Electron Paramagnetic Resonance Imaging Research Articles

EPR imaging of magnetic field effects on radiation dose distributions around millimeter-size air cavities

New hybrid radiotherapy treatment systems combining an MRI scanner with a source of ionizing radiation are being introduced in the clinic. The strong magnetic fields of MRI considerably affect radiation dose distributions, especially at tissue-air interfaces due to the electron return effect (ERE). Experimental investigation of the ERE within a sub-millimeter thick surface layer is still highly challenging.In the present work, we examine and quantify the magnetic field induced perturbations of dose distributions within a 0.5 mm layer surrounding millimeter-size air cavities by applying electron paramagnetic resonance imaging (EPRI).Air-filled fused quartz tubes (inner diameter 3 or 4 mm) mimic small air cavities and serve as model systems. The tubes were irradiated inside a PMMA phantom by a 6 MV photon beam. The irradiations were performed in the presence or absence of a transverse, magnetic field providing a magnetic field strength of 1.0 Tesla. The spatial distributions of radiation induced paramagnetic defects in the quartz tubes were subsequently determined by applying field-swept echo-detected EPRI and were then converted to relative dose distributions.The transverse magnetic field leads to considerable local dose enhancements and reductions (up to 35%) with respect to the mean dose within the quartz tubes. The experimentally determined dose distributions are in good quantitative agreement with Monte Carlo radiation transport simulations.The results of this work demonstrate the feasibility of field-swept echo-detected EPRI to measure magnetic field induced perturbations of dose distributions within a sub-millimeter thick surface layer at the dosimeter-air interface.

Merging Preclinical EPR Tomography with other Imaging Techniques

This paper presents a survey of electron paramagnetic resonance (EPR) image registration. Image registration is the process of overlaying images (two or more) of the same scene taken at different times, from different viewpoints and/or different techniques. EPR-imaging (EPRI) techniques belong to the functional-imaging modalities and therefore suffer from a lack of anatomical reference which is mandatory in preclinical imaging. For this reason, it is necessary to merging EPR images with other modalities which allow for obtaining anatomy images. Methodological analysis and review of the literature were done, providing a summary for developing a good foundation for research study in this field which is crucial in understanding the existing levels of knowledge. Out of these considerations, the aim of this paper is to enhance the scientific community’s understanding of the current status of research in EPR preclinical image registration and also communicate to them the contribution of this research in the field of image processing.

Simultaneous T2* mapping of 14N- and 15N-labeled dicarboxy-PROXYLs using CW-EPR-based single-point imaging.

This article reports a method of simultaneous T2* mapping of 14N- and 15N-labeled dicarboxy-PROXYLs using 750-MHz continuous-wave electron paramagnetic resonance (CW-EPR) imaging. To separate the spectra of 14N- and 15N-labeled dicarboxy-PROXYLs under magnetic field gradients, an optimization problem for spectral projections was formulated with the spatial total variation as a regularization term and solved using a local search based on the gradient descent algorithm. Using the single-point imaging (SPI) method with spectral projections of each radical, simultaneous T2* mapping was performed for solution samples. Simultaneous T2* mapping enabled visualization of the response of T2* values to the level of dissolved oxygen in the solution. Simultaneous T2* mapping applied to a mouse tumor model demonstrated the feasibility of the reported method for potential application to in vivo oxygenation imaging.

Modular imaging system: Rapid scan EPR at 800 MHz.

