Enhanced EPR sensitivity from a ferroelectric cavity insert.

We report the development of a simple ferroelectric cavity insert that increases the electron paramagnetic resonance (EPR) sensitivity by an order of magnitude when a sample is placed within it. The insert is a hollow cylinder (length 4.8 mm, outside diameter 1.7 mm, inside diameter 0.6 mm) made from a single crystal of KTaO(3), which has a dielectric constant of 230 at X-band (9.5 GHz). Its outside dimensions were chosen to produce a resonant frequency in the X-band range, based on electromagnetic field modeling calculations. The insert increases the microwave magnetic field (H(1)) at the center of the insert by a factor of 7.4 when placed in an X-band TM(110) cavity. This increases the EPR signal for a small (volume 0.13 microL) unsaturated nitroxide spin label sample by a factor of 64 at constant microwave power, and by a factor of 9.8 at constant H(1). The insert does not significantly affect the cavity quality factor Q, indicating that this device simply redistributes the microwave fields within the cavity, focusing H(1) onto the sample inside the insert, thus increasing the filling factor. A similar signal enhancement is obtained in the TM(110) and TE(102) cavities, and when the insert is oriented either vertically (parallel to the microwave field) or horizontally (parallel to the DC magnetic field) in the TM(110) cavity. This order-of-magnitude sensitivity enhancement allows EPR spectroscopy to be performed in conventional high-Q cavities on small EPR samples previously only measurable in loop-gap or dielectric resonators. This is of particular importance for small samples of spin-labeled biomolecules.     

High-field/high-frequency EPR spectrometer operating in pulsed and continuous-wave mode at 180 GHz

A novel electron paramagnetic resonance (EPR) spectrometer is reported, which has been developed to allow pulsed EPR experiments with high sensitivity and time resolution at a microwave (MW) frequency of 180 GHz (G-band) and wavelengths of approximately 1.6 mm. This corresponds to a magnetic field of about 6.4 T forg ≈ 2 signals. The “hybrid” system architecture combines components of quasioptical as well as conventional MW techniques, making it possible to achieve excellent spectrometer performance with respect to sensitivity and time resolution. Quasioptical MW components have been used to design an MW circulator allowing high sensitivity and low bias operation in the reflection mode. A miniaturized, closed-type cylindrical cavity provides a high sample filling factor and an adequate MW field strength (B₁) enhancement and thus permits reasonably short MW pulses (60 ns for a π/2 pulse) even with a moderate MW input power (15 mW at the cavity). Commercial quartz capillaries (up to 0.5 mm internal diameter) can be used as sample holders for a broad range of applications.     

A tunable reentrant resonator with transverse orientation of electric field for in vivo EPR spectroscopy

There has been a need for development of microwave resonator designs optimized to provide high sensitivity and high stability for EPR spectroscopy and imaging measurements of in vivo systems. The design and construction of a novel reentrant resonator with transversely oriented electric field (TERR) and rectangular sample opening cross section for EPR spectroscopy and imaging of in vivo biological samples, such as the whole body of mice and rats, is described. This design with its transversely oriented capacitive element enables wide and simple setting of the center frequency by trimming the dimensions of the capacitive plate over the range 100-900 MHz with unloaded Q values of approximately 1100 at 750 MHz, while the mechanical adjustment mechanism allows smooth continuous frequency tuning in the range +/-50 MHz. This orientation of the capacitive element limits the electric field based loss of resonator Q observed with large lossy samples, and it facilitates the use of capacitive coupling. Both microwave performance data and EPR measurements of aqueous samples demonstrate high sensitivity and stability of the design, which make it well suited for in vivo applications.     

