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

Reconstruction for Time-Domain In Vivo EPR 3D Multigradient Oximetric Imaging—A Parallel Processing Perspective

Three-dimensional Oximetric Electron Paramagnetic Resonance Imaging using the Single Point Imaging modality generates unpaired spin density and oxygen images that can readily distinguish between normal and tumor tissues in small animals. It is also possible with fast imaging to track the changes in tissue oxygenation in response to the oxygen content in the breathing air. However, this involves dealing with gigabytes of data for each 3D oximetric imaging experiment involving digital band pass filtering and background noise subtraction, followed by 3D Fourier reconstruction. This process is rather slow in a conventional uniprocessor system. This paper presents a parallelization framework using OpenMP runtime support and parallel MATLAB to execute such computationally intensive programs. The Intel compiler is used to develop a parallel C++ code based on OpenMP. The code is executed on four Dual-Core AMD Opteron shared memory processors, to reduce the computational burden of the filtration task significantly. The results show that the parallel code for filtration has achieved a speed up factor of 46.66 as against the equivalent serial MATLAB code. In addition, a parallel MATLAB code has been developed to perform 3D Fourier reconstruction. Speedup factors of 4.57 and 4.25 have been achieved during the reconstruction process and oximetry computation, for a data set with 23 × 23 × 23 gradient steps. The execution time has been computed for both the serial and parallel implementations using different dimensions of the data and presented for comparison. The reported system has been designed to be easily accessible even from low-cost personal computers through local internet (NIHnet). The experimental results demonstrate that the parallel computing provides a source of high computational power to obtain biophysical parameters from 3D EPR oximetric imaging, almost in real-time

