A Q-band pulse EPR/ENDOR spectrometer and the implementation of advanced one- and two-dimensional pulse EPR methodology

A versatile high-power pulse Q-band EPR spectrometer operating at 34.5--35.5 GHz and in a temperature range of 4--300 K is described. The spectrometer allows one to perform one- and two-dimensional multifrequency pulse EPR and pulse ENDOR experiments, as well as continuous wave experiments. It is equipped with two microwave sources and four microwave channels to generate pulse sequences with different amplitudes, phases, and carrier frequencies. A microwave pulse power of up to 100 W is available. Two channels form radiofrequency pulses with adjustable phases for ENDOR experiments. The spectrometer performance is demonstrated by single crystal pulse ENDOR experiments on a copper complex. A HYSCORE experiment demonstrates that the advantages of high-field EPR and correlation spectroscopy can be combined and exploited at Q-band. Furthermore, we illustrate how this combination can be used in cases where the HYSCORE experiment is no longer effective at 35 GHz because of the shallow modulation depth. Even in cases where the echo modulation is virtually absent in the HYSCORE experiment at Q-band, matched microwave pulses allow one to get HYSCORE spectra with a signal-to-noise ratio as good as at X-band. Finally, it is shown that the high microwave power, the short pulses, and the broad resonator bandwidth make the spectrometer well suited to Fourier transform EPR experiments.     

Dynamic nuclear polarization in pulsed ENDOR experiments

Properly prepared pulse sequences of microwave and radio frequency have been employed to investigate the effect of polarization transfer from the polarized photo excited triplet state of pentacene in p-terphenyl crystals to the surrounding protons in pulsed ENDOR experiments. The ENDOR signal, measured as the change of electron spin echo (ESE) amplitude, is affected by the mode of RF pulses. When B0 parallelx (the long molecular axis), the ESE amplitude of the high-field transition of the triplet state changes from the maximum positive to zero with a pi RF pulse, and to the maximum negative with a 2pi pulse, while that of the low-field transition changes from nearly zero to the maximum negative as the RF pulse width increases. The effect is attributed to the strong electron spin polarization produced in the creation of the photoexcited triplet state and the subsequent efficient electron- nuclear polarization transfer process.     

Application of Q-band electron spin echo spectrometer to investigation of glasses doped with rare earth ions

The Q-band electron spin echo (ESE) spectrometer which was created using modern microwave components is described. This simple incoherent apparatus was used with the X-band one for the study of phosphate and silicate glasses doped with non-Kramers rare earth Tb³⁺ ions. the EPR spectra measured by the ESE method have frequency independent peaks. The experimental results presumably show the existence of several types of paramagnetic centers in studied systems.     

Peculiarities of free induction and primary spin echo signals for spin-correlated radical pairs

Keeping in mind ion-radical pairs in a photosynthesis reaction centre first of all, we calculated free induction and spin echo (ESE) signals for an ensemble of radical pairs which initially start in a singlet state. It was shown that the intensity of signals should oscillate depending on the time interval τ between the start of a pair and a microwave pulse forming free induction (FI) or between the start of a pair and the first of two microwave pulses forming primary ESE signal. ESE phase of spin-correlated pairs does not coincide with the corresponding ESE phase of radical pairs in thermal equilibrium. One should also note an interesting feature of FI: immediately after the microwave pulse free induction signal equals zero, and non-zero free induction signal appears only due to spin evolution. This behaviour formally resembles the situation occurring when the primary ESE is formed: a light pulse which creates spin-correlated radical pairs acts as the first microwave pulse in conventional spin echo experiments. Analysis of FI and ESE in experiments on pulse photolysis or radiolysis may provide useful information about the contribution of spin-correlated radical pairs.     

