Pulsed EPR Spectrometer Research Articles

A broadband pulse EPR spectrometer for high-throughput measurements in the X-band

We present an X-band pulse EPR spectrometer with high throughput and excellent sensitivity in the 8.5-11.5GHz range. It is designed for high stability and low noise Fourier Transform measurements for applications in pulse dipolar spectroscopy, pulse hyperfine spectroscopy, and spin relaxation from cryogenic temperatures to room temperature. An arbitrary waveform generator is used to generate pulses of any frequency and shape for multiple resonance experiments or for uniform broadband excitation with bandwidths exceeding 350 MHz. We illustrate the capabilities and performance of the spectrometer by measurements on free radicals and biradicals in solids and liquids. Relaxation times of radicals in liquid solution are measured for fewer than 30,000,000 spins (less than 3 nanomoles per liter). Non-uniform acquisition provides higher throughput for mixtures of radicals with quite different relaxation rates. Conventional DEER measurements on a rigid biradical have good modulation depth. Broadband SIFTER with chirped adiabatic WURST pulses demonstrates versatility for the latest broadband pulse schemes. A broadband ESEEM measurement correlates ESEEM and EPR frequencies which characterize the conformation of a nitroxide radical. The entire EPR spectrum with a width approaching 300 MHz was excited and detected throughout the measurement. The spectrometer supports the operator in tuning, setting up experiments and monitoring their progress so that even novice users consistently can obtain optimal results.

Towards single-cell pulsed EPR using VCO-based EPR-on-a-chip detectors

AbstractElectron paramagnetic resonance (EPR) is the gold standard for studying paramagnetic species. As an example, in structural biology, it allows to extract information about distance distributions on the nanometer scale via site-directed spin labeling. Conventional pulsed EPR of biological samples is currently limited to relatively large sample concentrations and cryogenic temperatures, mainly due to low sensitivity and the significant dead time associated with conventional resonator-based EPR setups, essentially precluding in-cell EPR under physiological conditions. This paper presents our latest progress toward single-cell pulsed EPR using VCO-based EPR-on-a-chip (EPRoC) sensors. Together with an analytical model for VCO-based pulsed EPR, we present an experimental scheme to perform dead-time-free pulsed EPR measurements using EPRoC detectors. The proposed scheme is validated using extensive numerical simulations and proof-of-concept experiments on the spin dynamics of an organic radical at room temperature using a custom-designed EPRoC detector operating in the Ka-band around 30.4 GHz. Additionally, we discuss methods to improve the excitation field homogeneity and sample handling through chip post-processing and custom-designed microfluidics. Finally, we present our progress towards compact, portable pulsed EPR spectrometers incorporating EPRoC detectors, microfluidics, and custom-designed permanent magnets. Such portable EPR spectrometers can pave the way toward new EPR applications, including point-of-care diagnostics.

PELDOR Measurements on Nitroxide-Labeled Oligonucleotides.

In the past decades, pulsed dipolar electron paramagnetic resonance spectroscopy (PDS) has emerged as a powerful tool in biophysical chemistry to study the structure, dynamics, and function of biomolecules like oligonucleotides and proteins. Structural information is obtained from PDS methods in form of a distribution of distances between spin centers. Such spin centers can either be intrinsically present paramagnetic metal ions and organic radicals or may be attached to the biomolecule by means of site-directed spin labeling. The most common PDS experiment for probing interspin distances in the nanometer range is pulsed electron-electron double resonance (PELDOR or DEER). In the protocol presented here, we provide a step-by-step workflow on how to set up a PELDOR experiment on a commercially available pulsed EPR spectrometer, outline the data analysis, and highlight potential pitfalls. We suggest PELDOR measurements on nitroxide-labeled oligonucleotides to study the structure of either RNA-cleaving DNAzymes in complex with their RNA targets or modified DNAzymes with different functions and targets, in which deoxynucleotides are substituted by nitroxide-labeled nucleotides.

