Assessment of blood-brain barrier leakage and brain oxygenation in Connexin-32 knockout mice with systemic neuroinflammation using pulse electron paramagnetic resonance imaging techniques.

PURPOSE: The determination of blood-brain barrier (BBB) integrity and partial pressure of oxygen (pO2) in the brain is of substantial interest in several neurological applications. This study aimed to assess the feasibility of using trityl OX071-based pulse electron paramagnetic resonance imaging (pEPRI) to provide a quantitative estimate of BBB integrity and pO2 maps in mouse brains as a function of neuroinflammatory disease progression. METHODS: Five Connexin-32 (Cx32)-knockout (KO) mice were injected with lipopolysaccharide to induce neuroinflammation for imaging. Three wild-type mice were also used to optimize the imaging procedure and as control animals. An additional seven Cx32-KO mice were used to establish the BBB leakage of trityl using the colorimetric assay. All pEPRI experiments were performed using a preclinical instrument, JIVA-25 (25 mT/720 MHz), at times t = 0, 4, and 6 h following lipopolysaccharide injection. Two pEPRI imaging techniques were used: (a) single-point imaging for obtaining spatial maps to outline the brain and calculate BBB leakage using the signal amplitude, and (b) inversion-recovery electron spin echo for obtaining pO2 maps. RESULTS: A statistically significant change in BBB leakage was found using pEPRI with the progression of inflammation in Cx32 KO animals. However, the change in pO2 values with the progression of inflammation for these animals was not statistically significant. CONCLUSIONS: For the first time, we show the ability of pEPRI to provide pO2 maps in mouse brains noninvasively, along with a quantitative assessment of BBB leakage. We expect this study to open new queries from the field to explore the pathology of many neurological diseases and provide a path to new treatments.

The optimal 18F-fluoromisonidazole PET threshold to define tumor hypoxia in preclinical squamous cell carcinomas using pO2 electron paramagnetic resonance imaging as reference truth.

PURPOSE: To identify the optimal threshold in 18F-fluoromisonidazole (FMISO) PET images to accurately locate tumor hypoxia by using electron paramagnetic resonance imaging (pO2 EPRI) as ground truth for hypoxia, defined by pO2 [Formula: see text] 10 mmHg. METHODS: Tumor hypoxia images in mouse models of SCCVII squamous cell carcinoma (n = 16) were acquired in a hybrid PET/EPRI imaging system 2 h post-injection of FMISO. T2-weighted MRI was used to delineate tumor and muscle tissue. Dynamic contrast enhanced (DCE) MRI parametric images of Ktrans and ve were generated to model tumor vascular properties. Images from PET/EPR/MRI were co-registered and resampled to isotropic 0.5 mm voxel resolution for analysis. PET images were converted to standardized uptake value (SUV) and tumor-to-muscle ratio (TMR) units. FMISO uptake thresholds were evaluated using receiver operating characteristic (ROC) curve analysis to find the optimal FMISO threshold and unit with maximum overall hypoxia similarity (OHS) with pO2 EPRI, where OHS = 1 shows perfect overlap and OHS = 0 shows no overlap. The means of dice similarity coefficient, normalized Hausdorff distance, and accuracy were used to define the OHS. Monotonic relationships between EPRI/PET/DCE-MRI were evaluated with the Spearman correlation coefficient ([Formula: see text]) to quantify association of vasculature on hypoxia imaged with both FMISO PET and pO2 EPRI. RESULTS: FMISO PET thresholds to define hypoxia with maximum OHS (both OHS = 0.728 [Formula: see text] 0.2) were SUV [Formula: see text] 1.4 [Formula: see text] SUVmean and SUV [Formula: see text] 0.6 [Formula: see text] SUVmax. Weak-to-moderate correlations (|[Formula: see text]|< 0.70) were observed between PET/EPRI hypoxia images with vascular permeability (Ktrans) or fractional extracellular-extravascular space (ve) from DCE-MRI. CONCLUSION: This is the first in vivo comparison of FMISO uptake with pO2 EPRI to identify the optimal FMISO threshold to define tumor hypoxia, which may successfully direct hypoxic tumor boosts in patients, thereby enhancing tumor control.

Going Low in a World Going High: The Physiologic Use of Lower Frequency Electron Paramagnetic Resonance.