Radio frequencies in EPR: Conventional and advanced use

This mini-review focuses on various aspects of the application of radio frequency (rf) irradiation in electron paramagnetic resonance (EPR). The development of the electron-nuclear double resonance (ENDOR) technique is briefly described, and we highlight the use of circularly polarized rf fields and pulse ENDOR methodology in one- and two-dimensional experiments. The capability of pulse ENDOR at Q-band is illustrated with interesting experimental examples. Electron spin echo envelope modulation effects induced by an rf field in liquid samples demonstrate another role which rf fields can play. Technical achievements in the design of ENDOR resonators are illustrated by the example of a bridged loop-gap resonator. Finally, the influence of longitudinal rf fields on the dynamics of EPR transitions is explained using a dressed spin resonance treatment.     

A Q-band pulsed ENDOR spectrometer for the study of transition metal ion complexes in solids

We describe the design of a pulsed electron nuclear double resonance (ENDOR) spectrometer operating at Q-band frequencies (35 GHz) for studies of transition metal ion complexes in the temperature range between 4.2 and 297 K. Specific features of the spectrometer are a microwave IMPATT generator, a homebuilt cavity, and a commercial Bruker magnet. Standard Davies and Mims ENDOR sequences have been implemented. The performance of the spectrometer is demonstrated for a broad radio frequency range by¹H,¹⁴N,³¹P,¹³³Cs, and²⁰⁷Pb pulsed ENDOR experiments of Cu²⁺, Cr⁵⁺, and V⁴⁺ transition metal ion complexes in both single crystals and disordered materials.     

Pulsed-ESR Study of Spin Solitons as a Probe of Neutral-to-Ionic Phase Transition of TTF-CA Single Crystal

Ionic phase of charge-transfer crystal composed of tetrathiafulvalene (TTF) and p -chloranil (CA) has been studied by two-pulse electron-spin echo (ESE) at 4 K. By measuring the angular dependence of the field-swept ESE spectrum, at least three kinds of paramagnetic centers have been resolved, which are presumably ascribed to spin solitons formed at domain boundary. With varying the time separation between the microwave pulses, envelope modulations due to anisotropic hyperfine interaction have been detected. By analyzing the modulation, we have firstly obtained ENDOR spectrum of the spin solitons. Response of the ESE intensity for the optical excitation has been also examined.     

A tunable general purpose Q-band resonator for CW and pulse EPR/ENDOR experiments with large sample access and optical excitation

We describe a frequency tunable Q-band cavity (34 GHz) designed for CW and pulse Electron Paramagnetic Resonance (EPR) as well as Electron Nuclear Double Resonance (ENDOR) and Electron Electron Double Resonance (ELDOR) experiments. The TE(011) cylindrical resonator is machined either from brass or from graphite (which is subsequently gold plated), to improve the penetration of the 100 kHz field modulation signal. The (self-supporting) ENDOR coil consists of four 0.8mm silver posts at 2.67 mm distance from the cavity center axis, penetrating through the plunger heads. It is very robust and immune to mechanical vibrations. The coil is electrically shielded to enable CW ENDOR experiments with high RF power (500 W). The top plunger of the cavity is movable and allows a frequency tuning of ±2 GHz. In our setup the standard operation frequency is 34.0 GHz. The microwaves are coupled into the resonator through an iris in the cylinder wall and matching is accomplished by a sliding short in the coupling waveguide. Optical excitation of the sample is enabled through slits in the cavity wall (transmission ～60%). The resonator accepts 3mm o.d. sample tubes. This leads to a favorable sensitivity especially for pulse EPR experiments of low concentration biological samples. The probehead dimensions are compatible with that of Bruker flexline Q-band resonators and it fits perfectly into an Oxford CF935 Helium flow cryostat (4-300 K). It is demonstrated that, due to the relatively large active sample volume (20-30 μl), the described resonator has superior concentration sensitivity as compared to commercial pulse Q-band resonators. The quality factor (Q(L)) of the resonator can be varied between 2600 (critical coupling) and 1300 (over-coupling). The shortest achieved π/2-pulse durations are 20 ns using a 3 W microwave amplifier. ENDOR (RF) π-pulses of 20 μs ((1)H @ 51 MHz) were obtained for a 300 W amplifier and 7 μs using a 2500 W amplifier. Selected applications of the resonator are presented.     

