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.

Fabrication of oxygen-carrying microparticles functionalized with liver ECM-proteins to improve phenotypic three-dimensional in vitro liver assembly, function, and responses.

Oxygen and extracellular matrix (ECM)-derived biopolymers play vital roles in regulating many cellular functions in both the healthy and diseased liver. This study highlights the significance of synergistically tuning the internal microenvironment of three-dimensional (3D) cell aggregates composed of hepatocyte-like cells from the HepG2 human hepatocellular carcinoma cell line and hepatic stellate cells (HSCs) from the LX-2 cell line to enhance oxygen availability and phenotypic ECM ligand presentation for promoting the native metabolic functions of the human liver. First, fluorinated (PFC) chitosan microparticles (MPs) were generated with a microfluidic chip, then their oxygen transport properties were studied using a custom ruthenium-based oxygen sensing approach. Next, to allow for integrin engagements the surfaces of these MPs were functionalized using liver ECM proteins including fibronectin, laminin-111, laminin-511, and laminin-521, then they were used to assemble composite spheriods along with HepG2 cells and HSCs. After in vitro culture, liver-specific functions and cell adhesion patterns were compared between groups and cells showed enhanced liver phenotypic responses to laminin-511 and 521 as evidenced via enhanced E-cadherin and vinculin expression, as well as albumin and urea secretion. Furthermore, hepatocytes and HSCs exhibited more pronounced phenotypic arrangements when cocultured with laminin-511 and 521 modified MPs providing clear evidence that specific ECM proteins have distinctive roles in the phenotypic regulation of liver cells in engineering 3D spheroids. This study advances efforts to create more physiologically relevant organ models allowing for well-defined conditions and phenotypic cell signaling which together improve the relevance of 3D spheroid and organoid models.

Oxygen Imaging for Non-Invasive Metastasis Detection.

Sentinel lymph node (SLN) biopsy is an integral part of treatment planning for a variety of cancers as it evaluates whether a tumor has metastasized, an event that significantly reduces survival probability. However, this invasive procedure is associated with patient morbidity, and misses small metastatic deposits, resulting in the removal of additional nodes for tumors with high metastatic probability despite a negative SLN biopsy. To prevent this over-treatment and its associated morbidities for patients that were truly negative, we propose a tissue oxygen imaging method called Photoacoustic Lifetime Imaging (PALI) as an alternative or supplementary tool for SLN biopsy. As the hyper-metabolic state of cancer cells significantly depresses tissue oxygenation compared to normal tissue even for small metastatic deposits, we hypothesize that PALI can sensitively and specifically detect metastases. Before this hypothesis is tested, however, PALI's maximum imaging depth must be evaluated to determine the cancer types for which it is best suited. To evaluate imaging depth, we developed and simulated a phantom composed of tubing in a tissue-mimicking, optically scattering liquid. Our simulation and experimental results both show that PALI's maximum imaging depth is 16 mm. As most lymph nodes are deeper than 16 mm, ways to improve imaging depth, such as directly delivering light to the node using penetrating optical fibers, must be explored.

Dendrimer porphyrins as the oxygen sensor for intracellular imaging to suppress interaction towards biological molecules.

Optical methods using phosphorescence quenching by oxygen are suitable for the measurement of oxygen concentration within cells. In cells, however, the dyes such as Pt-porphyrins interact with biological components so that their optical properties are changed. Therefore, the absolute oxygen concentration determination in cells is difficult. To suppress this interaction, we focussed on porphyrin-cored dendrimers (dendrimer-porphyrins) and synthesized 2nd-4th generation dendrimer-porphyrins with various surface functional groups (G2-G4, ARG, αGLU and γGLU). These dendrimer-porphyrins showed oxygen sensing property and the change of their spectroscopic properties by biomolecules was supressed. Additionally, the dendrimer-porphyrins were accumulated in cells even in the presence of serum, so oxygen concentration imaging without the effect of serum starvation was also achieved.

Quantitative analysis of mucosal oxygenation using ex vivo imaging of healthy and inflamed mammalian colon tissue.

