MRI log file analysis for workflow improvement.

Magnetic Resonance Imaging (MRI) is an important diagnostic scanning tool for the detection and monitoring of specific diseases and conditions. However, the equipment cost, maintenance and specialty training of the technologists make the examination expensive. Consequently, unnecessary scanner time caused by poor scheduling, repeated sequences, aborted sequences, scanner idleness, or capture of non-diagnostic or low-value sequences is an opportunity to reduce costs and increase efficiency. This paper analyzes data collected from log files on 29 scanners over several years. 'Wasted' time is defined and key performance indicators (KPIs) are identified. A decrease in exam duration results when actively modifying and monitoring the number of sequences that comprise the exam card for a protocol.

Predicting intratumoral fluid pressure and liposome accumulation using physics informed deep learning.

Liposome-based anticancer agents take advantage of the increased vascular permeability and transvascular pressure gradients for selective accumulation in tumors, a phenomenon known as the enhanced permeability and retention(EPR) effect. The EPR effect has motivated the clinical use of nano-therapeutics, with mixed results on treatment outcome. High interstitial fluid pressure (IFP) has been shown to limit liposome drug delivery to central tumour regions. Furthermore, high IFP is an independent prognostic biomarker for treatment efficacy in radiation therapy and chemotherapy for some solid cancers. Therefore, accurately measuring spatial liposome accumulation and IFP distribution within a solid tumour is crucial for optimal treatment planning. In this paper, we develop a model capable of predicting voxel-by-voxel intratumoral liposome accumulation and IFP using pre and post administration imaging. Our approach is based on physics informed machine learning, a novel technique combining machine learning and partial differential equations. through application to a set of mouse data and a set of synthetically-generated tumours, we show that our approach accurately predicts the spatial liposome accumulation and IFP for an individual tumour while relying on minimal information. This is an important result with applications for forecasting tumour progression and designing treatment.

Classifying MRI motion severity using a stacked ensemble approach.

Motion artifacts are a common occurrence in Magnetic Resonance Imaging exam. Motion during acquisition has a profound impact on workflow efficiency, often requiring a repeat of sequences. Furthermore, motion artifacts may escape notice by technologists, only to be revealed at the time of reading by the radiologists, affecting their diagnostic quality. There is a paucity of clinical tools to identify and quantitatively assess the severity of motion artifacts in MRI. An image with subtle motion may still have diagnostic value, while severe motion may be uninterpretable by radiologists and requires the exam to be repeated. Therefore, a tool for the automatic identification of motion artifacts would aid in maintaining diagnostic quality, while potentially driving workflow efficiencies. Here we aim to quantify the severity of motion artifacts from MRI images using deep learning. Impact of subject movement parameters like displacement and rotation on image quality is also studied. A state-of-the-art, stacked ensemble model was developed to classify motion artifacts into five levels (no motion, slight, mild, moderate and severe) in brain scans. The stacked ensemble model is able to robustly predict rigid-body motion severity across different acquisition parameters, including T1-weighted and T2-weighted slices acquired in different anatomical planes. The ensemble model with XGBoost metalearner achieves 91.6% accuracy, 94.8% area under the curve, 90% Cohen's Kappa, and is observed to be more accurate and robust than the individual base learners.

Radiation and Heat Improve the Delivery and Efficacy of Nanotherapeutics by Modulating Intratumoral Fluid Dynamics.

Nanomedicine drug delivery systems are capable of transporting significant payloads to solid tumors. However, only a modest increase in antitumor efficacy relative to the standard of care has been observed. In this study, we demonstrate that a single dose of radiation or mild hyperthermia can substantially improve tumor uptake and distribution of nanotherapeutics, resulting in improved treatment efficacy. The delivery of nanomedicine was driven by a reduction in interstitial fluid pressure (IFP) and small perturbation of steady-state fluid flow. The transient effects on fluid dynamics in tumors with high IFP was also shown to dominate over immune cell endocytic capacity, another mechanism suspected of improving drug delivery. Furthermore, we demonstrate the specificity of this mechanism by showing that delivery of nanotherapeutics to low IFP tumors with high leukocyte infiltration does not benefit from pretreatment with radiation or heat. These results demonstrate that focusing on small perturbations to steady-state fluid dynamics, rather than large sustained effects or uncertain immune cell recruitment strategies, can impart a vulnerability to tumors with high IFP and enhance nanotherapeutic drug delivery and treatment efficacy.

Spatial frequency performance limitations of radiation dose optimization and beam positioning.

The flexibility and sophistication of modern radiotherapy treatment planning and delivery methods have advanced techniques to improve the therapeutic ratio. Contemporary dose optimization and calculation algorithms facilitate radiotherapy plans which closely conform the three-dimensional dose distribution to the target, with beam shaping devices and image guided field targeting ensuring the fidelity and accuracy of treatment delivery. Ultimately, dose distribution conformity is limited by the maximum deliverable dose gradient; shallow dose gradients challenge techniques to deliver a tumoricidal radiation dose while minimizing dose to surrounding tissue. In this work, this 'dose delivery resolution' observation is rigorously formalized for a general dose delivery model based on the superposition of dose kernel primitives. It is proven that the spatial resolution of a delivered dose is bounded by the spatial frequency content of the underlying dose kernel, which in turn defines a lower bound in the minimization of a dose optimization objective function. In addition, it is shown that this optimization is penalized by a dose deposition strategy which enforces a constant relative phase (or constant spacing) between individual radiation beams. These results are further refined to provide a direct, analytic method to estimate the dose distribution arising from the minimization of such an optimization function. The efficacy of the overall framework is demonstrated on an image guided small animal microirradiator for a set of two-dimensional hypoxia guided dose prescriptions.