Yakov Sergeevich Lebedev was a pioneer in high frequency EPR, taking advantage of the separation of g-factor anisotropy effects from nuclear hyperfine splitting and the higher frequency molecular motion sensitivity from higher frequency measurements8. This article celebrates a second EPR subfield in which Prof. Lebedev pioneered, EPR imaging. 9 We celebrate the clinical enhancements that are suggested in this low frequency work and imaging application to animal physiology at lower-than-standard EPR frequencies.

Investigation of the preconditioner-parameter in the preconditioned Chambolle-Pock algorithm applied to optimization-based image reconstruction.

The optimization-based image reconstruction methods have been thoroughly investigated in the field of medical imaging. The Chambolle-Pock (CP) algorithm may be employed to solve these convex optimization image reconstruction programs. The preconditioned CP (PCP) algorithm has been shown to have much higher convergence rate than the ordinary CP (OCP) algorithm. This algorithm utilizes a preconditioner-parameter to tune the implementation of the algorithm to the specific application, which ranges from 0 and 2, but is often set to 1. In this work, we investigated the impact of the preconditioner-parameter on the convergence rate of the PCP algorithm when it is applied to the TV constrained, data-divergence minimization (TVDM) optimization based image reconstruction. We performed the investigations in the context of 2D computed tomography (CT) and 3D electron paramagnetic resonance imaging (EPRI). For 2D CT, we used the Shepp-Logan and two FORBILD phantoms. For 3D EPRI, we used a simulated 6-spheres phantom and a physical phantom. Study results showed that the optimal preconditioner-parameter depends on the specific imaging conditions. Simply setting the parameter equal to 1 cannot guarantee a fast convergence rate. Thus, this study suggests that one should adaptively tune the preconditioner-parameter to obtain the optimal convergence rate of the PCP algorithm.

Three novel accurate pixel-driven projection methods for 2D CT and 3D EPR imaging.

OBJECTIVES: This work aims to explore more accurate pixel-driven projection methods for iterative image reconstructions in order to reduce high-frequency artifacts in the generated projection image. METHODS: Three new pixel-driven projection methods namely, small-pixel-large-detector (SPLD), linear interpolation based (LIB) and distance anterpolation based (DAB), were proposed and applied to reconstruct images. The performance of these methods was evaluated in both two-dimensional (2D) computed tomography (CT) images via the modified FORBILD phantom and three-dimensional (3D) electron paramagnetic resonance (EPR) images via the 6-spheres phantom. Specifically, two evaluations based on projection generation and image reconstruction were performed. For projection generation, evaluation was using a 2D disc phantom, the modified FORBILD phantom and the 6-spheres phantom. For image reconstruction, evaluations were performed using the FORBILD and 6-spheres phantom. During evaluation, 2 quantitative indices of root-mean-square-error (RMSE) and contrast-to-noise-ratio (CNR) were used. RESULTS: Comparing to the use of ordinary pixel-driven projection method, RMSE of the SPLD based least-square algorithm was reduced from 0.0701 to 0.0384 and CNR was increased from 5.6 to 19.47 for 2D FORBILD phantom reconstruction. For 3D EPRI, RMSE of SPLD was also reduced from 0.0594 to 0.0498 and CNR was increased from 3.88 to 11.58. In addition, visual evaluation showed that images reconstructed in both 2D and 3D images suffered from high-frequency line-shape artifacts when using the ordinary pixel-driven projection method. However, using 3 new methods all suppressed the artifacts significantly and yielded more accurate reconstructions. CONCLUSIONS: Three proposed pixel-driven projection methods achieved more accurate iterative image reconstruction results. These new and more accurate methods can also be easily extended to other imaging modalities. Among them, SPLD method should be recommended to 3D and four dimensional (4D) EPR imaging.

Electron Paramagnetic Resonance pO2 Image Tumor Oxygen-Guided Radiation Therapy Optimization.