An electron paramagnetic resonance (EPR) imaging system has been custom built for use in pre-clinical and, potentially, clinical studies. Commercial standalone modules have been used in the design that are MATLAB-controlled. The imaging system combines digital and analog technologies. It was designed to achieve maximum flexibility and versatility and to perform standard and novel user-defined experiments. This design goal is achieved by frequency mixing of an arbitrary waveform generator (AWG) output at the intermediate frequency (IF) with a constant source frequency (SF). Low noise SF at 250, 750, and 1000 MHz are available in the system. A wide range of frequencies from near-baseband to L-band can be generated as a result. Two-stage downconversion at the signal detection side is implemented that enables multi-frequency EPR capability. In the first stage, the signal frequency is converted to IF. A novel AWG-enabled digital auto-frequency control method that operates at IF is described that is used for automatic resonator tuning. Quadrature baseband EPR signal is generated in the second downconversion step. The semi-digital approach of mixing low-noise frequency sources with an AWG permits generation of arbitrary excitation patterns that include but are not limited to frequency sweeps for resonator tuning and matching, continuous-wave, and pulse sequences. Presented in this paper is the demonstration of rapid scan (RS) EPR imaging implemented at 800 MHz. Generation of stable magnetic scan waveforms is critical for the RS method. A digital automatic scan control (DASC) system was developed for sinusoidal magnetic field scans. DASC permits tight control of both amplitude and phase of the scans. A surface loop resonator was developed using 3D printing technology. RS EPR imaging system was validated using sample phantoms. In vivo imaging of a breast cancer mouse model is demonstrated.

Acupuncture attenuates cognitive impairment, oxidative stress and NF-κB activation in cerebral multi-infarct rats.

Patients with multiple infarct dementia (MID) have subtle deficits that commonly go unnoticed, and are at risk of developing Alzheimer's disease. Oxidative stress induced by ischaemic injury results in intracellular calcium accumulation and neuronal apoptosis, leading to cognitive impairment by triggering various cellular signal transduction pathways. Several studies have suggested that NF-κB in the presence of p53 has a pro-apoptotic function in various models, but the mechanism is unclear. The aim of this study was to investigate whether acupuncture could protect cognitive function against cerebral multi-infarction (CMi) induced oxidative stress by inhibiting the activation of NF-κB and its target gene p53. An animal model of CMi was established by injecting homologous blood emboli into the right internal carotid artery of male Wistar rats. After 2 weeks of acupuncture treatment, cognitive function was detected by novel object recognition. Electron spin resonance and Fluo-3 fuorescence imaging were used to test the generation of ROS and intracellular calcium accumulation, respectively. Expression of NF-κB and p53 was examined by Western blot analysis and immunofluorescence. CMi induced spatial learning and memory impairment, overproduction of intracellular hydroxyl radicals, and elevations of Ca2+, which were ameliorated by verum acupuncture treatment. Acupuncture inhibited activation of NF-κB and its downstream target gene p53. These findings suggest that acupuncture could protect cognitive function against oxidative stress induced by CMi, which is partially associated with suppression of NF-κB-p53 activation.

X-band EPR Spectroscopic Investigation of Seborrheic Keratosis.

Seborrheic keratosis (SK) is a common, noncancerous growth on the skin. However, the paramagnetic species (radicals) in pigmented SK have not been investigated yet. X-band (9 GHz) electron paramagnetic resonance (EPR) and EPR imaging (EPRI) were used to nondestructively investigate paraffin-embedded SK. Paramagnetic species in SK specimens were analyzed using linewidth, spectral pattern, and X-band EPRI. The EPR spectra of the SK showed a single line pattern. The EPR results revealed that the peak-to-peak linewidths (ΔHpp) of paraffin-embedded SK samples were 0.58 ± 0.02 mT. The g-value was 2.004 for those samples using EPR standards. EPR signal intensities of the SK samples reasonably corresponded to those for permeability values that are directly related to pigment color tone. Moreover, the two-dimensional (2D) EPRI of the SK showed the distribution of paramagnetic species in the samples with different magnitudes for the first time. The distribution corresponds to the pigmented region. We established that the paramagnetic species was melanin radicals, based on the EPR results obtained in addition to in vivo oxidation of melanin pigments. The present results suggest that EPR and 2D EPRI techniques can be useful for the radical characterization and evaluation of various types of SK.

Synthesis and Characterization of Hydrophilic Trityl Radical TFO for Biomedical and Biophysical Applications.