Two-dimensional imaging of two types of radicals by the CW-EPR method

New EPR resonators were developed by using a ceramic material with a high dielectric constant, epsilon=160. The resonators have a high quality factor, Q=10(3), and enhance the sensitivity of an EPR spectrometer up to 170 times. Some advantages of the new ceramic resonators are: (1) cheaper synthesis and simplified fabricating technology; (2) wider temperature range; and (3) ease of use. The ceramic material is produced with a titanate of complex oxides of rare-earth and alkaline metals, and has a perovskite type structure. The resonators were tested with X-band EPR spectrometers with cylindrical (TE(011)) and rectangular (TE(102)) cavities at 300 and 77K. We discovered that EPR signal strength enhancement depends on the dielectric constant of the material, resonator geometry and the size of the sample. Also, an unusual resonant mode was found in the dielectric resonator-metallic cavity structure. In this mode, the directions of microwave magnetic fields of the coupled resonators are opposite and the resonant frequency of the structure is higher than the frequency of empty metallic cavity.     

Microwave frequency modulation in CW EPR at W-band using a loop-gap resonator

Loop-gap resonator (LGR) technology has been extended to W-band (94GHz). One output of a multiarm Q-band (35GHz) EPR bridge was translated to W-band for sample irradiation by mixing with 59 GHz; similarly, the EPR signal was translated back to Q-band for detection. A cavity resonant in the cylindrical TE011 mode suitable for use with 100 kHz field modulation has also been developed. Results using microwave frequency modulation (FM) at 50 kHz as an alternative to magnetic field modulation are described. FM was accomplished by modulating a varactor coupled to the 59 GHz oscillator. A spin-label study of sensitivity was performed under conditions of overmodulation and gamma2H1(2)T1T2<1. EPR spectra were obtained, both absorption and dispersion, by lock-in detection at the fundamental modulation frequency (50 kHz), and also at the second and third harmonics (100 and 150 kHz). Source noise was deleterious in first harmonic spectra, but was very low in second and third harmonic spectra. First harmonic microwave FM was transferred to microwave modulation at second and third harmonics by the spins, thus satisfying the "transfer of modulation" principle. The loaded Q-value of the LGR with sample was 90 (i.e., a bandwidth between 3 dB points of about 1 GHz), the resonator efficiency parameter was calculated to be 9.3 G at one W incident power, and the frequency deviation was 11.3 MHz p-p, which is equivalent to a field modulation amplitude of 4 G. W-band EPR using an LGR is a favorable configuration for microwave FM experiments.     

Rectangular loop-gap resonator with the light access to the sample

A modified rectangular loop-gap resonator for X-band electron paramagnetic resonance (EPR) studies of aqueous samples, enabling the light access, is described. Changes introduced into rectangular resonator geometry, previously presented in Piasecki et al. (1998) [1], and redesigned coupling structure lead to the better thermal and mechanical stability. The modified structure makes provision for the controlled light access to the sample placed in a flat cell during an EPR experiment. The sensitivity of the resonator for aqueous samples as well as an experimentally tested microwave magnetic field homogeneity are presented. Results of simulations and experimental tests indicate that the presence of light access holes in the resonator's front side does not disturb the uniformity of microwave magnetic field distribution in the nodal plane. The optimal flat cell thickness for unsaturable and saturable aqueous samples has been calculated for this new structure. A modified rectangular geometry of the loop-gap resonator ensures a good performance for aqueous samples allowing its convenient and efficient light illumination during EPR signal recording .     

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.     

Single loop multi-gap resonator for whole body EPR imaging of mice at 1.2 GHz

For whole body EPR imaging of small animals, typically low frequencies of 250-750 MHz have been used due to the microwave losses at higher frequencies and the challenges in designing suitable resonators to accommodate these large lossy samples. However, low microwave frequency limits the obtainable sensitivity. L-band frequencies can provide higher sensitivity, and have been commonly used for localized in vivo EPR spectroscopy. Therefore, it would be highly desirable to develop an L-band microwave resonator suitable for in vivo whole body EPR imaging of small animals such as living mice. A 1.2 GHz 16-gap resonator with inner diameter of 42 mm and 48 mm length was designed and constructed for whole body EPR imaging of small animals. The resonator has good field homogeneity and stability to animal-induced motional noise. Resonator stability was achieved with electrical and mechanical design utilizing a fixed position double coupling loop of novel geometry, thus minimizing the number of moving parts. Using this resonator, high quality EPR images of lossy phantoms and living mice were obtained. This design provides good sensitivity, ease of sample access, excellent stability and uniform B(1) field homogeneity for in vivo whole body EPR imaging of mice at 1.2 GHz.     