Modern standards for radiation treatment do not take into account tumor oxygenation for radiation treatment planning. Strong correlation between tumor oxygenation and radiation treatment success suggests that oxygen-guided radiation therapy (OGRT) may be a promising enhancement of cancer radiation treatment. We have developed an OGRT protocol for rodents. Electron paramagnetic resonance (EPR) imaging is used for recording oxygen maps with high spatial resolution and excellent accuracy better than 1 torr. Radiation is delivered with an animal intensity modulated radiation therapy (IMRT) XRAD225Cx micro-CT/ therapy system. The radiation plan is delivered in two steps. First, a uniform 15% tumor control dose (TCD15) is delivered to the whole tumor. In the second step, an additional booster dose amounting to the difference between TCD98 and TCD15 is delivered to radio-resistant, hypoxic tumor regions. Delivery of the booster dose is performed using a multiport conformal beam protocol. For radiation beam shaping we used individual radiation blocks 3D-printed from tungsten infused ABS polymer. Calculation of beam geometry and the production of blocks is performed next to the EPR imager, immediately after oxygen imaging. Preliminary results demonstrate the sub-millimeter precision of the radiation delivery and high dose accuracy. The efficacy of the radiation treatment is currently being tested on syngeneic FSa fibrosarcoma tumors grown in the legs of C3H mice.

Correlation Between Hypoxia Proteins and EPR-Detected Hypoxia in Tumors.

Rapid expansion of tumor cells that outpace existing vasculature essential for nutrient and oxygen support as well as waste removal, correlates with profound changes in the microenvironment including angiogenesis, vasodilation, glucose metabolism, and cell cycle perturbations. Since hypoxic cells are up to three times more radioresistant than normoxic cells, identification of hypoxic populations to predict radiotherapeutic outcome is important. The consequences of hypoxia and activated proteins contribute to radioresistant tumors and radiotherapeutic failure. Stereotactic MCa4 tumor tissue biopsies from mouse tumors that were guided by electron paramagnetic resonance (EPR) O2 imaging were examined for hypoxia-induced proteins. The oxygen broadening of narrow EPR spectral lines or, equivalently, the increase in relaxation rates of electron magnetization, report pO2 with 1-2 torr resolution in image voxels less than 1 mm3. The pO2 reporter molecule OX063d64 (trityl) was used to acquire the data described here. Trityl appears to be selectively retained in tumors with a half-life of ~30 min. We used an inversion recovery electron spin echo (IRESE) to measure the T1 rate of the trityl inside the tumor bearing leg. We estimate our uncertainty in pO2 measurement to be 1-3 torr per voxel. Three hypoxic cell biomarkers, hypoxic-induced factor 1-alpha (HIF-1α), vascular endothelial growth factor (VEGF), and carbonic anhydrase IX (CA9), were examined using the ELISA assay. Quantification of these proteins based on results from the ELISA immunoassay kits indicate a strong correlation between EPR pO2-identified hypoxic fractions (<10 torr) and HIF-1α, VEGF, and CA9. We clearly demonstrate that hypoxic regions in tumors generate substantial amounts of HIF- 1α, VEGF, and CA9 protein.

Triarylmethyl Radical OX063d24 Oximetry: Electron Spin Relaxation at 250 MHz and RF Frequency Dependence of Relaxation and Signal-to-Noise.

The triarylmethyl radical OX063d24 is currently used for pulsed electron paramagnetic resonance oximetry at 250 MHz. Both 1/T 1 and 1/T 2 increase with increasing oxygen concentration. The dependence of 1/T 1 on probe concentration is smaller than for 1/T 2. To inform the selection of the optimum frequency for in vivo oximetry 1/T 1, 1/T 2 and signal-to-noise were measured as a function of frequency between 400 and 1000 MHz on a variable-frequency spectrometer with an adjustable-frequency cross-loop resonator. 1/T 1 and 1/T 2 decrease with increasing frequency and signal-to-noise increases with increasing frequency, which are all favourable for imaging at higher frequencies. However, depth of penetration of the radio frequency (RF) into an animal decreases with increasing frequency. Assuming that the RF loss in the animal to be studied determines the resonator Q, our results indicate that the optimum frequency for in vivo imaging will be determined by the desired depth of penetration in the tissue.

A Pulse EPR 25 mT magnetometer with 10ppm resolution.

A magnetometer designed for permanent magnet manufacturing and operated around 25 mT with 10ppm absolute accuracy is described. The magnetometer uses pulse electron paramagnetic resonance (EPR) methodology. The use of a pulsed broadband acquisition allowed reliable measurements in the presence of the magnetic field gradient and in relatively inhomogeneous magnetic fields of un-shimmed magnets.

Resonators for In Vivo Imaging: Practical Experience.