Colonic inflammation is associated with decreased tissue oxygenation, significantly affecting gut homeostasis. However, the crosstalk between O2 consumption and supply in the inflamed tissue are not fully understood. Using a murine model of colitis, we analysed O2 in freshly prepared samples of healthy and inflamed colon tissue. We developed protocols for efficient ex vivo staining of mouse distal colon mucosa with a cell-penetrating O2 sensitive probe Pt-Glc and high-resolution imaging of O2 concentration in live tissue by confocal phosphorescence lifetime-imaging microscopy (PLIM). Microscopy analysis revealed that Pt-Glc stained mostly the top 50-60 µm layer of the mucosa, with high phosphorescence intensity in epithelial cells. Measured O2 values in normal mouse tissue ranged between 5 and 35 µM (4-28 Torr), tending to decrease in the deeper tissue areas. Four-day treatment with dextran sulphate sodium (DSS) triggered colon inflammation, as evidenced by an increase in local IL6 and mKC mRNA levels, but did not affect the gross architecture of colonic epithelium. We further observed an increase in oxygenation, partial activation of hypoxia inducible factor (HIF) 1 signalling, and negative trends in pyruvate dehydrogenase activity and O2 consumption rate in the colitis mucosa, suggesting a decrease in mitochondrial respiration, which is known to be regulated via HIF-1 signalling and pyruvate oxidation rate. These results along with efficient staining with Pt-Glc of rat and human colonic mucosa reveal high potential of PLIM platform as a powerful tool for the high-resolution analysis of the intestinal tissue oxygenation in patients with inflammatory bowel disease and other pathologies, affecting tissue respiration.

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.

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.

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.

Imaging of oxygen gradients in giant umbrella cells: an ex vivo PLIM study.

O2 plays a pivotal role in aerobic metabolism and regulation of cell and tissue function. Local differences and fluctuations in tissue O2 levels are well documented; however, the physiological significance of O2 microgradients, particularly at the subcellular level, remains poorly understood. Using the cell-penetrating phosphorescent O2 probe Pt-Glc and confocal fluorescence microscopy, we visualized O2 distribution in individual giant (>100-µm) umbrella cells located superficially in the urinary bladder epithelium. We optimized conditions for in vivo phosphorescent staining of the inner surface of the mouse bladder and subsequent ex vivo analysis of excised live tissue. Imaging experiments revealed significant (≤85 µM) and heterogeneous deoxygenation within respiring umbrella cells, with radial O2 gradients of up to 40 µM across the cell, or ∼0.6 µM/µm. Deeply deoxygenated (5-15 µM O2) regions were seen to correspond to the areas enriched with polarized mitochondria. Pharmacological activation of mitochondrial respiration decreased oxygenation and O2 gradients in umbrella cells, while inhibition with antimycin A dissipated the gradients and caused gradual reoxygenation of the tissue to ambient levels. Detailed three-dimensional maps of O2 distribution potentially can be used for the modeling of intracellular O2-dependent enzymatic reactions and downstream processes, such as hypoxia-inducible factor signaling. Further ex vivo and in vivo studies on intracellular and tissue O2 gradients using confocal imaging can shed light on the molecular mechanisms regulating O2-dependent (patho)physiological processes in the bladder and other tissues.

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.

Validation of the design of a high-sensitivity and high-resolution PET system for a preclinical PET/EPR hybrid scanner.

We report the development of a high-sensitivity and high-resolution PET subsystem for a next-generation preclinical PET/EPR hybrid scanner for investigating and improving hypoxia imaging with PET. The PET subsystem consists of 14 detector modules (DM) installed within a cylindrical supporting frame whose outer and inner diameters are 115mm and 60mm, respectively. Each DM contains eight detector units (DU) in a row and each DU is made of a 12×12 array of 1×1×10mm3 LYSO crystals (with a 1.05mm pitch) coupled to a 4×4 silicon photomultiplier (SiPM) array that has a 3.2mm pitch (Hamamatsu multi-pixel photon counter (MPPC) array 14161-3050HS-04). The PET subsystem has a 104mm axial field-of-view (AFOV) that is sufficient for full-body mouse imaging, therefore enabling temporal and spatial correlation studies of tumor hypoxia between PET and EPR. It employs 1mm-width crystals to support sub-millimeter image resolution that is desired for mouse imaging. Al-though a DM contains 1,152 LYSO crystals, by use of a newly devised signal readout method only six outputs are produced. Recently a partial prototype of this subsystem consisting of four DMs is built. In this paper, we present performance measurement results obtained for the developed DMs and initial imaging results obtained by the prototype. The developed DMs show uniformly superior performance in identifying the hit crystal and detector unit, in energy resolution, and in coincidence time resolution. The images obtained for a 22Na point source and a 18F-filled U-shaped tube source show an image resolution of about 1.1mm and 1.2mm FWHM in the transverse and axial directions respectively, and demonstrate successful imaging over the entire 104mm AFOV of the prototype. This estimated image resolution however includes the contribution by the source size.

Atomically Resolved Phase Coexistence in VO2 Thin Films.