Tetrathiatriarylmethyl (TAM, trityl) radicals have found wide applications as spin probes/labels for EPR spectroscopy and imaging, and as polarizing agents for dynamic nuclear polarization. The high hydrophilicity of TAM radicals is essential for their biomedical applications. However, the synthesis of hydrophilic TAM radicals (e.g., OX063) is extremely challenging and has only been reported in the patent literature, to date. Herein, an efficient synthesis of a highly water-soluble TAM radical bis(8-carboxyl-2,2,6,6-tetramethylbenzo[1,2-d:4,5-d']bis([1,3]dithiol-4-yl)-mono-(8-carboxyl-2,2,6,6-tetrakis(2-hydroxyethyl)benzo[1,2-d:4,5-d']bis([1,3]dithiol-4-yl)methyl (TFO), which contains four additional hydroxylethyl groups, relative to the Finland trityl radical CT-03, is reported. Similar to OX063, TFO exhibits excellent properties, including high water solubility in phosphate buffer, low log P, low pKa , long relaxation times, and negligible binding with bovine serum albumin. On the other hand, TFO has a sharper EPR line and higher O2 sensitivity than those of OX063. Therefore, in combination with its facile synthesis, TFO should find wide applications in magnetic resonance related fields and this synthetic approach would shed new light on the synthesis of other hydrophilic TAM radicals.

Dextran-conjugated tetrathiatriarylmethyl radicals as biocompatible spin probes for EPR spectroscopy and imaging.

Tetrathiatriarylmethyl (TAM) radicals represent soluble paramagnetic probes for biomedical electron paramagnetic resonance (EPR)-based spectroscopy and imaging. There is an increasing demand in the development of multifunctional, biocompatible and targeted trityl probes hampered by the difficulties in derivatization of the TAM structure. We proposed a new straightforward synthetic strategy using click chemistry for the covalent conjugation of the TAM radical with a water-soluble biocompatible carrier exemplified here by dextran. A set of dextran-grafted probes varied in the degrees of Finland trityl radical loading and dextran modification by polyethelene glycol has been synthesized. The EPR spectrum of the optimized macromolecular probe exhibits a single narrow line with high sensitivity to oxygen and has advantages over the unbound Finland trityl of being insensitive to interactions with albumin. In vivo EPR imaging of tissue oxygenation performed in breast tumor-bearing mouse using dextran-grafted probe demonstrates its utility for preclinical oximetric applications.

Development of an L-band rapid scan EPR digital console.

The development of a digital console for in-vivo rapid scan electron paramagnetic resonance (RS-EPR) spectroscopy and imaging is described in detail. The console was build using field programmable gate array (FGPA) technology that permits real-time control of the resonator and scanning magnetic fields during the measurements. Automatic resonator tuning and matching are achieved by implementing a digital feedback control system and using voltage-tunable capacitors. A band-pass subsampling method is used to directly digitize EPR signals at the carrier frequencies of about 1.2 GHz. The magnetic field scan waveforms, excitation EPR frequency, and sampling clock are all internally synchronized. Full-cycle RS-EPR signals are accumulated in the FPGA in real time without any time gaps. The result is the elimination of the re-arm time, during which data are not acquired. The proposed design in this manuscript has a small footprint and is relatively low cost. The FPGA-based RS-EPR system was tested using standard LiNc-BuO and tempone-d16 samples. The RS-EPR linewidth of the LiNc-BuO sample was consistent with an independent pulsed EPR measurement.