Multibore sample cell increases EPR sensitivity for aqueous samples

We have performed calculations, verified by experiment, to explain why the sensitivity of biological EPR can be dramatically increased by dividing the aqueous sample into separate compartments. In biological EPR, the major factor affecting sensitivity is the number of spins in the sample. For an aqueous sample at ambient temperature, this is limited by the requirement for a small volume, due to strong non-resonant absorption of microwaves by water. However, recent empirical studies have shown that this volume limitation can be greatly relieved by dividing the aqueous sample into separate volumes, which allows much more aqueous sample to be loaded into a resonant cavity without significant degradation of the cavity quality factor. Calculations, based on the Bruggeman mixing rule, show quantitatively that the composite aqueous sample has a permittivity much less than that of bulk water, depending on the aqueous volume fraction f. Analysis for X-band EPR spectroscopy shows that the optimal volume fraction of an aqueous composite sample, producing maximum sensitivity, is f=0.15, increasing the sensitivity by a factor of 8.7, compared with an aqueous sample in a single tube.     

Dielectric resonator-based side-access probe for muscle fiber EPR study

We present a novel dielectric resonator (DR)-based resonant structure that accommodates aqueous sample capillaries in orientations that are either parallel (i.e., side-access) or perpendicular to the direction of an external (Zeeman) magnetic field, B(0). The resonant structure consists of two commercially available X-band DRs that are separated by a Rexolite spacer and resonate in the fundamental TE(01delta) mode. The separator between the DRs is used to tune the resonator to the desired frequency and, by appropriately drilled sample holes, to provide access for longitudinal samples, notably capillaries containing oriented, spin-labeled muscle fibers. In contrast to the topologically similar cylindrical TE(011) cavity, the DR-based structure has distinct microwave properties that favor its use for parallel orientation of lossy aqueous samples. For perpendicular orientation of a dilute (6.25 microM) aqueous solution of IASL spin label, the S/N ratio was at least one order of magnitude better for the side-access DR-based structure than for a standard TE(102) cavity. EPR spectra acquired for maleimide spin-labeled myosin filaments also revealed ca. 10 times better S/N ratio than those obtained with a standard TE(102) cavity. For the side-access DR with sample capillaries oriented either parallel or perpendicular to the external magnetic field, the Q- and filling factors are in good agreement with the theoretical estimates derived from the distribution of magnetic (H(1)) and electric (E(1)) components.     

A continuous-wave and pulsed electron spin resonance spectrometer operating at 275 GHz

An electron paramagnetic resonance (EPR) spectrometer is described which allows for continuous-wave and pulsed EPR experiments at 275 GHz (wavelength 1.1 mm). The related magnetic field of 9.9 T for g approximately 2 is supplied by a superconducting solenoid. The microwave bridge employs quasi-optical as well as conventional waveguide components. A cylindrical, single-mode cavity provides a high filling factor and a high sensitivity for EPR detection. Even with the available microwave power of 1 mW incident at the cavity a high microwave magnetic field B1 is obtained of about 0.1 mT which permits pi/2-pulses as short as 100 ns. The performance of the spectrometer is illustrated with the help of spectra taken with several samples.     