Measurement of drug-target engagement in live cells by two-photon fluorescence anisotropy imaging.

The ability to directly image and quantify drug-target engagement and drug distribution with subcellular resolution in live cells and whole organisms is a prerequisite to establishing accurate models of the kinetics and dynamics of drug action. Such methods would thus have far-reaching applications in drug development and molecular pharmacology. We recently presented one such technique based on fluorescence anisotropy, a spectroscopic method based on polarization light analysis and capable of measuring the binding interaction between molecules. Our technique allows the direct characterization of target engagement of fluorescently labeled drugs, using fluorophores with a fluorescence lifetime larger than the rotational correlation of the bound complex. Here we describe an optimized protocol for simultaneous dual-channel two-photon fluorescence anisotropy microscopy acquisition to perform drug-target measurements. We also provide the necessary software to implement stream processing to visualize images and to calculate quantitative parameters. The assembly and characterization part of the protocol can be implemented in 1 d. Sample preparation, characterization and imaging of drug binding can be completed in 2 d. Although currently adapted to an Olympus FV1000MPE microscope, the protocol can be extended to other commercial or custom-built microscopes.

Radiation therapy primes tumors for nanotherapeutic delivery via macrophage-mediated vascular bursts.

Efficient delivery of therapeutic nanoparticles (TNPs) to tumors is critical in improving efficacy, yet strategies that universally maximize tumoral targeting by TNP modification have been difficult to achieve in the clinic. Instead of focusing on TNP optimization, we show that the tumor microenvironment itself can be therapeutically primed to facilitate accumulation of multiple clinically relevant TNPs. Building on the recent finding that tumor-associated macrophages (TAM) can serve as nanoparticle drug depots, we demonstrate that local tumor irradiation substantially increases TAM relative to tumor cells and, thus, TNP delivery. High-resolution intravital imaging reveals that after radiation, TAM primarily accumulate adjacent to microvasculature, elicit dynamic bursts of extravasation, and subsequently enhance drug uptake in neighboring tumor cells. TAM depletion eliminates otherwise beneficial radiation effects on TNP accumulation and efficacy, and controls with unencapsulated drug show that radiation effects are more pronounced with TNPs. Priming with combined radiation and cyclophosphamide enhances vascular bursting and tumoral TNP concentration, in some cases leading to a sixfold increase of TNP accumulation in the tumor, reaching 6% of the injected dose per gram of tissue. Radiation therapy alters tumors for enhanced TNP delivery in a TAM-dependent fashion, and these observations have implications for the design of next-generation tumor-targeted nanomaterials and clinical trials for adjuvant strategies.

Design and Development of Fluorescent Vemurafenib Analogs for In Vivo Imaging.

Herein we describe fluorescent derivatives of vemurafenib to probe therapeutic BRAF inhibition in live cells and in vivo. The compounds were evaluated and compared by determining target binding, inhibition of mutant BRAF melanoma cell lines and live cell imaging. We show that vemurafenib-BODIPY is a superior imaging drug to visualize the targets of vemurafenib in live cells and in vivo in non-resistant and resistant melanoma tumors.

Radiation effects on the tumor microenvironment: Implications for nanomedicine delivery.

The tumor microenvironment has an important influence on cancer biological and clinical behavior and radiation treatment (RT) response. However, RT also influences the tumor microenvironment in a complex and dynamic manner that can either reinforce or inhibit this response and the likelihood of long-term disease control in patients. It is increasingly evident that the interplay between RT and the tumor microenvironment can be exploited to enhance the accumulation and intra-tumoral distribution of nanoparticles, mediated by changes to the vasculature and stroma with secondary effects on hypoxia, interstitial fluid pressure (IFP), solid tissue pressure (STP), and the recruitment and activation of bone marrow-derived myeloid cells (BMDCs). The use of RT to modulate nanoparticle drug delivery offers an exciting opportunity to improve antitumor efficacy. This review explores the interplay between RT and the tumor microenvironment, and the integrated effects on nanoparticle drug delivery and efficacy.

Spatial Measurements of Perfusion, Interstitial Fluid Pressure and Liposomes Accumulation in Solid Tumors.