Development of a fast‐scan EPR imaging system for highly accelerated free radical imaging

In continuous wave EPR imaging, the acquisition of high-quality images was previously limited by the requisite long acquisition times of each image projection that was typically greater than 1 second. To accelerate the process of image acquisition facilitating greater numbers of projections and higher image resolution, instrumentation was developed to greatly accelerate the magnetic field scan that is used to obtain each EPR image projection. A low-inductance solenoidal coil for field scanning was used along with a spherical solenoid air core magnet, and scans were driven by triangular symmetric waves, allowing forward and reverse spectrum acquisition as rapid as 3.8 ms. The uniform distribution of projections was used to optimize the contribution of projections for 3D image reconstruction. Using this fast-scan EPR system, high-quality EPR images of phantoms and perfused rat hearts were performed using trityl or nanoparticulate LiNcBuO (lithium octa-n-butoxy-substituted naphthalocyanine) probes with fast-scan EPR imaging at L-band, achieving spatial resolutions of up to 250 micrometers in 1 minute. Fast-scan EPR imaging can greatly facilitate the efficient and precise mapping of the spatial distribution of free radical and other paramagnetic probes in living systems.

Highly sensitive electron paramagnetic resonance nanoradicals for quantitative intracellular tumor oxymetric images.

Purpose: Tumor oxygenation is a critical parameter influencing the efficacy of cancer therapy. Low levels of oxygen in solid tumor have been recognized as an indicator of malignant progression and metastasis, as well as poor response to chemo- and radiation therapy. Being able to measure oxygenation for an individual’s tumor would provide doctors with a valuable way of identifying optimal treatments for patients.Methods: Electron paramagnetic resonance imaging (EPRI) in combination with an oxygen-measuring paramagnetic probe was performed to measure tumor oxygenation in vivo. Triarylmethyl (trityl) radical exhibits high specificity, sensitivity, and resolution for quantitative measurement of O2 concentration. However, its in vivo applications in previous studies have been limited by the required high dosage, its short half-life, and poor intracellular permeability. To address these limitations, we developed high-capacity nanoformulated radicals that employed fluorescein isothiocyanate-labeled mesoporous silica nanoparticles (FMSNs) as trityl radical carriers. The high surface area nanostructure and easy surface modification of physiochemical properties of FMSNs enable efficient targeted delivery of highly concentrated, nonself-quenched trityl radicals, protected from environmental degradation and dilution.Results: We successfully designed and synthesized a tumor-targeted nanoplatform as a carrier for trityl. In addition, the nanoformulated trityl does not affect oxygen-sensing capacity by a self-relaxation or broadening effect. The FMSN-trityl exhibited high sensitivity/response to oxygen in the partial oxygen pressure range from 0 to 155 mmHg. Furthermore, MSN-trityl displayed outstanding intracellular oxygen mapping in both in vitro and in vivo animal studies.Conclusion: The highly sensitive nanoformulated trityl spin probe can profile intracellular oxygen distributions of tumor in a real-time and quantitative manner using in vivo EPRI.

Molecular Probes for Evaluation of Oxidative Stress by In Vivo EPR Spectroscopy and Imaging: State-of-the-Art and Limitations

Oxidative stress, defined as a misbalance between the production of reactive oxygen species and the antioxidant defenses of the cell, appears as a critical factor either in the onset or in the etiology of many pathological conditions. Several methods of detection exist. However, they usually rely on ex vivo evaluation or reports on the status of living tissues only up to a few millimeters in depth, while a whole-body, real-time, non-invasive monitoring technique is required for early diagnosis or as an aid to therapy (to monitor the action of a drug). Methods based on electron paramagnetic resonance (EPR), in association with molecular probes based on aminoxyl radicals (nitroxides) or hydroxylamines especially, have emerged as very promising to meet these standards. The principles involve monitoring the rate of decrease or increase of the EPR signal in vivo after injection of the nitroxide or the hydroxylamine probe, respectively, in a pathological versus a control situation. There have been many successful applications in various rodent models. However, current limitations lie in both the field of the technical development of the spectrometers and the molecular probes. The scope of this review will mainly focus on the latter.

