A broadband pulsed radio frequency electron paramagnetic resonance spectrometer for biological applications

A time-domain radio frequency (rf) electron paramagnetic resonance (EPR) spectrometer/imager (EPRI) capable of detecting and imaging free radicals in biological objects is described. The magnetic field was 10 mT which corresponds to a resonance frequency of 300 MHz for paramagnetic species. Short pulses of 20-70 ns from the signal generator, with rise times of less than 4 ns, were generated using high speed gates, which after amplification to 283 Vpp, were deposited into a resonator containing the object of interest. Cylindrical resonators containing parallel loops at uniform spacing were used for imaging experiments. The resonators were maintained at the resonant frequency by tuning and matching capacitors. A parallel resistor and overcoupled circuit was used to achieve Q values in the range 20-30. The transmit and receive arms were isolated using a transmit/receive diplexer. The dead time following the trailing edge of the pulse was about 450 ns. The first stage of the receive arm contained a low noise, high gain and fast recovery amplifier, suitable for detection of spin probes with spin-spin relaxation times (T2) in the order of μs. Detection of the induction signal was carried out by mixing the signals in the receiver arm centered around 300 MHz with a local oscillator at a frequency of 350 MHz. The amplified signals were digitized and summed using a 1 GHz digitizer/summer to recover the signals and enhance the signal-to-noise ratio (SNR). The time-domain signals were transformed into frequency-domain spectra, using Fourier transformation (FT). With the resonators used, objects of size up to 5 cm3 could be studied in imaging experiments. Spatial encoding of the spins was accomplished by volume excitation of the sample in the presence of static field gradients in the range of 1.0-1.5 G/cm. The spin densities were produced in the form of plane integrals and images were reconstructed using standard back-projection methods. The image resolution of the phantom objects containing the spin probe surrounded by lossy biologic medium was better than 0.2 mm with the gradients used. To examine larger objects at local sites, surface coils were used to detect and image spin probes successfully. The results from this study indicate the potential of rf FT EPR for in vivo applications. In particular, rf FT EPR may provide a means to obtain physiologic information such as tissue oxygenation and redox status

Increased Resolution and Improved Spectral Quality in Four-Dimensional \u3csup\u3e13\u3c/sup\u3eC/\u3csup\u3e13\u3c/sup\u3eC-Separated HMQC-NOESY-HMQC Spectra Using Pulsed Field Gradients

Pulsed field gradients ( PFG) make it possible to record two-- and three-dimensional spectra using experiments with only a single scan per increment (1-10) . In these experiments PFGs are employed to select the desired coherence-transfer pathway. However, each time a combination of PFGs is used to select a coherence transfer from order -n to m, a second desired pathway, from -n to m , is eliminated, resulting in an intrinsic sensitivity loss of a factor √2. Consequently, coherence pathway selection by means of PFGs is suitable only for applications where the signal-to-noise ratio is not a limiting factor. However, for 3D and 4D experiments applied to dilute protein samples, the intrinsic loss in signal-to-noise of √2 for each PFG coherence selection step is highly undesirable. To circumvent this problem, a different strategy which relies on suppression of undesired coherence-transfer pathways can be used (JI). In this latter approach, coherences associated with spurious magnetization transfer, which frequently result from pulse imperfections, can be eliminated, thereby reducing the number of phase --cycling steps required. A shorter phase cycle allows a reduction in measuring time for experiments that are not limited by the signal-to-noise ratio. Alternatively, it permits an increase in resolution in the indirectly detected dimensions of experiments where measuring time limits the number of increments that can be executed

High-speed digitizer/averager data-acquisition system for fourier transform electron paramagnetic resonance spectroscopy

A high-speed digitizer/averager data-acquisition system designed and built as part of a 300-MHz Fourier transform electron paramagnetic resonance spectrometer is described. There are two key features of the system: (1) the maximum digitizing rate is 300 Msamples/s and (2) a 256-point free-induction-decay signal running summation can be updated in less than 3 μ s. At the maximum digitizing rate, the system can sum 65 536 FIDs in 220 ms. The system consists of an analog-to-digital converter/adder unit (ADCA) and an IBM compatible personal computer. The ADCA is comprised of a digitizer, high-speed sample buffers, high-speed adders/memory, and control hardware. Design techniques, such as parallel processing, utilized to meet the high-speed performance requirements are described. Trigger and timing signals for the system are derived from the spectrometer. System efficiency, synchronization, and time base stability are demonstrated in the spectrometer at a sampling frequency of 200 MHz. Signa-to-noise ratio enhancements are shown using a lithium phthalocyanine test sample

In vivo imaging of a stable paramagnetic probe by pulsed-radiofrequency electron paramagnetic resonance spectroscopy

Imaging of free radicals by electron paramagnetic resonance (EPR) spectroscopy using time domain acquisition as in nuclear magnetic resonance (NMR) has not been attempted because of the short spin-spin relaxation times, typically under 1 μs, of most biologically relevant paramagnetic species. Recent advances in radiofrequency (RF) electronics have enabled the generation of pulses of the order of 10-50 ns. Such short pulses provide adequate spectral coverage for EPR studies at 300 MHz resonant frequency. Acquisition of free induction decays (FID) of paramagnetic species possessing inhomogenously broadened narrow lines after pulsed excitation is feasible with an appropriate digitizer/averager. This report describes the use of time-domain RF EPR spectrometry and imaging for in vivo applications. FID responses were collected from a water-soluble, narrow line width spin probe within phantom samples in solution and also when infused intravenously in an anesthetized mouse. Using static magnetic field gradients and back-projection methods of image reconstruction, two-dimensional images of the spin-probe distribution were obtained in phantom samples as well as in a mouse. The resolution in the images was better than 0.7 mm and devoid of motional artifacts in the in vivo study. Results from this study suggest a potential use for pulsed RF EPR imaging (EPRI) for three-dimensional spatial and spectral-spatial imaging applications. In particular, pulsed EPRI may find use in in vivo studies to minimize motional artifacts from cardiac and lung motion that cause significant problems in frequency-domain spectral acquisition, such as in continuous wave (cw) EPR techniques