Analysis of the Movement of Line-like Samples of Variable Length along theX-Axis of a Double TE104and a Single TE102Rectangular Cavity

Movement of line-like samples with lengths from 5 to 50 mm along thex-axis of the double TE₁₀₄rectangular cavity has been analyzed. The observed dependencies of the EPR signal intensity versus sample position showed: (i) a sharp maximum for sample lengths from 5 to 20 mm; (ii) a plateau, over which the EPR signal intensity remained constant within experimental errors of 0.26–1.07%, for lengths from 30 to 40 mm; and (iii) a “sloping plateau,” which could be approximated by the linear function (correlation,r= 0.98) for sample length 50 mm. Theoretical values of the experimentally observed dependencies of the intensity versus sample position were calculated using the modified sine-squared function and the correlation between observed and theoretically predicted dependencies is very good. The experimental dependence of the EPR signal intensity versus the sample length for samples situated at the same point in the cavity was nonlinear with a maximum for the 40-mm sample. The dependence of the EPR signal intensity upon the movement of a large cylindrical sample (o.d. 4 mm and length 100 mm) along thex-axis of the cavity was similar to that found for the 50-mm sample. However, an additional oscillating signal superimposed on the sloping plateau was observed. The presence of a large sample fixed in the complementary cavity of the double TE₁₀₄cavity caused an additional deformation of the signal intensity for a 30-mm sample which was moving in the first cavity. The primary effect was that the plateau was replaced by a region in which the intensity increased linearly with sample position,r= 0.99. Each of the above phenomena may be a source of significant errors in quantitative EPR spectroscopy. Cylindrical samples to be compared should be of identical length and internal diameter. Accurate and precise positioning of each sample in the microwave cavity is essential.     

Influence of the diameter and wall thickness of a quartz pipe inserted in the EPR cavity on the signal intensity

Some dependences between the EPR signal intensity of point samples and the diameter and wall thickness of the quartz tubes with which a standard rectangular TE₁₀₂ EPR cavity is loaded are reported. It is found that the EPR signal intensity linearly depends on the wall thickness of the container and can be increased approximately twice when thick sample tubes are used. The microwave power in the location of the sample increases similarly whereas the normalized bell-shape distribution of the signal intensity along thez-axis of the cavity remains unchanged. The effective microwave power necessary to saturate the sample decreases and the cavity filling factor increases.     

Analysis of two stacked cylindrical dielectric resonators in a TE₁₀₂ microwave cavity for magnetic resonance spectroscopy

The frequency, field distributions and filling factors of a DR/TE??? probe, consisting of two cylindrical dielectric resonators (DR1 and DR2) in a rectangular TE??? cavity, are simulated and analyzed by finite element methods. The TE(+++) mode formed by the in-phase coupling of the TE??(δ)(DR1), TE??(δ)(DR2) and TE??? basic modes, is the most appropriate mode for X-band EPR experiments. The corresponding simulated B(+++) fields of the TE(+++) mode have significant amplitudes at DR1, DR2 and the cavity's iris resulting in efficient coupling between the DR/TE??? probe and the microwave bridge. At the experimental configuration, B(+++) in the vicinity of DR2 is much larger than that around DR1 indicating that DR1 mainly acts as a frequency tuner. In contrast to a simple microwave shield, the resonant cavity is an essential component of the probe that affects its frequency. The two dielectric resonators are always coupled and this is enhanced by the cavity. When DR1 and DR2 are close to the cavity walls, the TE(+++) frequency and B(+++) distribution are very similar to that of the empty TE??? cavity. When all the experimental details are taken into account, the agreement between the experimental and simulated TE(+++) frequencies is excellent. This confirms that the resonating mode of the spectrometer's DR/TE??? probe is the TE(+++) mode. Additional proof is obtained from B?(x), which is the calculated maximum x component of B(+++). It is predominantly due to DR2 and is approximately 4.4 G. The B?(x) maximum value of the DR/TE??? probe is found to be slightly larger than that for a single resonator in a cavity because DR1 further concentrates the cavity's magnetic field along its x axis. Even though DR1 slightly enhances the performance of the DR/TE??? probe its main benefit is to act as a frequency tuner. A waveguide iris can be used to over-couple the DR/TE??? probe and lower its Q to ≈150. Under these conditions, the probe has a short dead time and a large bandwidth. The DR/TE??? probe's calculated conversion factor is approximately three times that of a regular cavity making it a good candidate for pulsed EPR experiments.     

