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.

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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 pulse DEER (Double Electron-Electron Resonance) technique is frequently applied for measuring nanometer distances between specific sites in biological macromolecules. In this work we extend the applicability of this method to high field distance measurements in a protein assembly with mixed spin labels, i.e. a nitroxide spin label and a Gd(3+) tag. We demonstrate the possibility of spectroscopic selection of distance distributions between two nitroxide spin labels, a nitroxide spin label and a Gd(3+) ion, and two Gd(3+) ions. Gd(3+)-nitroxide DEER measurements possess high potential for W-band long range distance measurements (6 nm) by combining high sensitivity with ease of data analysis, subject to some instrumental improvements.

DNP on heteronuclear spin systems often results in interesting phenomena such as the polarization enhancement of one nucleus during MW irradiation at the "forbidden" transition frequencies of another nucleus or the polarization transfer between the nuclei without MW irradiation. In this work we discuss the spin dynamics in a four-spin model system of the form {ea-eb-((1)H,(13)C)}, with the Larmor frequencies ωa, ωb, ωH and ωC, by performing Liouville space simulations. This spin system exhibits the common (1)H solid effect (SE), (13)C cross effect (CE) and in addition high order CE-DNP enhancements. Here we show, in particular, the "proton shifted (13)C-CE" mechanism that results in (13)C polarization when the model system, at one of its (13)C-CE conditions, is excited by a MW field at the zero quantum or double quantum electron-proton transitions ωMW = ωa ± ωH and ωMW = ωb ± ωH. Furthermore, we introduce the "heteronuclear" CE mechanism that becomes efficient when the system is at one of its combined CE conditions |ωa - ωb| = |ωH ± ωC|. At these conditions, simulations of the four-spin system show polarization transfer processes between the nuclei, during and without MW irradiation, resembling the polarization exchange effects often discussed in the literature. To link the "microscopic" four-spin simulations to the experimental results we use DNP lineshape simulations based on "macroscopic" rate equations describing the electron and nuclear polarization dynamics in large spin systems. This approach is applied based on electron-electron double resonance (ELDOR) measurements that show strong (1)H-SE features outside the EPR frequency range. Simulated ELDOR spectra combined with the indirect (13)C-CE (iCE) mechanism, result in additional "proton shifted (13)C-CE" features that are similar to the experimental ones. These features are also observed experimentally in (13)C-DNP spectra of a sample containing 15 mM of trityl in a glass forming solution of (13)C-glycerol/H2O and are analyzed by calculating the basic (13)C-SE and (13)C-iCE shapes using simulated ELDOR spectra that were fitted to the experimental ones.