Concurrent structural and electronic transformations in VO2 thin films are of 2-fold importance: enabling fine-tuning of the emergent electrical properties in functional devices, yet creating an intricate interfacial domain structure of transitional phases. Despite the importance of understanding the structure of VO2 thin films, a detailed real-space atomic structure analysis in which the oxygen atomic columns are also resolved is lacking. Moreover, intermediate atomic structures have remained elusive due to the lack of robust atomically resolved quantitative analysis. Here, we directly resolve both V and O atomic columns and discover the presence of intermediate monoclinic (M2) phase nanolayers (less than 2 nm thick) in epitaxially grown VO2 films on a TiO2 (001) substrate, where the dominant part of VO2 undergoes a transition from the tetragonal (rutile) phase to the monoclinic M1 phase. Strain analysis suggests that the presence of the M2 phase is related to local strain gradients near the TiO2/VO2 interface. We unfold the crucial role of imaging the spatial configurations of the oxygen anions (in addition to V cations) by utilizing atomic-resolution electron microscopy. Our approach can be used to unravel the structural transitions in a wide range of correlated oxides, offering substantial implications for, e.g., optoelectronics and ferroelectrics.

Metabolic Imaging of Acute Ischemic Stroke (PET, 1Hydrogen Spectroscopy, 17Oxygen Imaging, 23Sodium MRI, pH Imaging).

Acute stroke imaging plays a vital and time-sensitive role in therapeutic decision-making. Current clinical workflows widely use computed tomography (CT) and magnetic resonance (MR) techniques including CT and MR perfusion to estimate the volume of ischemic penumbra at risk for infarction without acute intervention. The use of imaging techniques aimed toward evaluating the metabolic derangements underlying a developing infarct may provide additional information for differentiating the penumbra from benign oligemia and infarct core. The authors review several modalities of metabolic imaging including PET, hydrogen and oxygen spectroscopy, sodium MRI, and pH-weighted MRI.

Oxygen Imaging of a Rabbit Tumor Using a Human-Sized Pulse Electron Paramagnetic Resonance Imager.

PURPOSE: Spatial heterogeneity in tumor hypoxia is one of the most important factors regulating tumor growth, development, aggressiveness, metastasis, and affecting treatment outcome. Most solid tumors are known to have hypoxia or low oxygen levels (pO2 ≤10 torr). Electron paramagnetic resonance oxygen imaging (EPROI) is an emerging oxygen mapping technology. EPROI utilizes the linear relationship between the relaxation rates of the injectable OX071 trityl spin probe and the partial oxygen pressure (pO2). However, most of the EPROI studies have been limited to mouse models of solid tumors because of the instrument-size limitations. The purpose of this work was to develop a human-sized 9-mT (250 MHz resonance frequency, 60 cm bore size) pulse EPROI instrument and evaluate its performance with rabbit VX-2 tumor oxygen imaging. METHODS: A New Zealand white rabbit with a 3.2-cm VX-2 tumor in the calf muscle was imaged using the human-sized EPROI instrument and a 2.25-in. ID volume coil. The animal received a ~8-min intravenous injection of OX071 (5.2 mL total volume at 72 mM concentration) and, after 75 min, an intratumoral injection (120 µL total at 5 mM OX071 concentration) and underwent EPROI. At the end of the experiments, MRI was performed using a preclinical 9.4-T MRI system to outline the tumor boundaries. RESULTS: For the first time, a human-sized pulse EPROI instrument with a 60-cm bore size/250-MHz frequency was built and evaluated using rabbit tumor oxygen imaging. For the first time, the systemic IV injection of the oxygen-sensitive trityl OX071 spin probe was used for an animal of this size. The resulting EPROI image from the IV injection showed complete tumor coverage. The image obtained after intratumoral injection showed localized coverage in the upper lobe of the tumor, demonstrating the need for improved intratumoral injection protocol. CONCLUSIONS: This study demonstrates the performance of the world's first human-sized pulse EPROI instrument. It also demonstrates that the EPROI of larger animals can be performed using the systemic injection of a manageable amount of the spin probe. This brings EPROI one step closer to clinical applications in cancer therapies. Oxygen imaging is a platform technology, and the instrument and techniques developed here will also be useful for other clinical applications.

Personalized Oncology by In Vivo Chemical Imaging: Photoacoustic Mapping of Tumor Oxygen Predicts Radiotherapy Efficacy.