Impact of the Characteristic Impedance of Coaxial Lines on the Sensitivity of a 750-MHz Electronically Tunable EPR Resonator

A 750-MHz electronically tunable resonator was investigated in terms of the sensitivity of electron paramagnetic resonance (EPR) signal detection. The conversion efficiency of the radio-frequency magnetic field was calculated for resonators with 50- and 100-Ω coaxial coupling lines using three-dimensional (3D) microwave field and microwave circuit simulators. Based on the simulation results, two tunable resonators were physically constructed and compared in terms of EPR signal sensitivity using a nitroxyl radical solution. While the resonator with 100-Ω coaxial lines provided 14% greater signal intensity, its signal-to-noise ratio was lower than that of the resonator with 50-Ω lines. To demonstrate the capability of the constructed tunable resonator for EPR imaging experiments, a solution of nitroxyl radical and the leg of a tumor-bearing mouse were visualized.     

Aqueous flat cells perpendicular to the electric field for use in electron paramagnetic resonance spectroscopy

An analytic solution of the Maxwell equations for aqueous flat cells in rectangular TE(102) cavities has led to the prediction of significant (3-6 times) X-band EPR signal improvement over the standard flat cell for a new sample configuration consisting of many flat cells oriented perpendicular to the electric field nodal plane. Analytic full wave solutions in the presence of sample and wall losses have been obtained and numerically evaluated. Observation of the predicted fields led to a classification of three distinct types of sample loss mechanisms, which, in turn inspired sample designs that minimize each loss type. The resulting EPR signal enhancement is due to the presence and centering of a tangential electric field node within each individual sample region. Samples that saturate with the available RF magnetic field and those that do not are considered. Signal enhancement appears in both types. These observations, done for the TE(102) mode, carry over to the uniform field (UF) modes, a relatively new class of microwave cavities for use in EPR spectroscopy developed in this laboratory. Rectangular UF modes have an RF magnetic field magnitude that is uniform in a plane. Based on this analysis, a practical multiple flat-cell design is proposed.     

Transverse oriented electric field re-entrant resonator (TERR) with automatic tuning and coupling control for EPR spectroscopy and imaging of the beating heart

Sample motion, particularly that of a beating heart, induces baseline noise and spectral distortion on an EPR spectrum. In order to quench motional noise and restore the EPR signal amplitude and line-width, an L-band transverse oriented electric field re-entrant resonator (TERR) was designed and constructed with provisions for automatic tuning control (ATC) and automatic coupling control (ACC) suited for studies of isolated beating rat hearts. Two sets of electronic circuits providing DC biased voltage to two varactor diodes were implemented to electronically adjust coupling and tuning. The resonator has a rectangular cross-sectional sample arm of 25 mm diameter with a Q value of 1100 without sample. Once inserted with lossy aqueous samples of 0.45% NaCl, Q value drops to 400 with a volume of 0.5 ml and 150 with 5 ml. The ATC/ACC functions were tested with a moving phantom and isolated beating rat hearts with the improvement of signal to noise ratio (S/N, peak amplitude of signal over peak amplitude of baseline noise) of 6.7-, and 4 to 6-fold, respectively. With these improvements, EPR imaging could be performed on an isolated beating rat heart. Thus, this TERR resonator with ATC/ACC enables application of EPR spectroscopy and imaging for the measurement and imaging of radical metabolism, redox state, and oxygenation in the isolated beating rat heart.     