Resonators for preclinical electron paramagnetic resonance imaging have been designed primarily for rodents and rabbits and have internal diameters between 16 and 51 mm. Lumped circuit resonators include loop-gap, Alderman-Grant, and saddle coil topologies and surface coils. Bimodal resonators are useful for isolating the detected signal from incident power and reducing dead time in pulse experiments. Resonators for continuous wave, rapid scan, and pulse experiments are described. Experience at the University of Chicago and University of Denver in design of resonators for in vivo imaging is summarized.

Triarylmethyl Radical: EPR Signal to Noise at Frequencies between 250 MHz and 1.5 GHz and Dependence of Relaxation on Radical and Salt Concentration and on Frequency.

In vivo oximetry by pulsed electron paramagnetic resonance is based on measurements of changes in electron spin relaxation rates of probe molecules, such as the triarylmethyl radicals. A series of experiments was performed at frequencies between 250 MHz and 1.5 GHz to assist in the selection of an optimum frequency for oximetry. Electron spin relaxation rates for the triarylmethyl radical OX063 as a function of radical concentration, salt concentration, and resonance frequency were measured by electron spin echo 2-pulse decay and 3-pulse inversion recovery in the frequency range of 250 MHz-1.5 GHz. At constant OX063 concentration, 1/T1 decreases with increasing frequency because the tumbling dependent processes that dominate relaxation at 250 MHz are less effective at higher frequency. 1/T2 also decreases with increasing frequency because 1/T1 is a significant contribution to 1/T2 for trityl radicals in fluid solution. 1/T2-1/T1, the incomplete motional averaging contribution to 1/T2, increases with increasing frequency. At constant frequency, relaxation rates increase with increasing radical concentration due to contributions from collisions that are more effective for 1/T2 than 1/T1. The collisional contribution to relaxation increases as the concentration of counter-ions in solution increases, which is attributed to interactions of cations with the negatively charged radicals that decrease repulsion between trityl radicals. The Signal-to-Noise ratio (S/N) of field-swept echo-detected spectra of OX063 were measured in the frequency range of 400 MHz-1 GHz. S/N values, normalized by √Q, increase as frequency increases. Adding salt to the radical solution decreased S/N because salt lowers the resonator Q. Changing the temperature from 19 to 37 °C caused little change in S/N at 700 MHz. Both slower relaxation rates and higher S/N at higher frequencies are advantageous for oximetry. The potential disadvantage of higher frequencies is the decreased depth of penetration into tissue.

Towards Human Oxygen Images with Electron Paramagnetic Resonance Imaging.

Electron paramagnetic resonance imaging (EPRI) has been used to noninvasively provide 3D images of absolute oxygen concentration (pO2) in small animals. These oxygen images are well resolved both spatially (~1 mm) and in pO2 (1-3 mmHg). EPRI preclinical images of pO2 have demonstrated extremely promising results for various applications investigating oxygen related physiologic and biologic processes as well as the dependence of various disease states on pO2, such as the role of hypoxia in cancer. Recent developments have been made that help to progress EPRI towards the eventual goal of human application. For example, a bimodal crossed-wire surface coil has been developed. Very preliminary tests demonstrated a 20 dB isolation between transmit and receive for this coil, with an anticipated additional 20 dB achievable. This could potentially be used to image local pO2 in human subjects with superficial tumors with EPRI. Local excitation and detection will reduce the specific absorption rate limitations on images and eliminate any possible power deposition concerns. Additionally, a large 9 mT EPRI magnet has been constructed which can fit and provide static main and gradient fields for imaging local anatomy in an entire human. One potential obstacle that must be overcome in order to use EPRI to image humans is the approved use of the requisite EPRI spin probe imaging agent (trityl). While nontoxic, EPRI trityl spin probes have been injected intravenously when imaging small animals, and require relatively high total body injection doses that would not be suitable for human imaging applications. Work has been done demonstrating the alternative use of intratumoral (IT) injections, which can reduce the amount of trityl required for imaging by a factor of 2000- relative to a whole body intravenous injection. The development of a large magnet that can accommodate human subjects, the design of a surface coil for imaging of superficial pO2, and the reduction of required spin probe using IT injections all are crucial steps towards the eventual use of EPRI to image pO2 in human subjects. In the future this can help investigate the oxygenation status of superficial tumors (e.g., breast tumors). The ability to image pO2 in humans has many other potential applications to diseases such as peripheral vascular disease, heart disease, and stroke.

Approaching Oxygen-Guided Intensity-Modulated Radiation Therapy.