Time-domain EPR imaging with slice selection

Synopsis: The slice selection imaging has advantages of reducing imaging time and obtaining optimum dynamic range in image for EPR imaging as well as for MRI. However, the slice selection using a selective pulse, which is used in MRI, is difficult to implement in EPR imaging because of ultra-fast relaxation time compared to gradient settling time. Therefore, we used a modulated gradient field to achieve slice selection in pulsed EPR imaging in this study. We demonstrated the slice selection imaging with tubes and a living mouse to show the effect of slice selection in pulsed EPR imaging. Introduction: In MRI, slice-selection is accomplished using a selective pulse in presence of a slice selective gradient. The spatial encoding and other functional properties of the spins in the selected slice are carried out by the subsequent refocusing pulses and phase or frequency encoding gradients. Such slice selection is difficult in pulsed EPR imaging, due the ~microsecond relaxation times of unpaired electrons which are shorter than gradient settling times. An alternative mode of slice-selection however is feasible by applying a modulated gradient along one of the directions1,2. The selected slice is located at the ‘zero-crossing’ of the modulated gradient. Its thickness depends on the modulation amplitude and frequency. Such slice selection can reduce the imaging time by an order of magnitude since only 2D images are measured, and the slice location can be changed by physically translating the resonator or electrically off-setting the center of the oscillatory gradient. Phantom and in vivo results are shown. Modalities which image a small number of m slices We have incorporated this approach of slice-selection using sinusoidally modulated gradients to generate a set of 2D images of slices that can greatly reduce the measurement time and can thus allow improvement in the SNR and resolution in the selected slices without additional measurement time. Methods: We employ the single point imaging (SPI) scheme, by which two and three dimensional in vivo EPR imaging and relaxation based oximetry have been carried out routinely in our laboratory. In this development, we use the same imaging equipment operating at 300 MHz, with an additional provision of applying a low frequency (100 Hz) sinusoidal field along one of the gradient axes at nominal AC amplitude of about 10 mT/m. The modulation of the gradient along a particular axis introduces inhomogeneity along that axis everywhere except around the midpoint at which the amplitude is zero. A two-dimensional phase encoding in a plane perpendicular to the axis of the modulated gradient retains coherent phase information only from the narrow slice at the center with spin distribution on either side of the slice undergoing total loss of coherence and does not contribute to the detected signal (Fig. 1). As proof of principle we made a phantom consisting of three tubes(4 mm i.d) filled to different levels with 3 mM Oxo633 (a stable trityl radical with a narrow single ESR absorption) and placed at a spacing interval of 10 mm as shown in Fig. 2A. Two dimensional images were obtained by single point imaging with a maximum gradient of 15 mT/m along the three planes.In addition to above phantom experiment, we performed in vivo experiments to investigate how dynamic range was improved using a mouse hind leg. Along with the mouse leg, we placed a TCNQ tube which produces strong signal as shown in Fig. 3A. The mouse was injected 75 mM oxo63 intravenously. Two dimensional images were obtained by single point imaging with a maximum gradient of 8 mT/m. In order to investigate the minimum slice thickness that can be achieved, we filled a 14 mm glass cuvette (the ones used in optical spectroscopy) with 2 mM Oxo63 and placed the cuvette at the center of the resonator along Y-direction. The EPR spectra were obtained when the Z-gradient was modulated at 100 Hz with a gradual increase in amplitude from 0 to 2 volt. Results and Discussion: When we carried out the 2D phase encoding in the XY plane with the Z-gradient being modulated at 100 Hz at amplitude of 1.4 volt, we saw only the tube centered at z-coordinate of zero (Fig. 2D). The other two tubes did not produce any signal due to the inhomogeneity imposed by the modulated Z-gradient. By shifting the resonator such that the other tubes were brought to the center sequentially, we could get slices showing exclusive images of each tube. Figure 3B and 3C shows the comparison between images acquired with conventional 2D projection and slice-selection methods. The distribution of Oxo63 acquired by 2D projection imaging was interfered by strong signal of TCNQ, while the slice selection image showed only the distribution of Oxo63. This suggested the optimum dynamic range in signal intensity was achieved by slice selection technique. The minimum slice thickness that could be achieved was around 1.7 mm at and above 1.8 volt. Conclusion: With modulation gradient, we have demonstrated the slice selection in pulsed EPR imaging and succeeded to obtain slice selected images with optimum dynamic range in signal intensity. The method will also enable the study functional dynamics in the images with improved temporal resolution.References:1. Hinshaw WS. Spin mapping:application of moving gradients to NMR. Phys Lett A. 1974; 48: 87–88. 2.Sato-Akaba H, Abe H, Fujii H, Hirata H. Slice-selective images of free radicals in mice with modulated field gradient electron paramagnetic resonance (EPR) imaging. Magn Reson Med. 2008; 59: 885-890. 3.Ardenkjaer-Larsen JH, Laursen I, Leunbach I, Ehnholm G, Wistrand LG, Petersson JS, Golman K. EPR and DNP properties of certain novel single electron contrast agents intended for oximetric imaging. J Magn Reson 1998; 133: 1–12.Joint Annual Meeting ISMRM-ESMRMB 201

Quantitative imaging of pO2 in orthotopic murine gliomas : hypoxia correlates with resistance to radiation

Hypoxia is considered one of the microenvironmental factors associated with the malignant nature of glioblastoma. Thus, evaluating intratumoural distribution of hypoxia would be useful for therapeutic planning as well as assessment of its effectiveness during the therapy. Electron paramagnetic resonance imaging (EPRI) is an imaging technique which can generate quantitative maps of oxygen in vivo using the exogenous paramagnetic compound, triarylmethyl and monitoring its line broadening caused by oxygen. In this study, the feasibility of EPRI for assessment of oxygen distribution in the glioblastoma using orthotopic U87 and U251 xenograft model is examined. Heterogeneous distribution of pO2 between 0 and 50 mmHg was observed throughout the tumours except for the normal brain tissue. U251 glioblastoma was more likely to exhibit hypoxia than U87 for comparable tumour size (median pO2; 29.7 and 18.2 mmHg, p = .028, in U87 and U251, respectively). The area with pO2 under 10 mmHg on the EPR oximetry (HF10) showed a good correlation with pimonidazole staining among tumours with evaluated size. In subcutaneous xenograft model, irradiation was relatively less effective for U251 compared with U87. In conclusion, EPRI is a feasible method to evaluate oxygen distribution in the brain tumour