75 Years of EPR. EPR Milestones in 60 Years Bruker History

After the discovery of EPR by E.K. Zavoiski 1944 in Kazan (E.K. Zavoiski Zhurn Eksperimentalnoi i teoreticheskoi fiziki 15:344, 1945; E.K. Zavoiski J Phys USSR 9:211–245, 1945) EPR spectrometers were home-built and mostly dedicated to a special application. But commercial instruments in the contrary must be universal and cover a broad range of methods and applications. Therefore they should be compatible with any valuable accessory and offer optimal sensitivity and resolution and they should be easily convertible to other frequency bands and new methods of investigation. The sensitivity of the EPR has increased in consecutive steps, from a S/N of 200 for the week pitch in 1970 up to 3000 with a new microwave source and a new super high Q cavity (SHQE). So far all the main requirements for a universal cw-EPR spectrometer for scientific applications have been met successfully. In 1987 with the ESP380 the first commercial pulse EPR spectrometer was introduced. This was no more an improvement of the previous EPR, this was a revolution in EPR instrumentation. Some necessary microwave-components were not available on the market and had to be developed and produced by the company itself. To cope with the extremely short relaxation times, necessitating high bandwidths, a new range of resonators had to be developed. In cooperation with the Eaton-group Bruker has introduced a rapid scan accessory for the ELEXSYS spectrometers increasing sensitivity for almost two orders of magnitude. The rapid scan may also give new possibilities for EPR imaging in the much less sensitive S- and L-bands for biomedical research.

Hybrid nanophotonic-nanomagnonic SiC-YiG quantum sensor: II/dark spins quantum sensing with V2 spins and fiber based OP-PELDOR/ODMR

First experiments like optically detected (OD) electron paramagnetic resonance (ODMR), photoluminescence detected RABI oscillations, and optical pumping (OP) assisted pulsed EPR measurements of T2 and T1 of V2 spins in bulk SiC, which were previously demonstrated on various home build EPR spectrometers with free space optics, are here all demonstrated for the first time using a commercial X band pulsed EPR spectrometer combined with a single optical fiber and a standard external photoluminescence setup. Quantum sensing of bulk dark spins dipolar coupled to V2 spins in SiC is also demonstrated here for the first time using single fiber based OP assisted pulsed electron electron double resonance spectroscopy (PELDOR). A spin wave resonance study of model permalloy nanostripes is also presented allowing to check the ferromagnetic nanostripes design. These experiments are first key steps towards the fiber-based integration of the recently proposed SiC-YiG quantum sensor device [J. Tribollet, Eur. Phys. J. Appl. Phys.90, 20102 (2020)], to a commercially available and worldwide used pulsed EPR spectrometer, with important applications expected in structural biology, surface chemistry, and quantum computing.

Hybrid nanophotonic-nanomagnonic SiC-YiG quantum sensor: I/theoretical design and properties

Here I present the theory of a new hybrid paramagnetic-ferrimagnetic SiC-YiG quantum sensor. It is designed to allow sub-nanoscale single external spin sensitivity optically detected pulsed electron electron double resonance spectroscopy, using an X band pulsed EPR spectrometer and an optical fiber. The sensor contains one single V2 negatively charged silicon vacancy color center in 4H-SiC, whose photoluminescence is waveguided by a 4H-SiC nanophotonic structure towards an optical fiber. This V2 spin probe is created by ion implantation at a depth of few nanometers below the surface, determined by optically detected paramagnetic resonance under the strong magnetic field gradient of a YiG ferrimagnetic nanostripe located on the back-side of the nanophotonic structure. This gradient also allow the study, slice by slice at nanoscale, of the target paramagnetic sample. The fabrication process of this quantum sensor, its magnetic and optical properties, its external spins sensing properties in a structural biology context, and its integration to a standard commercially available pulsed EPR spectrometer are all presented here.

Frequency-Swept Integrated and Stretched Solid Effect Dynamic Nuclear Polarization.

We investigate a new time domain approach to dynamic nuclear polarization (DNP), the frequency-swept integrated solid effect (FS-ISE), utilizing a high power, broadband 94 GHz (3.35 T) pulse EPR spectrometer. The bandwidth of the spectrometer enabled measurement of the DNP Zeeman frequency/field profile that revealed two dominant polarization mechanisms, the expected ISE, and a recently observed mechanism, the stretched solid effect (S2E). At 94 GHz, despite the limitations in the microwave chirp pulse length (10 μs) and the repetition rate (2 kHz), we obtained signal enhancements up to ∼70 for the S2E and ∼50 for the ISE. The results successfully demonstrate the viability of the FS-ISE and S2E DNP at a frequency 10 times higher than previous studies. Our results also suggest that these approaches are candidates for implementation at higher magnetic fields.