The outcome of cancer radiation treatment is strongly correlated with tumor oxygenation. The aim of this study is to use oxygen tension distributions in tumors obtained using Electron Paramagnetic Resonance (EPR) imaging to devise better tumor radiation treatment. The proposed radiation plan is delivered in two steps. In the first step, a uniform 50% tumor control dose (TCD50) is delivered to the whole tumor. For the second step an additional dose boost is delivered to radioresistant, hypoxic tumor regions. FSa fibrosarcomas grown in the gastrocnemius of the legs of C3H mice were used. Oxygen tension images were obtained using a 250 MHz pulse imager and injectable partially deuterated trityl OX63 (OX71) spin probe. Radiation was delivered with a novel animal intensity modulated radiation therapy (IMRT) XRAD225Cx microCT/radiation therapy delivery system. In a simplified scheme for boost dose delivery, the boost area is approximated by a sphere, whose radius and position are determined using an EPR O2 image. The sphere that irradiates the largest fraction of hypoxic voxels in the tumor was chosen using an algorithm based on Receiver Operator Characteristic (ROC) analysis. We used the fraction of irradiated hypoxic volume as the true positive determinant and the fraction of irradiated normoxic volume as the false positive determinant in the terms of that analysis. The most efficient treatment is the one that demonstrates the shortest distance from the ROC curve to the upper left corner of the ROC plot. The boost dose corresponds to the difference between TCD90 and TCD50 values. For the control experiment an identical radiation dose to the normoxic tumor area is delivered.

Maximally spaced projection sequencing in electron paramagnetic resonance imaging.

Electron paramagnetic resonance imaging (EPRI) provides 3D images of absolute oxygen concentration (pO2) in vivo with excellent spatial and pO2 resolution. When investigating such physiologic parameters in living animals, the situation is inherently dynamic. Improvements in temporal resolution and experimental versatility are necessary to properly study such a system. Uniformly distributed projections result in efficient use of data for image reconstruction. This has dictated current methods such as equal-solid-angle (ESA) spacing of projections. However, acquisition sequencing must still be optimized to achieve uniformity throughout imaging. An object-independent method for uniform acquisition of projections, using the ESA uniform distribution for the final set of projections, is presented. Each successive projection maximizes the distance in the gradient space between itself and prior projections. This maximally spaced projection sequencing (MSPS) method improves image quality for intermediate images reconstructed from incomplete projection sets, enabling useful real-time reconstruction. This method also provides improved experimental versatility, reduced artifacts, and the ability to adjust temporal resolution post factum to best fit the data and its application. The MSPS method in EPRI provides the improvements necessary to more appropriately study a dynamic system.

Real-Time Image Reconstruction for Pulse EPR Oxygen Imaging Using a GPU and Lookup Table Parameter Fitting.

The importance of tissue oxygenation has led to a great interest in methods for imaging pO2 in vivo. Electron paramagnetic resonance imaging (EPRI) provides noninvasive, near absolute 1 mm-resolved 3D images of pO2 in the tissues and tumors of living animals. Current EPRI image reconstruction methods tend to be time consuming and preclude real-time visualization of information. Methods are presented to significantly accelerate the reconstruction process in order to enable real-time reconstruction of EPRI pO2 images. These methods are image reconstruction using graphics processing unit (GPU)-based 3D filtered back-projection and lookup table parameter fitting. The combination of these methods leads to acceleration factors of over 650 compared to current methods and allows for real-time reconstruction of EPRI images of pO2 in vivo.

Implementation of GPU-accelerated back projection for EPR imaging.

Electron paramagnetic resonance (EPR) Imaging (EPRI) is a robust method for measuring in vivo oxygen concentration (pO2). For 3D pulse EPRI, a commonly used reconstruction algorithm is the filtered backprojection (FBP) algorithm, in which the backprojection process is computationally intensive and may be time consuming when implemented on a CPU. A multistage implementation of the backprojection can be used for acceleration, however it is not flexible (requires equal linear angle projection distribution) and may still be time consuming. In this work, single-stage backprojection is implemented on a GPU (Graphics Processing Units) having 1152 cores to accelerate the process. The GPU implementation results in acceleration by over a factor of 200 overall and by over a factor of 3500 if only the computing time is considered. Some important experiences regarding the implementation of GPU-accelerated backprojection for EPRI are summarized. The resulting accelerated image reconstruction is useful for real-time image reconstruction monitoring and other time sensitive applications.