The bruker high-frequency-EPR system

The design and performance of the first commercial 94 GHz continuous-wave (CW-)/Fourier transform (FT-) EPR and ENDOR spectrometer are described. The spectrometer design is based on a heterodyne microwave bridge using an X-band intermediate frequency (IF), a hybrid magnet system, a variable-temperature, top-loading TeraFlex probehead with a TE₀₁₁ cavity as well as the ELEXSYS-line digital electronics and the Xepr software package. The W-band bridge can be driven by a CW- or pulse-IF unit and delivers a microwave power of 5 mW at 94 GHz. In pulse mode the power is sufficient for a π/2 pulse of 100 ns at a resonatorQ-value of 3000. The magnet system consists of a 6 T split-coil superconducting magnet and a water-cooled room-temperature coil. The main coil can be swept over the full range from 0 to 6 T. The room-temperature coil has a 800 G sweep range around the persistent field of the main magnet. The ENDOR probe features a tuned circuit for¹H nuclei allowing an RF π-pulse of 8 μs with a 200 W amplifier. A broad-band setup is used for other nuclei. The E680 FT-EPR system utilizes the PatternJet pulse programmer and the SpecJet high-speed transient signal averager. The concerted action of these two devices results in a pulse EPR sensitivity equal or higher than in CW-EPR. Selected examples indicating the performance of the 94 GHz CW/FT-EPR and ENDOR systems are shown.     

时间域电子自旋共振

时间域电子自旋共振(ESR)是研究顺磁弛豫机理和动力学过程不可缺少的方法,也是提高检测信号的灵敏度和分辨率的重要途径之一。然而,目前在ESR技术中较常用的还是频率域,而时间域ESR(包括付里叶变换法)却远远不如脉冲付里叶变换核磁共振(PFT-NMR)那样迅速的发展。本文对此进行了讨论,认为:如采用与PFT-NMR稍为不同的方法,并在微波技术、快速脉冲电路和电子计算技术等不断改善的基础上,时间域ESR势将成为今后发展的大方向。近年来,在时间域ESR技术方面,最引人重视的是:饱和恢复法和电子自旋回波(ESE)法。本文着重对这两种方法的基本原理、实验方法、应用场合及其优越性和局限性进行了评述。例如,用付里叶变换法(包括二维付里叶变换)把电子自旋回波调制的包络自时间域变换成频率域,从而获得高分辨率的频谱,则可分析出取向或无规取向样品的微弱超精细结构。又如,ENDOR(电子-核双共振)自旋回波与通常的ENDOR相比,前者具有较高的灵敏度以及可检测较低的ENDOR频率等独特之处。此外,文中对瞬态顺磁中间产物的时间分辨ESR和三重态分子在零场中的ESR也分别进行了简短的评介。最后,对时间域ESR发展的远景作了预计。     

Defects in Nanodiamonds: Application of High-Frequency cw and Pulse EPR,ODMR

Different aspects of applications of electron paramagnetic resonance (EPR) based techniques including high frequency (HF) electron spin echo (ESE), electron-nuclear double resonance (ENDOR) and optically detected magnetic resonance (ODMR) approaches to study diamond nanostructures are examined.     

Selection of dipolar interaction by the “2 + 1” pulse train ESE

The new kind of electron spin echo (ESE), the “2 + 1” pulse train, is described. This method allows the measurement of dipole-dipole interactions between paramagnetic centers which are substantially weaker than those that can be measured by the ordinary two-pulse train. The dead time of ESE spectrometer response in this method is decreased to the duration of the first two pulses. The theory of dipole-dipole interaction in the ESE signal decay in the “2 + 1” pulse train is developed for different Flip rates and for different cases of spin spatial distribution. The theoretical data fit the experiment, carried out with model systems of H and D atoms, randomly distributed in the frozen solutions of sulfuric acid, and of biradicals. New data concerning the spatial distribution of radical clusters, resulting from y irradiation of the methanol, are given.     