Using rapid-scan EPR to improve the detection limit of quantitative EPR by more than one order of magnitude

X-band rapid-scan EPR was implemented on a commercially available Bruker ELEXSYS E580 spectrometer. Room temperature rapid-scan and continuous-wave EPR spectra were recorded for amorphous silicon powder samples. By comparing the resulting signal intensities the feasibility of performing quantitative rapid-scan EPR is demonstrated. For different hydrogenated amorphous silicon samples, rapid-scan EPR results in signal-to-noise improvements by factors between 10 and 50. Rapid-scan EPR is thus capable of improving the detection limit of quantitative EPR by at least one order of magnitude. In addition, we provide a recipe for setting up and calibrating a conventional pulsed and continuous-wave EPR spectrometer for rapid-scan EPR.

Adiabatic and fast passage ultra-wideband inversion in pulsed EPR

We demonstrate that adiabatic and fast passage ultra-wideband (UWB) pulses can achieve inversion over several hundreds of MHz and thus enhance the measurement sensitivity, as shown by two selected experiments. Technically, frequency-swept pulses are generated by a 12 GS/s arbitrary waveform generator and upconverted to X-band frequencies. This pulsed UWB source is utilized as an incoherent channel in an ordinary pulsed EPR spectrometer. We discuss experimental methodologies and modeling techniques to account for the response of the resonator, which can strongly limit the excitation bandwidth of the entire non-linear excitation chain. Aided by these procedures, pulses compensated for bandwidth or variations in group delay reveal enhanced inversion efficiency. The degree of bandwidth compensation is shown to depend critically on the time available for excitation. As a result, we demonstrate optimized inversion recovery and double electron electron resonance (DEER) experiments. First, virtually complete inversion of the nitroxide spectrum with an adiabatic pulse of 128ns length is achieved. Consequently, spectral diffusion between inverted and non-inverted spins is largely suppressed and the observation bandwidth can be increased to increase measurement sensitivity. Second, DEER is performed on a terpyridine-based copper (II) complex with a nitroxide-copper distance of 2.5nm. As previously demonstrated on this complex, when pumping copper spins and observing nitroxide spins, the modulation depth is severely limited by the excitation bandwidth of the pump pulse. By using fast passage UWB pulses with a maximum length of 64ns, we achieve up to threefold enhancement of the modulation depth. Associated artifacts in distance distributions when increasing the bandwidth of the pump pulse are shown to be small.

Liquid state DNP for water accessibility measurements on spin-labeled membrane proteins at physiological temperatures

We demonstrate the application of continuous wave dynamic nuclear polarization (DNP) at 0.35T for site-specific water accessibility studies on spin-labeled membrane proteins at concentrations in the 10–100μM range. The DNP effects at such low concentrations are weak and the experimentally achievable dynamic nuclear polarizations can be below the equilibrium polarization. This sensitivity problem is solved with an optimized home-built DNP probe head consisting of a dielectric microwave resonator and a saddle coil as close as possible to the sample. The performance of the probe head is demonstrated with both a modified pulsed EPR spectrometer and a dedicated CW EPR spectrometer equipped with a commercial NMR console. In comparison to a commercial pulsed ENDOR resonator, the home-built resonator has an FID detection sensitivity improvement of 2.15 and an electron spin excitation field improvement of 1.2. The reproducibility of the DNP results is tested on the water soluble maltose binding protein MalE of the ABC maltose importer, where we determine a net standard deviation of 9% in the primary DNP data in the concentration range between 10 and 100μM. DNP parameters are measured in a spin-labeled membrane protein, namely the vitamin B12 importer BtuCD in both detergent-solubilized and reconstituted states. The data obtained in different nucleotide states in the presence and absence of binding protein BtuF reveal the applicability of this technique to qualitatively extract water accessibility changes between different conformations by the ratio of primary DNP parameters ϵ. The ϵ-ratio unveils the physiologically relevant transmembrane communication in the transporter in terms of changes in water accessibility at the cytoplasmic gate of the protein induced by both BtuF binding at the periplasmic region of the transporter and ATP binding at the cytoplasmic nucleotide binding domains.