Principal component analysis enhances SNR for dynamic electron paramagnetic resonance oxygen imaging of cycling hypoxia in vivo.

PURPOSE: Low oxygen concentration (hypoxia) in tumors strongly affects their malignant state and resistance to therapy. These effects may be more deleterious in regions undergoing cycling hypoxia. Electron paramagnetic resonance imaging (EPRI) has provided a noninvasive, quantitative imaging modality to investigate static pO2 in vivo. However, to image changing hypoxia, EPRI images with better temporal resolution may be required. The tradeoff between temporal resolution and signal-to-noise ratio (SNR) results in lower SNR for EPRI images with imaging time short enough to resolve cycling hypoxia. METHODS: Principal component analysis allows for accelerated image acquisition with acceptable SNR by filtering noise in projection data, from which pO2 images are reconstructed. Principal component analysis is used as a denoising technique by including only low-order components to approximate the EPRI projection data. RESULTS: Simulated and experimental studies show that principal component analysis filtering increases SNR, particularly for small numbers of sub-volumes with changing pO2 , enabling an order of magnitude increase in temporal resolution with minimal deterioration in spatial resolution or image quality. CONCLUSION: The SNR necessary for dynamic EPRI studies with temporal resolution required to investigate cycling hypoxia and its physiological implications is enabled by principal component analysis filtering.

Absolute oxygen R1e imaging in vivo with pulse electron paramagnetic resonance.

PURPOSE: Tissue oxygen (O2) levels are among the most important and most quantifiable stimuli to which cells and tissues respond through inducible signaling pathways. Tumor O2 levels are major determinants of the response to cancer therapy. Developing more accurate measurements and images of tissue O2 partial pressure (pO2), assumes enormous practical, biological, and medical importance. METHODS: We present a fundamentally new technique to image pO2 in tumors and tissues with pulse electron paramagnetic resonance (EPR) imaging enabled by an injected, nontoxic, triaryl methyl (trityl) spin probe whose unpaired electron's slow relaxation rates report the tissue pO2. Heretofore, virtually all in vivo EPR O2 imaging measures pO2 with the transverse electron spin relaxation rate, R2e, which is susceptible to the self-relaxation confounding O2 sensitivity. RESULTS: We found that the trityl electron longitudinal relaxation rate, R1e, is an order of magnitude less sensitive to confounding self-relaxation. R1e imaging has greater accuracy and brings EPR O2 images to an absolute pO2 image, within uncertainties. CONCLUSION: R1e imaging more accurately determines oxygenation of cancer and normal tissue in animal models than has been available. It will enable enhanced, rapid, noninvasive O2 images for understanding oxygen biology and the relationship of oxygenation patterns to therapy outcome in living animal systems.

How in vivo EPR measures and images oxygen.

The partial pressure of oxygen (pO2) in tissues plays an important role in the pathophysiology of many diseases and influences outcome of cancer therapy, ischemic heart and cerebrovascular disease treatments and wound healing. Over the years a suite of EPR techniques for reliable oxygen measurements has been developed. This is a mini-review of pulse EPR in vivo oxygen imaging methods that utilize soluble spin probes. Recent developments in pulse EPR imaging technology have brought an order of magnitude increase in image acquisition speed, enhancement of sensitivity and considerable improvement in the precision and accuracy of oxygen measurements.

What we learn from in vivo EPR oxygen images.

Distributions of oxygen concentration (pO2) are a critical determinant of normal tissue health as well as tumor aggressiveness and response to therapy. A number of studies show the value of normal tissue and tumor tissue oxygenation images and some of these will be discussed here. A strong correlation between tumor hypoxic fraction as measured with electron paramagnetic resonance oxygen imaging and radiation treatment success or failure has been found in two separate cancer types. Oxygen images of the torso of wild type mice show initial reduction of lung, liver, visceral, and muscle pO2 with cyclic halving of fraction of inspired oxygen (FiO2), but variation is blunted over an hour. Spontaneous breast cancers in Mouse Mammary Tumor Viral (MMTV) promoted-polyoma middle T antigen (PyMT) mice with BNIP3, a major factor in promotion of mitochondrial autophagy, knocked out will be compared with wild type animals. Preliminary studies for the BNIP3 knock out animals show extremely low pO2. The wide variety of studies, in which oxygen images can play an integral role, serve to demonstrate the importance of oxygen images.