Pulsed ENDOR Spectroscopy at Large Thermal Spin Polarizations and the Absolute Sign of the Hyperfine Interaction

It is shown that in pulsed Mims-type ENDOR experiments performed at 95 GHz and 1.2 K the sign of the ENDOR signal can be positive, corresponding to an increase of the stimulated echo intensity, as well as negative, corresponding to a decrease of the stimulated echo. The positive “anomalous” sign is not observed at conventional EPR frequencies. It is explained that the effect arises through spin–lattice relaxation in the situation of large thermal spin polarizations and that it allows the determination of the absolute sign of the hyperfine interaction.     

Absolute signs of hyperfine coupling constants as determined by pulse ENDOR of polarized radical pairs

A novel method that allows the determination of absolute signs of hyperfine coupling constants in polarized radical pair (RP) pulse electron-nuclear double resonance (ENDOR) spectra is presented, The variable mixing time (VMT) ENDOR method used here leads to a separation of ENDOR transitions originating from different electron spin manifolds by employing their dependence on the time-dependent parameters of the pulse sequence. The simple kinetic model of the RP VMT ENDOR experiment shows very good agreement with the experiments performed on the P ₇₀₀ ^.+ A ₁ ^.- RP in photosystem I. This method relies on the selective excitation of absorptive or emissive lines of one radical in the RP EPR spectrum and therefore requires high spectral resolution. This condition was fulfilled for the system studied at the low-field edge of the RP EPR spectrum obtained at Q-band. The method presented here has a very high sensitivity and does not require any equipment additional to the one used for RP pulse ENDOR. The VMT ENDOR method offers the possibility for selective suppression of signals from different electron spin manifolds.     

Simultaneous acquisition of pulse EPR orientation selective spectra

High resolution pulse EPR methods are usually applied to resolve weak magnetic electron-nuclear or electron-electron interactions that are otherwise unresolved in the EPR spectrum. Complete information regarding different magnetic interactions, namely, principal components and orientation of principal axis system with respect to the molecular frame, can be derived from orientation selective pulsed EPR measurements that are performed at different magnetic field positions within the inhomogeneously broadened EPR spectrum. These experiments are usually carried out consecutively, namely a particular field position is chosen, data are accumulated until the signal to noise ratio is satisfactory, and then the next field position is chosen and data are accumulated. Here we present a new approach for data acquisition of pulsed EPR experiments referred to as parallel acquisition. It is applicable when the spectral width is much broader than the excitation bandwidth of the applied pulse sequence and it is particularly useful for orientation selective pulse EPR experiments. In this approach several pulse EPR measurements are performed within the waiting (repetition) time between consecutive pulse sequences during which spin lattice relaxation takes place. This is achieved by rapidly changing the main magnetic field, B(0), to different values within the EPR spectrum, performing the same experiment on the otherwise idle spins. This scheme represents an efficient utilization of the spectrometer and provides the same spectral information in a shorter time. This approach is demonstrated on W-band orientation selective electron-nuclear double resonance (ENDOR), electron spin echo envelope modulation (ESEEM), electron-electron double resonance (ELDOR)--detected NMR and double electron-electron resonance (DEER) measurements on frozen solutions of nitroxides. We show that a factors of 3-6 reduction in total acquisition time can be obtained, depending on the experiment applied.     

Abnormal electron spin echo and multiple-quantum coherence in a spin-correlated radical pair system

The phenomena of the abnormal “out-of-phase” electron spin echo in a photo-induced spin-correlated radical pair system are examined theoretically. It is shown that such abnormal phenomena are a consequence of initial non-Boltzmann distribution and zero-quantum coherence produced by laser excitation. The analysis of echo amplitude versus the pulse-angle of the microwave pulse reveals two sources for the formation of the echo. The method of excitation and detection of multiple-quantum coherence using a phase-cycled 2-pulse sequence is also discussed. Such a technique is complementary to the ESE method.     