Spin dynamics of paramagnetic centers with anisotropic g tensor and spin of 1/2

The influence of g tensor anisotropy on spin dynamics of paramagnetic centers having real or effective spin of 1/2 is studied. The g anisotropy affects both the excitation and the detection of EPR signals, producing noticeable differences between conventional continuous-wave (cw) EPR and pulsed EPR spectra. The magnitudes and directions of the spin and magnetic moment vectors are generally not proportional to each other, but are related to each other through the g tensor. The equilibrium magnetic moment direction is generally parallel to neither the magnetic field nor the spin quantization axis due to the g anisotropy. After excitation with short microwave pulses, the spin vector precesses around its quantization axis, in a plane that is generally not perpendicular to the applied magnetic field. Paradoxically, the magnetic moment vector precesses around its equilibrium direction in a plane exactly perpendicular to the external magnetic field. In the general case, the oscillating part of the magnetic moment is elliptically polarized and the direction of precession is determined by the sign of the g tensor determinant (g tensor signature). Conventional pulsed and cw EPR spectrometers do not allow determination of the g tensor signature or the ellipticity of the magnetic moment trajectory. It is generally impossible to set a uniform spin turning angle for simple pulses in an unoriented or ‘powder’ sample when g tensor anisotropy is significant.

A Dynamic Nuclear Polarization spectrometer at 95 GHz/144 MHz with EPR and NMR excitation and detection capabilities

A spectrometer specifically designed for systematic studies of the spin dynamics underlying Dynamic Nuclear Polarization (DNP) in solids at low temperatures is described. The spectrometer functions as a fully operational NMR spectrometer (144 MHz) and pulse EPR spectrometer (95 GHz) with a microwave (MW) power of up to 300 mW at the sample position, generating a MW B 1 field as high as 800 KHz. The combined NMR/EPR probe comprises of an open-structure horn-reflector configuration that functions as a low Q EPR cavity and an RF coil that can accommodate a 30–50 μl sample tube. The performance of the spectrometer is demonstrated through some basic pulsed EPR experiments, such as echo-detected EPR, saturation recovery and nutation measurements, that enable quantification of the actual intensity of MW irradiation at the position of the sample. In addition, DNP enhanced NMR signals of samples containing TEMPO and trityl are followed as a function of the MW frequency. Buildup curves of the nuclear polarization are recorded as a function of the microwave irradiation time period at different temperatures and for different MW powers.

A signal-to-noise standard for pulsed EPR

A 2 mm diameter by 10 mm long cylinder of fused SiO 2 (quartz) γ-irradiated to 1 kGy with 60Co contains about 2 × 10 16 spins/cm 3. 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.

Direct Evidence for Nitrogen Ligation to the High Stability Semiquinone Intermediate in Escherichia coli Nitrate Reductase A

The membrane-bound heterotrimeric nitrate reductase A (NarGHI) catalyzes the oxidation of quinols in the cytoplasmic membrane of Escherichia coli and reduces nitrate to nitrite in the cytoplasm. The enzyme strongly stabilizes a menasemiquinone intermediate at a quinol oxidation site (Q(D)) located in the vicinity of the distal heme b(D). Here molecular details of the interaction between the semiquinone radical and the protein environment have been provided using advanced multifrequency pulsed EPR methods. (14)N and (15)N ESEEM and HYSCORE measurements carried out at X-band ( approximately 9.7 GHz) on the wild-type enzyme or the enzyme uniformly labeled with (15)N nuclei reveal an interaction between the semiquinone and a single nitrogen nucleus. The isotropic hyperfine coupling constant A(iso)((14)N) approximately 0.8 MHz shows that it occurs via an H-bond to one of the quinone carbonyl group. Using (14)N ESEEM and HYSCORE spectroscopies at a lower frequency (S-band, approximately 3.4 GHz), the (14)N nuclear quadrupolar parameters of the interacting nitrogen nucleus (kappa = 0.49, eta = 0.50) were determined and correspond to those of a histidine N(delta), assigned to the heme b(D) ligand His-66 residue. Moreover S-band (15)N ESEEM spectra enabled us to directly measure the anisotropic part of the nitrogen hyperfine interaction (T((15)N) = 0.16 MHz). A distance of approximately 2.2 Abetween the carbonyl oxygen and the nitrogen could then be calculated. Mechanistic implications of these results are discussed in the context of the peculiar properties of the menasemiquinone intermediate stabilized at the Q(D) site of NarGHI.

Experimental Observation of Free Induction Decay of Phenoxyl-Type Radical

The free induction decay (FID) of the polycrystalline sample of the phenoxyl radical derived from α-(3,5-di-tert-butyl-4-hydroxyphenyl)-N-tert-butylnitrone, which is a novel spin-trapping reagent, in a corresponding diamagnetic matrix was successfully observed after one pulse even at room temperature, while FIDs of phenoxyl radicals usually are inaccessible due to their short relaxation times and the dead time of the pulse EPR spectrometer. This may be the first experimental observation in phenoxyl-type radicals. This study thus showed the unique character of the phenoxyl radical, that is, the distribution of the electron density relative to the usefulness for the spin-trapping techniques.