Brain Redox Imaging Using In Vivo Electron Paramagnetic Resonance Imaging and Nitroxide Imaging Probes

Reactive oxygen species (ROS) are produced by living organisms as a result of normal cellular metabolism. Under normal physiological conditions, oxidative damage is prevented by the regulation of ROS by the antioxidant network. However, increased ROS and decreased antioxidant defense may contribute to many brain disorders, such as stroke, Parkinson’s disease, and Alzheimer’s disease. Noninvasive assessment of brain redox status is necessary for monitoring the disease state and the oxidative damage. Continuous-wave electron paramagnetic resonance (CW-EPR) imaging using redox-sensitive imaging probes, such as nitroxides, is a powerful method for visualizing the redox status modulated by oxidative stress in vivo. For conventional CW-EPR imaging, however, poor signal-to-noise ratio, low acquisition efficiency, and lack of anatomic visualization limit its ability to achieve three-dimensional redox mapping of small rodent brains. In this review, we discuss the instrumentation and coregistration of EPR images to anatomical images and appropriate nitroxide imaging probes, all of which are needed for a sophisticated in vivo EPR imager for all rodents. Using new EPR imaging systems, site-specific distribution and kinetics of nitroxide imaging probes in rodent brains can be obtained more accurately, compared to previous EPR imaging systems. We also describe the redox imaging studies of animal models of brain disease using newly developed EPR imaging.

Data processing of 3D and 4D in-vivo electron paramagnetic resonance imaging co-registered with ultrasound. 3D printing as a registration tool

We present the concept of image registration using ultrasound (US) and electron paramagnetic resonance (EPR) imaging and discuss the benefits of this solution, as well as its limitations.Both phantoms and murine tumors were used to test US and EPR image co-registration. Comparison of dental molding cast immobilization and predesigned cradle revealed that the latter approach is more effective in stabilizing the fiducial position. In vivo imaging of mouse tumors, image registration and comparison of fiducials system for 3D spatial as well as 4D spatial-spectral EPR imaging supported by 3D US were demonstrated.Ultrasound may provide a convenient alternative to other anatomical imaging methods for image registration in preclinical research. Of particular interest is a fusion of US tissue structure, doppler vascular function and EPR oxygen or redox imaging.

In vivo melanoma imaging based on dynamic nuclear polarization enhancement in melanin pigment of living mice using in vivo dynamic nuclear polarization magnetic resonance imaging

Melanin is a pigment that includes free radicals and is widely distributed in living animals. Malignant melanoma is one of the most progressive tumors in humans with increasing incidence worldwide, and has shown resistance to chemotherapy, resulting in high mortality at the metastatic stage. In general, melanoma involves the abnormal accumulation of melanin pigment produced by malignant melanocytes. Electron paramagnetic resonance (EPR) spectroscopy and imaging is a powerful technique to directly visualize melanomas using endogenous free radicals in the melanin pigment. Because melanin radicals have a large linewidth, the low spatial resolution of EPR imaging results in blurred images and a lack of anatomical information. Dynamic nuclear polarization (DNP)-MRI is a noninvasive imaging method to obtain the spatio-temporal information of free radicals with MRI anatomical resolution. Proton signals in tissues, including free radicals, can be dramatically enhanced by EPR irradiation at the resonance frequency of the free radical prior to applying the MRI pulse sequence. However, the DNP effects of free radicals in the pigment of living organisms is unclear. Therefore, if endogenous free radicals in melanin pigment could be utilized as a bio-probe for DNP-MRI, this will be an advantage for the specific enhancement of melanoma tissues and might allow the separate noninvasive visualization of melanoma tissues without the need for probe administration. Here, we report that biological melanin pigment induced a in vivo DNP effect by interacting with water molecules. In addition, we demonstrated in vivo melanoma imaging based on the DNP effects of endogenous free radicals in the melanin pigment of living mice.