A Davies/Hahn multi-sequence for studies of spin relaxation in pulsed ENDOR

We extend earlier studies of the effects of relaxation on the intensities of pulsed ENDOR signals by introducing a Davies/Hahn (D/H) pulsed ENDOR multi-sequence that corresponds to a series of Davies sequences with the preparation pulse 'turned off'. In this pulse train, the Hahn [pi/2, pi] detection pulse pair of sequence n-1 both generates the echo detected for that sequence and acts as the preparation portion of sequence n, in effect replacing the pi preparation pulse of the Davies sequence. We show both theoretically, through a master-equation approach, and with both (1)H(I=1/2) and (14)N(I=1) ENDOR experiments on the non-heme Fe enzymes, superoxide reductase (SOR) (S=1/2) and AntDO (S=3/2), that under conditions of high electron-spin polarization (high microwave frequency/low temperature) the D/H multi-sequence allows simplification of ENDOR spectra by suppression of nuclear transitions associated with the m(S)=+1/2 (alpha) manifold. As such suppression depends on the sign of A, it allows determination of this sign. The suppression as a function of the time between individual sequences is found to exhibit behaviors that can be classified into three regimes of the ratio of cross-relaxation to spin-lattice relaxation rates: strong cross-relaxation (X-case); comparable rates (XL); negligible cross relaxation (L). Interestingly, the ENDOR behavior of the S=1/2 SOR center indicates it is an L case, while the S=3/2 AntDO is an L case. Overall, the D/H protocol appears to be a robust and general tool for using relaxation effects to manipulate ENDOR spectra.     

Pulsed Electron-Nuclear Double Resonance (ENDOR) at 140 GHz

We describe a spectrometer for pulsed ENDOR at 140 GHz, which is based on microwave IMPATT diode amplifiers and a probe consisting of a TE011 cavity with a high-quality resonance circuit for variable radiofrequency irradiation. For pulsed EPR we obtain an absolute sensitivity of 3x10(9) spins/Gauss at 20 K. The performance of the spectrometer is demonstrated with pulsed ENDOR spectra of a standard bis-diphenylene-phenyl-allyl (BDPA) doped into polystyrene and of the tyrosyl radical from E. coli ribonucleotide reductase (RNR). The EPR spectrum of the RNR tyrosyl radical displays substantial g-anisotropy at 5 T and is used to demonstrate orientation-selective Davies-ENDOR.     

Distance determination in human ubiquitin by pulsed double electron-electron resonance and double quantum coherence ESR methods

Recently, distance measurements by pulsed ESR (electron spin resonance) have been obtained using pulsed DEER (double electron-electron resonance) and DQC (double quantum coherence) in SDSL (site directed spin labeling) proteins. These methods can observe long range dipole interactions (15-80A). We applied these methods to human ubiquitin proteins. The distance between the 20th and the 35th cysteine was estimated in doubly spin labeled human ubiquitin. Pulsed DEER requires two microwave sources. However, a phase cycle is not usually required in this method. On the other hand, DQC-ESR at X-band ( approximately 9GHz) can acquire a large echo signal by using pulses of short duration and high power, but this method has an ESEEM (electron spin echo envelope modulation) problem. We used a commercial pulsed ESR spectrometer and compared these two methods.     

X- and Q-Band Electron Spin Echo Study of Stochastic Molecular Librations of Spin Labels in Lipid Bilayers

Electron spin echo (ESE) of nitroxide spin labels allows detecting fast nanosecond stochastic restricted rotations (stochastic molecular librations), which is a common property of molecules in disordered media including biological systems. Under the typical experimental conditions, the anisotropic electron paramagnetic resonance (EPR) spectrum of a nitroxide is only partly excited by microwave pulses, which allows selecting an anisotropic contribution to the transverse spin relaxation by comparing echo decays at different spectral positions. On the other hand, for low-amplitude orientational motion, the excitation bandwidth is large enough to cover the range of spectral diffusion occurring during the echo formation. To verify that the two-pulse echo decay is indeed related to fast motions, the stimulated electron spin echo can be used. In addition, theory predicts an increase of the relaxation rates at higher microwave resonance frequency. To check this prediction, in the present work we performed a comparative study of ESE decays at microwave X- and Q-bands, for spin-labeled lipids in the gel phase of a 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) bilayer. A good agreement found between experimental data and computer simulation provides additional justification for the model of fast stochastic molecular librations.