We hereby apply the approach of photoacoustic chemical imaging, performing an in vivo chemical analysis that is spatially resolved (200 µm) and in real time, to predict a given tumor's response to therapy. Using triple negative breast cancer as a model, we took photoacoustic images of tumors' oxygen distributions in patient-derived xenografts (PDXs) in mice using biocompatible, oxygen-sensitive tumor-targeted chemical contrast nanoelements (nanosonophores), which function as contrast agents for photoacoustic imaging. Following radiation therapy, we established a quantitatively significant correlation between the spatial distribution of the initial oxygen levels in the tumor and its spatial distribution of the therapy's efficacy: the lower the local oxygen, the lower the local radiation therapy efficacy. We thus provide a simple, noninvasive, and inexpensive method to both predict the efficacy of radiation therapy for a given tumor and identify treatment-resistant regions within the tumor's microenvironment.

Evaluating Tumor Hypoxia Radiosensitization Via Electron Paramagnetic Resonance Oxygen Imaging (EPROI).

PURPOSE: Tumor hypoxia contributes to aggressive phenotypes and diminished therapeutic responses to radiation therapy (RT) with hypoxic tissue being 3-fold less radiosensitive than normoxic tissue. A major challenge in implementing hypoxic radiosensitizers is the lack of a high-resolution imaging modality that directly quantifies tissue-oxygen. The electron paramagnetic resonance oxygen-imager (EPROI) was used to quantify tumor oxygenation in two murine tumor models: E0771 syngeneic transplant breast cancers and primary p53/MCA soft tissue sarcomas, with the latter autochthonous model better recapitulating the tumor microenvironment in human malignancies. We hypothesized that tumor hypoxia differs between these models. We also aimed to quantify the absolute change in tumor hypoxia induced by the mitochondrial inhibitor papaverine (PPV) and its effect on RT response. PROCEDURES: Tumor oxygenation was characterized in E0771 and primary p53/MCA sarcomas via EPROI, with the former model also being quantified indirectly via diffuse reflectance spectroscopy (DRS). After confirming PPV's effect on hypoxic fraction (via EPROI), we compared the effect of 0 versus 2 mg/kg PPV prior to 20 Gy on tumor growth delay and survival. RESULTS: Hypoxic sarcomas were more radioresistant than normoxic sarcomas (p=0.0057, 2-way ANOVA), and high baseline hypoxic fraction was a significant (p=0.0063, Cox Regression Model) hazard in survivability regardless of treatment. Pre-treatment with PPV before RT did not radiosensitize tumors in the sarcoma or E0771 model. In the sarcoma model, EPROI successfully identified baseline hypoxic tumors. DRS quantification of total hemoglobin, saturated hemoglobin, changes in mitochondrial potential and glucose uptake showed no significant difference in E0771 tumors pre- and post-PPV. CONCLUSION: EPROI provides 3D high-resolution pO2 quantification; EPR is better suited than DRS to characterize tumor hypoxia. PPV did not radiosensitize E0771 tumors nor p53/MCA sarcomas, which may be related to the complex pattern of vasculature in each tumor. Additionally, understanding model-dependent tumor hypoxia will provide a much-needed foundation for future therapeutic studies with hypoxic radiosensitizers.

In Vivo Partial Oxygen Pressure Assessment in Subcutaneous and Intraperitoneal Sites Using Imaging of Solid Oxygen Probe.

The purpose of this study was to assess the natural partial oxygen pressure (pO2) of subcutaneous (SC) and intraperitoneal (IP) sites in mice to determine their relative suitability as sites for placement of implants. The pO2 measurements were performed using oxygen imaging of solid probes using lithium phthalocyanine (LiPc) as the oxygen sensitive material. LiPc is a water-insoluble crystalline probe whose spin-lattice and spin-spin relaxation rates (R1 and R2) are sensitive to the local oxygen concentration. To facilitate direct in vivo oxygen imaging, we prepared a solid probe containing encapsulated LiPc crystals in polydimethylsiloxane (PDMS), an oxygen-permeable and bioinert polymer. Although LiPc-PDMS or similar probes have been used in repeated spectroscopic or average oxygen measurements using continuous wave electron paramagnetic resonance (EPR) since the late 1990s and now have advanced to clinical applications, they have not been used for pulse EPR oxygen imaging. One LiPc-PDMS probe of 2 mm diameter and 10 mm length was implanted in SC or IP sites (left or right side) in each animal. The pO2 imaging of implanted LiPc-PDMS probes was performed weekly for 6 weeks using O2M preclinical 25 mT oxygen imager, JIVA-25™, using the pulse inversion recovery electron spin echo method. At week 6, the probes were recovered, and histological examinations were performed. We report in this study, first-ever solid probe oxygen imaging of implanted devices and pO2 assessment of SC and IP sites.