EPR-based oximetric imaging : a combination of single point-based spatial encoding and T1 weighting

Purpose: Spin-lattice relaxation time (T1)-weighted time-domain EPR oximetry is reported for in vivo applications using a paramagnetic probe, a trityl-based Oxo71. Methods: The R1 dependence of the trityl probe Oxo71 on pO2 was assessed using single point imaging (SPI) mode of spatial encoding combined with rapid repetition, similar to T1-weighted MRI, where R1 was determined from 22 repetition times ranging from 2.1–40.0 μs at 300 MHz. The pO2 maps of a phantom with three tubes containing 2 mM Oxo71 solutions equilibrated at 0%, 2%, and 5% oxygen were determined by R1 and apparent spin-spin relaxation rate (R2*) simultaneously. Results: The pO2 maps derived from R1 and R2* agreed with the known pO2 levels in the tubes of Oxo71. However, the histograms of pO2 revealed that R1 offers better pO2 resolution than R2* in low pO2 regions. The standard deviations of pixels at 2% pO2 (15.2 mmHg) were about 5 times lower in R1-based estimation than R2*-based estimation (mean ± SD: 13.9 ± 1.77 mmHg and 18.3 ± 8.70 mmHg, respectively). The in vivo pO2 map obtained from R1-based assessment displayed a homogeneous profile in low pO2 regions in tumor xenografts, consistent with previous reports on R2*-based oximetric imaging. The scan time to obtain the R1 map can be significantly reduced using three repetition times ranging from 4.0‒12.0 μs. Conclusion: Using the SPI modality, R1-based oximetry imaging with useful spatial and oxygen resolutions for small animals was demonstrated

Direct detection and time-locked subsampling applied to pulsed electron paramagnetic resonance imaging

The application of direct time-locked subsampling (TLSS) to Fourier transform electron paramagnetic resonance (FT-EPR) spectroscopy at radio frequencies (rf) is described. With conventional FT-EPR spectroscopy, the high Larmor frequencies (L)often necessitate the use of intermediate frequency (IF) stages to down convert the received free induction decay (FID) signal to a frequency that can be acquired with common data acquisition technology. However, our research focuses on in vivo studies, and consequently utilizes a FT-EPR system with a L<SUB>f</SUB> of 300 MHz. This relatively low frequency L<SUB>f</SUB>, in conjunction with the advent of bandpass sampling analog-to-digital conversion and signal processing technologies, has enabled us to omit the I<SUB>F</SUB> stage in our FT-EPR system. With this in mind, TLSS techniques have been developed to directly sample the 300 MHz FID signal at a sampling rate of 80 MHz providing a signal bandwidth of 20 MHz. The required modifications to the data acquisition and processing system specific to this application are described. Custom software developed to control the EPR system setup, acquire the signals, and post process the data, is outlined. Data was acquired applying both coherent averaging and stochastic excitation sequences. The results of these experiments demonstrate digital down conversion of the 300 MHz FID signal to quadrature baseband. Direct FID TLSS eliminates many noise sources common in EPR systems employing traditional analog receiver techniques, such as the IF mixer stage in single channel systems, and the quadrature baseband mixer stage in dual channel systems

Evaluation of sub-microsecond recovery resonators for in vivo electron paramagnetic resonance imaging