HYSCORE and DEER with an upgraded 95 GHz pulse EPR spectrometer

The set-up of a new microwave bridge for a 95 GHz pulse EPR spectrometer is described. The virtues of the bridge are its simple and flexible design and its relatively high output power (0.7 W) that generates π pulses of 25 ns and a microwave field, B 1 = 0.71 mT. Such a high B 1 enhances considerably the sensitivity of high field double electron-electron resonance (DEER) measurements for distance determination, as we demonstrate on a nitroxide biradical with an interspin distance of 3.6 nm. Moreover, it allowed us to carry out HYSCORE (hyperfine sublevel-correlation) experiments at 95 GHz, observing nuclear modulation frequencies of 14N and 17O as high as 40 MHz. This opens a new window for the observation of relatively large hyperfine couplings, yet not resolved in the EPR spectrum, that are difficult to observe with HYSCORE carried out at conventional X-band frequencies. The correlations provided by the HYSCORE spectra are most important for signal assignment, and the improved resolution due to the two dimensional character of the experiment provides 14N quadrupolar splittings.

A CW and pulse EPR spectrometer operating at 122 and 244 GHz using a quasi-optical bridge and a cryogen-free 12 T superconducting magnet

A high-field continuous-wave (CW) and pulse electron paramagnetic resonance spectrometer operating at 122 and 244 GHz is described. The instrument is based on a millimeter-wave bridge built from quasi-optical components. To improve the sensitivity, a cryo-cooled detector/mixer is used. The magnetic field is generated using a cryogen-free superconducting 12 T magnet (warm bore, 88 mm) equipped with a helium-flow cryostat for sample cooling. The advantages of this spectrometer are described and first results (obtained in CW mode) on different types of samples at 122 and 244 GHz are presented. The extensions to pulse operation as well as double resonance techniques (electron-electron and electron-nuclear) are briefly discussed.

Hydrogen Bonds between Nitrogen Donors and the Semiquinone in the Qi-site of the bc1 Complex

The ubisemiquinone stabilized at the Qi-site of the bc1 complex of Rhodobacter sphaeroides forms a hydrogen bond with a nitrogen from the local protein environment, tentatively identified as ring N from His-217. The interactions of 14N and 15N have been studied by X-band (approximately 9.7 GHz) and S-band (3.4 GHz) pulsed EPR spectroscopy. The application of S-band spectroscopy has allowed us to determine the complete nuclear quadrupole tensor of the 14N involved in H-bond formation and to assign it unambiguously to the Nepsilon of His-217. This tensor has distinct characteristics in comparison with H-bonds between semiquinones and Ndelta in other quinone-processing sites. The experiments with 15N showed that the Nepsilon of His-217 was the only nitrogen carrying any considerable unpaired spin density in the ubiquinone environment, and allowed calculation of the isotropic and anisotropic couplings with the Nepsilon of His-217. From these data, we could estimate the unpaired spin density transferred onto 2s and 2p orbitals of nitrogen and the distance from the nitrogen to the carbonyl oxygen of 2.38+/-0.13A. The hyperfine coupling of other protein nitrogens with semiquinone is <0.1 MHz. This did not exclude the nitrogen of the Asn-221 as a possible hydrogen bond donor to the methoxy oxygen of the semiquinone. A mechanistic role for this residue is supported by kinetic experiments with mutant strains N221T, N221H, N221I, N221S, N221P, and N221D, all of which showed some inhibition but retained partial turnover.

An open volume, high isolation, radio frequency surface coil system for pulsed magnetic resonance