Imaging the snorkel effect during submerged germination in rice: Oxygen supply via the coleoptile triggers seminal root emergence underwater.

Submergence during germination impedes aerobic metabolisms and limits the growth of most higher plants. However, some wetland plants including rice can germinate under submerged conditions. It has long been hypothesized that the first elongating shoot tissue, the coleoptile, acts as a snorkel to acquire atmospheric oxygen (O2) to initiate the first leaf elongation and seminal root emergence. Here, we obtained direct evidence for this hypothesis by visualizing the spatiotemporal O2 dynamics during submerged germination in rice using a planar O2 optode system. In parallel with the O2 imaging, we tracked the anatomical development of shoot and root tissues in real-time using an automated flatbed scanner. Three hours after the coleoptile tip reached the water surface, O2 levels around the embryo transiently increased. At this time, the activity of alcohol dehydrogenase (ADH), an enzyme critical for anaerobic metabolism, was significantly reduced, and the coleorhiza covering the seminal roots in the embryo was broken. Approximately 10 h after the transient burst in O2, seminal roots emerged. A transient O2 burst around the embryo was shown to be essential for seminal root emergence during submerged rice germination. The parallel application of a planar O2 optode system and automated scanning system can be a powerful tool for examining how environmental conditions affect germination in rice and other plants.

Implantable sensor for local Cherenkov-excited luminescence imaging of tumor pO2 during radiotherapy.

SIGNIFICANCE: The necessity to use exogenous probes for optical oxygen measurements in radiotherapy poses challenges for clinical applications. Options for implantable probe biotechnology need to be improved to alleviate toxicity concerns in human use and facilitate translation to clinical trial use. AIM: To develop an implantable oxygen sensor containing a phosphorescent oxygen probe such that the overall administered dose of the probe would be below the Federal Drug Administration (FDA)-prescribed microdose level, and the sensor would provide local high-intensity signal for longitudinal measurements of tissue pO2. APPROACH: PtG4, an oxygen quenched dendritic molecule, was mixed into an agarose matrix at 100 µM concentration, allowing for local injection into tumors at the total dose of 10 nmol per animal, forming a gel at the site of injection. Cherenkov-excited luminescence imaging (CELI) was used to acquire the phosphorescence and provide intratumoral pO2. RESULTS: Although PtG4 does not form covalent bonds with agarose and gradually leaches out into the surrounding tissue, its retention time within the gel was sufficiently long to demonstrate the capability to measure intratumoral pO2 with the implantable gel sensors. The sensor's performance was first evaluated in vitro in tissue simulation phantoms, and then the sensor was used to measure changes in oxygen in MDA-MB-231 tumors during hypofractionated radiotherapy. CONCLUSIONS: Our study demonstrates that implantable oxygen sensors in combination with CELI present a promising approach for quantifying oxygen changes during the course of radiation therapy and thus for evaluating the tumor response to radiation. By improving the design of the gel-probe composition in order to prevent leaching of the probe into the tissue, biosensors can be created that should allow longitudinal oxygen measurements in tumors by means of CELI while using FDA-compliant microdose levels of the probe and thus lowering toxicity concerns.

Dual-emissive, oxygen-sensing boron nanoparticles quantify oxygen consumption rate in breast cancer cells.

SIGNIFICANCE: Decreasing the oxygen consumption rate (OCR) of tumor cells is a powerful method for ameliorating tumor hypoxia. However, quantifying the change in OCR is challenging in complex experimental systems. AIM: We present a method for quantifying the OCR of two tumor cell lines using oxygen-sensitive dual-emissive boron nanoparticles (BNPs). We hypothesize that our BNP results are equivalent to the standard Seahorse assay. APPROACH: We quantified the spectral emissions of the BNP and accounted for external oxygen diffusion to quantify OCR over 24 h. The BNP-computed OCR of two breast cancer cell lines, E0771 and 4T07, were compared with their respective Seahorse assays. Both cell lines were also irradiated to quantify radiation-induced changes in the OCR. RESULTS: Using a Bland-Altman analysis, our BNPs OCR was equivalent to the standard Seahorse assay. Moreover, in an additional experiment in which we irradiated the cells at their 50% survival fraction, the BNPs were sensitive enough to quantify 24% reduction in OCR after irradiation. CONCLUSIONS: Our results conclude that the BNPs are a viable alternative to the Seahorse assay for quantifying the OCR in cells. The Bland-Altman analysis showed that these two methods result in equivalent OCR measurements. Future studies will extend the OCR measurements to complex systems including 3D cultures and in vivo models, in which OCR measurements cannot currently be made.