Time-domain (TD) electron paramagnetic resonance (EPR) imaging at 300 MHz for in vivo applications requires resonators with recovery times less than 1 us after pulsed excitation to reliably capture the rapidly decaying free induction decay (FID). In this study, we tested the suitability of the Litz foil coil resonator (LCR), commonly used in MRI, for in vivo EPR/EPRI applications in the TD mode and compared with parallel coil resonator (PCR). In TD mode, the sensitivity of LCR was lower than that of the PCR. However, in continuous wave (CW) mode, the LCR showed better sensitivity. The RF homogeneity was similar in both the resonators. The axis of the RF magnetic field is transverse to the cylindrical axis of the LCR, making the resonator and the magnet co-axial. Therefore, the loading of animals, and placing of the anesthesia nose cone and temperature monitors was more convenient in the LCR compared to the PCR whose axis is perpendicular to the magnet axis

Metabolic and physiologic imaging biomarkers of the tumor microenvironment predict treatment outcome with radiation or a hypoxia-activated prodrug in mice.

Pancreatic ductal adenocarcinoma (PDAC) is characterized by hypoxic niches that lead to treatment resistance. Therefore, studies of tumor oxygenation and metabolic profiling should contribute to improved treatment strategies. Here we define two imaging biomarkers that predict differences in tumor response to therapy: 1) partial oxygen pressure (pO2), measured by EPR imaging; and 2) [1- 13C] pyruvate metabolism rate, measured by hyperpolarized 13C MRI. Three human PDAC xenografts with varying treatment sensitivity (Hs766t, MiaPaCa-2, and Su.86.86) were grown in mice. The median pO2 of the mature Hs766t, MiaPaCa-2, and Su.86.86 tumors was 9.1±1.7, 11.1±2.2, and 17.6±2.6 mmHg, and the rate of pyruvate-to-lactate conversion was 2.72±0.48, 2.28±0.26, and 1.98±0.51 min-1 , respectively (n=6, each). These results are in agreement with steady state data of matabolites quantified by mass spectroscopy and histological analysis indicating glycolytic and hypoxia profile in Hs766t, MiaPaca-2, and Su.86.86 tumors. Fractionated radiation therapy (5 Gy x 5) resulted in a tumor growth delay of 16.7±1.6 and 18.0±2.7 days in MiaPaca-2 and Su.86.86 tumors, respectively, compared to 6.3±2.7 days in hypoxic Hs766t tumors. Treatment with gemcitabine, a first-line chemotherapeutic agent, or the hypoxia-activated prodrug TH-302 was more effective against Hs766t tumors (20.0±3.5 and 25.0±7.7 days increase in survival time, respectively) than MiaPaCa-2 (2.7±0.4 and 6.7±0.7 days) and Su.86.86 (4.7±0.6 and 0.7±0.6 days) tumors. Collectively, these results demonstrate the ability of molecular imaging biomarkers to predict the response of PDAC to treatment with radiation therapy and TH-30

Strategies for improved temporal and spectral resolution in in vivo oximetric imaging using time-domain EPR

A radiofrequency (RF) time-domain electron paramagnetic resonance (EPR) instrument operating at 300, 600, and 750 MHz was used to image tumor hypoxia with high spatial and temporal resolution. A high-speed signal-averaging Peripheral Component Interconnect (PCI) board with flexibility in the input signal level and the number of digitized samples per free induction decay (FID) was incorporated into the receive arm of the spectrometer. This enabled effective and fast averaging of FIDs. Modification of the phase-encoding protocol, and replacement of the General Purpose Interface Bus (GPIB)-based handshake with a PCI-based D/A board for direct control of the gradient amplifier decreased the gradient settling and communication overhead times by nearly two orders of magnitude. Cyclically-ordered phase sequence (CYCLOPS) phase cycling was implemented to correct for pulse imperfections and cancel out unwanted constant signals. These upgrades considerably enhanced the performance of the imager in terms of image collection time, sensitivity, and temporal resolution. We demonstrated this by collecting a large number of 2D images successively and rapidly. The results show that it is feasible to achieve accurate, 2D pO2 maps of tumor hypoxia with 1-mm2 resolution and minimal artifacts using a set of multigradient images within an acceptable measuring time of about 3 s, and 3D maps can be obtained in less than 1 min