Optimization-Based Image Reconstruction From Fast-Scanned, Noisy Projections in EPR Imaging

Tumor oxygen concentration image is essential to oxygen-image guided, precise radiation therapy. Electron paramagnetic resonance imaging is an advanced oxygen imaging technique. However, the scanning time is still comparatively long, leading to motion artifacts for static imaging and low time resolution for dynamic imaging. Usually, a projection signal at a specific angle is obtained by averaging thousands of repeatedly collected projections to suppress random noise and achieve a high signal-to-noise ratio (SNR). Reducing the repetition times of projection collecting at a specific angle may effectively speed up the whole scanning process. However, the EPR images reconstructed by the conventional three-dimensional filtered backprojection (FBP) algorithm from these fast-scanned, low SNR projections are too noisy to be used for further image postprocessing. In the paper, we investigate the capability of an optimization-based algorithm in accurate reconstruction from noisy projections. We designed a total variation constrained, data divergence minimization model, derived its Chambolle-Pock (CP) solving algorithm, and then validated and evaluated the CP algorithm via mathematical and physical phantoms. The studies show that the CP algorithm may accurately reconstruct EPR images from fast-scanned, noisy projections, and thus the whole scanning process may be speeded up four times compared with the full scan time demanded by the FBP algorithm in the image reconstruction of the complex physical phantom.

Counterion influence on dynamic spin properties in a V(iv) complex.

Using transition metal ions for spin-based applications, such as electron paramagnetic resonance imaging (EPRI) or quantum computation, requires a clear understanding of how local chemistry influences spin properties. Herein we report a series of four ionic complexes to provide the first systematic study of one aspect of local chemistry on the V(iv) spin - the counterion. To do so, the four complexes (Et3NH)2[V(C6H4O2)3] (1), (n-Bu3NH)2[V(C6H4O2)3] (2), (n-Hex3NH)2[V(C6H4O2)3] (3), and (n-Oct3NH)2[V(C6H4O2)3] (4) were probed by EPR spectroscopy in solid state and solution. Room temperature, solution X-band (ca. 9.8 GHz) continuous-wave electron paramagnetic resonance (CW-EPR) spectroscopy revealed an increasing linewidth with larger cations, likely a counterion-controlled tumbling in solution via ion pairing. In the solid state, variable-temperature (5-180 K) X-band (ca. 9.4 GHz) pulsed EPR studies of 1-4 in o-terphenyl glass demonstrated no effect on spin-lattice relaxation times (T 1), indicating little role for the counterion on this parameter. However, the phase memory time (T m) of 1 below 100 K is markedly smaller than those of 2-4. This result is counterintuitive, as 2-4 are relatively richer in 1H nuclear spin, hence, expected to have shorter T m. Thus, these data suggest an important role for counterion methyl groups on T m, and moreover provide the first instance of a lengthening T m with increasing nuclear spin quantity on a molecule.

Membrane-specific spin trap, 5-dodecylcarbamoyl-5-N-dodecylacetamide-1-pyroline-N-oxide (diC12PO): theoretical, bioorthogonal fluorescence imaging and EPR studies.

Membranous organelles are major endogenous sources of reactive oxygen and nitrogen species. When present at high levels, these species can cause macromolecular damage and disease. To better detect and scavenge free radical forms of the reactive species at their sources, we investigated whether nitrone spin traps could be selectively targeted to intracellular membranes using a bioorthogonal imaging approach. Electron paramagnetic resonance imaging demonstrated that the novel cyclic nitrone 5-dodecylcarbamoyl-5-N-dodecylacetamide-1-pyroline-N-oxide (diC12PO) could be used to target the nitrone moiety to liposomes composed of phosphatidyl choline. To test localization with authentic membranes in living cells, fluorophores were introduced via strain-promoted alkyne-nitrone cycloaddition (SPANC). Two fluorophore-conjugated alkynes were investigated: hexynamide-fluoresceine (HYA-FL) and dibenzylcyclooctyne-PEG4-5/6-sulforhodamine B (DBCO-Rhod). Computational and mass spectrometry experiments confirmed the cycloadduct formation of DBCO-Rhod (but not HYA-FL) with diC12PO in cell-free solution. Confocal microscopy of bovine aortic endothelial cells treated sequentially with diC12PO and DBCO-Rhod demonstrated clear localization of fluorescence with intracellular membranes. These results indicate that targeting of nitrone spin traps to cellular membranes is feasible, and that a bioorthogonal approach can aid the interrogation of their intracellular compartmentalization properties.