Impact of High-Dielectric-Loss Materials on the Microwave Field in EPR Experiments

A dielectric material distorts the microwave field inside an EPR resonator, which results in distortion of the EPR signal from spins inside the material. In this paper, the effects of a spherical bulb filled with a dielectric liquid such as water or a water–ethanol mixture were examined. EPR spectra were recorded for small samples inside and outside of the sphere. The studies include CW and ESE experiments at two microwave frequencies, X band (9.2 GHz) and L band (1.03 GHz). The double integral (area) of an EPR signal depends on[formula]at the position of the sample, causing a large difference in EPR signal intensities between samples in regions of different dielectrics. The phase of the EPR signal also is affected by the presence of the dielectric. These results were compared with three methods of calculating electromagnetic fields (quasi-static method, plane-wave-superposition method, and numerical analysis). Good agreement was found between experimental and calculated results.     

Influence of the movement of cylindrical samples with variable internal diameter and variable length along thex-axis of a double TE104 and a single TE102 rectangular cavity on the EPR signal intensity: A sample shape study

The response of the cavity to the movement of cylindrical samples with internal diameters from 0.7 to 4 mm and lengths from 5 to 50 mm along thex-axis of the Bruker double TE₁₀₄ and single TE₁₀₂ rectangular cavity has been analyzed. Independently of sample internal diameter, the experimentally observed dependences of the electron paramagnetic resonance (EPR) signal intensity versus sample position in the cavity showed the following: (i) a sharp maximum for sample lengths from 5 to 20 mm; (ii) a “plateau”, over which the signal intensity remained constant within experimental errors of 0.47–1.16%, for lengths from 30 to 40 mm; and (iii) a “sloping plateau” region, which could be approximated by the linear function (correlationr = 0.96–0.98) for the 50 mm sample. Theoretical predictions of the experimental dependences of the signal intensity versus sample position in the cavity were calculated with the “modified” and “revised” sine-squared function, and the correlation between observed and theoretically computed dependences is very good. Additionally, the experimental dependence of the signal intensity versus the sample internal diameter and length for cylindrical samples situated at the position in the cavity at which the signal intensity was a maximum was likewise numerically approximated by the surface fitting with the Lorentzian cumulative additive function (correlationr = 0.999). The experimental dependence of the signal intensity versus the sample internal diameter for the given sample length is nonlinear. The samples with internal diameters of 0.7 and 1 mm gave the total maximum of signal intensity for the 40 mm sample, however, the samples with internal diameters of 2, 3 and 4 mm gave the total maximal value of signal intensity, which was identical for both the 30 and 40 mm samples. The experimental dependence of the EPR signal intensity versus the sample volume clearly showed that the samples with identical volumes, however, with different shapes, can give significantly different signal intensities (with differences ca. 200–400%). Then, the comparison of cylindrical samples with identical volumes but different shapes may be a serious source of significant errors in quantitative EPR spectroscopy. Cylindrical samples to be compared should be of identical shape. Accurate and precise positioning of each sample in the microwave cavity is essential.     

A New Q-Band EPR Probe for Quantitative Studies of Even Electron Metalloproteins

Existing Q-band (35 GHz) EPR spectrometers employ cylindrical cavities for more intense microwave magnetic fields B₁, but are so constructed that only one orientation between the external field B and B₁is allowed, namely the B B₁orientation, thus limiting the use of the spectrometer to measurements on Kramers spin systems (odd electron systems). We have designed and built a Q-band microwave probe to detect EPR signals in even electron systems, which operates in the range 2 K ≤ T ≤ 300 K for studies of metalloprotein samples. The cylindrical microwave cavity operates in the TE₀₁₁mode with cylindrical wall coupling to the waveguide, thus allowing all orientations of the external magnetic field B relative to the microwave field B₁. Such orientations allow observation of EPR transitions in non-Kramers ions (even electron) which are either forbidden or significantly weaker for B B₁. Rotation of the external magnetic field also permits easy differentiation between spin systems from even and odd electron oxidation states. The cavity consists of a metallic helix and thin metallic end walls mounted on epoxy supports, which allows efficient penetration of the modulation field. The first quantitative EPR measurements from a metalloprotein (Hemerythrin) at 35 GHz with B₁ B are presented.