries.

Combined k-space q-space pulsed ESR imaging: mapping of restricted diffusion in (FA)(2)PF(6).

A (FA)(2)PF(6) crystal from the family of the quasi-one-dimensional organic conductors was selectively damaged by a beam of Helium ions with a slitted mask placed in the beam's trajectory. Pulsed ESR density weighted imaging of the damaged crystal revealed the appearance of regions where the ESR signal was absent. The one-dimensional motion of the charge carriers was thus restricted to the undamaged sections. The local charge carrier spin dynamics in these restricted areas was probed by combined k-space q-space pulsed ESR imaging. The local expected appearance of the restricted pulsed gradient spin echo (PGSE) "diffusive diffraction" effect is shown. The position of the diffraction minima is compatible with the density imaging results.

Spatial mapping of mobility and density of the conduction electrons in (FA)2PF6

Novel implementation of the Fourier imaging technique on conduction electron spins in the one-dimensional organic conductors (FA)2PF6 (FA: fluoranthene) is reported. Two-dimensional spatial imaging of resolution 30 &mgr;m(2) is combined with the pulsed-gradient spin-echo technique, to derive maps revealing the local properties of the electron spin density and mobility. The maps generally show pronounced inhomogeneity of both density and mobility on the scale of approximately 30-300 &mgr;m. Highly mobile regions were identified to exist, and the mobility in these was quantitatively evaluated by a basic theoretical model of restricted diffusion.

Three-dimensional pulsed ESR Fourier imaging.

Three-dimensional pulsed ESR imaging was performed on a (FA)(2)PF(6) crystal using a three-dimensional Fourier imaging sequence. The best resolution achieved was of 20 microm(3). Comparison with images obtained using the filtered back-projection method shows the superiority of this method under the given conditions.