Towards reduction of SAR in scaling up in vivo pulsed EPR imaging to larger objects.

An excessive RF power requirement is one of the main obstacles in the clinical translation of EPR imaging. The radio frequency (RF) pulses used in EPR imaging to excite electron spins must be very short to match their fast relaxation. With traditional pulse schemes and ninety degree flip angles, this can lead to either unsafe specific absorption rate (SAR) levels or unfeasibly long repetition times. In spectroscopy experiments, it has been shown that stochastic excitation and correlation detection can reduce the power while maintaining sensitivity but have yet to be applied to imaging experiments. Stochastic excitation is implemented using a pseudo-random phase modulation of the input stimulus. Using a crossed coil resonator assembly comprised of an outer saddle coil and an inner surface coil, it was possible to obtain a minimum isolation of ~50 dB across a 12 MHz bandwidth. An incident peak RF power of 5 mW was used to excite the system. The low background signal obtained from this resonator allowed us to generate images with 32 dB (>1000:1) signal-to-noise ratio (SNR) while exciting with a traditional pulse sequence in a phantom containing the solid paramagnetic probe NMP-TCNQ (N-methyl pyridinium tetracyanoquinodimethane). Using two different stochastic excitation schemes, we were able to achieve a greater than 4-fold increase in SNR at the same peak power and number of averages, compared to single pulse excitation. This procedure allowed imaging at significantly lower RF power levels than used in conventional EPR imaging system configurations. Similar techniques may enable clinical applications for EPR imaging by facilitating the use of larger RF coils while maintaining a safe SAR level.

A Multimodal Molecular Imaging Study Evaluates Pharmacological Alteration of the Tumor Microenvironment to Improve Radiation Response.

: Hypoxic zones in solid tumors contribute to radioresistance, and pharmacologic agents that increase tumor oxygenation prior to radiation, including antiangiogenic drugs, can enhance treatment response to radiotherapy. Although such strategies have been applied, imaging assessments of tumor oxygenation to identify an optimum time window for radiotherapy have not been fully explored. In this study, we investigated the effects of α-sulfoquinovosylacyl-1,3-propanediol (SQAP or CG-0321; a synthetic derivative of an antiangiogenic agent) on the tumor microenvironment in terms of oxygen partial pressure (pO2), oxyhemoglobin saturation (sO2), blood perfusion, and microvessel density using electron paramagnetic resonance imaging, photoacoustic imaging, dynamic contrast-enhanced MRI with Gd-DTPA injection, and T2*-weighted imaging with ultrasmall superparamagnetic iron oxide (USPIO) contrast. SCCVII and A549 tumors were grown by injecting tumor cells into the hind legs of mice. Five days of daily radiation (2 Gy) combined with intravenous injection of SQAP (2 mg/kg) 30 minutes prior to irradiation significantly delayed growth of tumor xenografts. Three days of daily treatment improved tumor oxygenation and decreased tumor microvascular density on T2*-weighted images with USPIO, suggesting vascular normalization. Acute effects of SQAP on tumor oxygenation were examined by pO2, sO2, and Gd-DTPA contrast-enhanced imaging. SQAP treatment improved perfusion and tumor pO2 (ΔpO2: 3.1 ± 1.0 mmHg) and was accompanied by decreased sO2 (20%-30% decrease) in SCCVII implants 20-30 minutes after SQAP administration. These results provide evidence that SQAP transiently enhanced tumor oxygenation by facilitating oxygen dissociation from oxyhemoglobin and improving tumor perfusion. Therefore, SQAP-mediated sensitization to radiation in vivo can be attributed to increased tumor oxygenation. SIGNIFICANCE: A multimodal molecular imaging study evaluates pharmacological alteration of the tumor microenvironment to improve radiation response.