We present an open volume, high isolation, RF system suitable for pulsed NMR and EPR spectrometers with reduced dead time. It comprises a set of three RF surface coils disposed with mutually parallel RF fields and a double-channel receiver (RX). Theoretical and experimental results obtained with a prototype operating at about 100 MHz are reported. Each surface RF coil (diameter 5.5 cm) was tuned to f 0 = 100.00 ± 0.01 MHz when isolated. Because of the mutual coupling and the geometry of the RF coils, only two resonances at f 1 = 97.94 MHz and f 2 = 101.85 MHz were observed. We show they are associated with two different RF field spatial distributions. In continuous mode (CW) operation the isolation between the TX coil and one of the RX coils (single-channel) was about −10 dB. By setting the double-channel RF assembly in subtraction mode the isolation values at f 1 or f 2 could be optimised to about −75 dB. Following a TX RF pulse (5 μs duration) an exponential decay with time constant of about 600 ns was observed. The isolation with single-channel RX coil was about −11 dB and it increased to about −47 dB with the double-channel RX in subtraction mode. Similar results were obtained with the RF pulse frequency selected to f 2 and also with shorter (500 ns) RF pulses. The above geometrical parameters and operating frequency of the RF assembly were selected as a model for potential applications in solid state NMR and in free radical EPR spectroscopy and imaging.

EPR and Pulsed ENDOR Study of El6 and Related Defects in 4H-SiC

The EI5 and EI6 centers are typical intrinsic defects in radiation-damaged and semi-insulating SiC. So far, the origins of EI5 and EI6 have been identified as positively-charged carbon vacancies (VC) and silicon antisites (SiC), respectively. However, our complete set of Si hyperfine (HF) data changes these identifications. Our EPR data clearly show that both centers can be well described by VC but their locations should be different (cubic sites for EI5 and hexagonal sites for EI6), as recently proposed by other groups. It was also found that both defects have similar high thermal stability over 1000 oC. In addition to EI5 and EI6, we found a new thermally-stable center, labeled HEI1, in n-type radiation-damaged 4H-SiC. Introduction Electron or neutron irradiation to SiC has been widely studied to explore a variety of intrinsic defects (vacancies, antisites, etc.) in this material. These radiation experiments were performed at relatively low temperatures (4 K or room temperature ~ 450 oC). From a technological point of view, defects that have high thermal stability are of crucial importance. Also, the origin of high thermal stability is of great interest to science. Therefore, we investigate thermally-stable defects in SiC by means of electron irradiation at a high temperature (850 oC) and identify their atomic structures by using electron paramagnetic resonance (EPR). In our study, 4H polytype was selected because of its importance for electronic-device applications. In our high-temperature radiation experiments, only selected (i.e., thermally-stable) defects were found in a specimen. There were at least three types of thermally-stable defects; EI5, EI6, and a new center (we label it “HEI1”). Although the identification of EI5 and EI6 had been published [1,2], more recent EPR experiment [3] and theoretical calculation [4] suggested an alternative model that both EI5 and EI6 are VC defects but they are located at cubic and hexagonal sites, respectively [4]. The conclusion has not been obtained yet, because of a lack of HF interaction data. Thus, we here report complete angular dependence of Si HF interactions of EI5 and EI6. Then, we show that EI6 is well compatible with a hexagonal VC rather than a simple SiC. In addition, EPR data of HEI1 are briefly reported. Experimental SiC substrates are commercial 4H-SiC(0001) wafers supplied by Nippon steel corporation. Dopant concentrations are 1×10 and 1×10 cm for the pand n-type wafers, respectively. Electron irradiation was performed with an electron energy of 3 MeV and a dose of 4×10 cm. During the irradiation, substrates were located in a high vacuum and were heated at a constant temperature of 850 oC. Then, they were measured by X-band EPR and pulsed-EPR spectrometers (Bruker E500 and E580 systems, respectively). Pulsed-EPR was used to deconvolute HF structures by means of pulsed electron-nuclear-double-resonance (ENDOR) techniques. Results and Discussions EI5 and EI6 in p-type 4H-SiC. Figure 1(b) shows EPR spectrum of a p-type sample measured for B (magnetic field) || c. As is indicated in the figure, EPR spectrum consists mainly of central lines of EI5 and EI6, and many HF satellites (labeled a to g). Before starting the analyses of this spectrum, we should mention that EI5 (VC) was stable over 1000 °C, although its anneal-out temperature was known to be much lower (450 ~ 600 °C [1,2,4]) than that of EI6 (> 1000 °C [1]). In our anneal study, both EI5 and EI6 were fully stable at 1000 °C, started to be annihilated at 1200 °C, and almost disappeared at 1500 °C. Thus, we have to change our mind with respect to the thermal stability of EI5. Figure 1(a) shows an angular-dependence map when B was rotated from the c axis ([0001]) to the c-normal direction ([112 _ 0]). The HF satellites a, b and c were already reported by Son et al. [1,2]. EN D O R in te ns ity [a .u .]