The heterogeneous intra-tumoral accumulation of liposomes is a critical determinant of their efficacy. Both the chaotic tumor microcirculation and elevated IFP are linked to the heterogeneous intra-tumoral distribution of nanotechnology-based drug delivery systems such as liposomes. In the present study, the relationship between tumor microcirculation, elevated IFP, and accumulation of nanoparticles was investigated through in vivo experimentation. This was accomplished by evaluation of the tumor microcirculation using dynamic contrast enhanced computed tomography (DCE-CT) and measurement of tumor IFP using a novel image-guided robotic needle placement system connected to the micro-CT scanner. The intra-tumoral accumulation of liposomes was determined by CT image-based assessment of a nanoparticle liposomal formulation that stably encapsulate the contrast agent iohexol (CT-liposomes). CT imaging allowed for co-localization of the spatial distribution of tumor hemodynamics, IFP and CT-liposome accumulation in an individual subcutaneous xenograft mouse model of breast cancer. Measurements led to the discovery that perfusion and plasma volume fraction are strong mediators of the intra-tumoral distribution of liposomes. Furthermore, the results suggest that IFP plays an indirect role in mediating liposome distribution through modulating blood flow.

Integration of imaging into clinical practice to assess the delivery and performance of macromolecular and nanotechnology-based oncology therapies.

Functional and molecular imaging has become increasingly used to evaluate interpatient and intrapatient tumor heterogeneity. Imaging allows for assessment of microenvironment parameters including tumor hypoxia, perfusion and proliferation, as well as tumor metabolism and the intratumoral distribution of specific molecular markers. Imaging information may be used to stratify patients for targeted therapies, and to define patient populations that may benefit from alternative therapeutic approaches. It also provides a method for non-invasive monitoring of treatment response at earlier time-points than traditional cues, such as tumor shrinkage. Further, companion diagnostic imaging techniques are becoming progressively more important for development and clinical implementation of targeted therapies. Imaging-based companion diagnostics are likely to be essential for the validation and FDA approval of targeted nanotherapies and macromolecular medicines. This review describes recent clinical advances in the use of functional and molecular imaging to evaluate the tumor microenvironment. Additionally, this article focuses on image-based assessment of distribution and anti-tumor effect of nano- and macromolecular systems.

The intra-tumoral relationship between microcirculation, interstitial fluid pressure and liposome accumulation.

The heterogeneous intra-tumoral accumulation of liposomes has been linked to both the chaotic tumor microcirculation and to elevated interstitial fluid pressure (IFP). Here, we explored the relationship between tumor microcirculation, IFP, and the intra-tumoral accumulation of liposomes. Measurements of the tumor microcirculation using perfusion imaging, IFP using a novel image-guided robotic needle positioning system, and the intra-tumoral distribution of liposomes using volumetric micro-CT imaging were performed in mice bearing subcutaneous and orthotopic MDA-MB-231 tumors. Intra-tumoral perfusion and IFP were substantially different between the two tumor implantation sites. Tumor perfusion and not vascular permeability was found to be the primary mediator of the intra-tumoral accumulation of CT-liposomes. A strong relationship was observed between the radial distribution of IFP, metrics of tumor perfusion, and the intra-tumoral accumulation of liposomes. Tumors with elevated central IFP that decreased at the periphery had low perfusion and low levels of CT-liposome accumulation that increased towards the periphery. Conversely, tumors with low and radially uniform IFP exhibited higher levels of tumor perfusion and CT-liposome accumulation. Both tumor perfusion and elevated IFP exhibit substantial intra-tumoral heterogeneity and both play an integral role in mediating the intra-tumoral accumulation of liposomes through a complex interactive effect. Measuring IFP in the clinical setting remains challenging and these results demonstrate that tumor perfusion imaging alone provides a robust non-invasive method to identify factors that contribute to poor liposome accumulation and may allow for pre-selection of patients that are more likely to respond to nanoparticle therapy.

Spatial and temporal mapping of heterogeneity in liposome uptake and microvascular distribution in an orthotopic tumor xenograft model.

Existing paradigms in nano-based drug delivery are currently being challenged. Assessment of bulk tumor accumulation has been routinely considered an indicative measure of nanomedicine potency. However, it is now recognized that the intratumoral distribution of nanomedicines also impacts their therapeutic effect. At this time, our understanding of the relationship between the bulk (i.e., macro-) tumor accumulation of nanocarriers and their intratumoral (i.e., micro-) distribution remains limited. Liposome-based drug formulations, in particular, suffer from diminished efficacy in vivo as a result of transport-limiting properties, combined with the heterogeneous nature of the tumor microenvironment. In this report, we perform a quantitative image-based assessment of macro- and microdistribution of liposomes. Multi-scalar assessment of liposome distribution was enabled by a stable formulation which co-encapsulates an iodinated contrast agent and a near-infrared fluorescence probe, for computed tomography (CT) and optical microscopy, respectively. Spatio-temporal quantification of tumor uptake in orthotopic xenografts was performed using CT at the bulk tissue level, and within defined sub-volumes of the tumor (i.e., rim, periphery and core). Tumor penetration and relative distribution of liposomes were assessed by fluorescence microscopy of whole tumor sections. Microdistribution analysis of whole tumor images exposed a heterogeneous distribution of both liposomes and tumor vasculature. Highest levels of liposome uptake were achieved and maintained in the well-vascularized tumor rim over the study period, corresponding to a positive correlation between liposome and microvascular density. Tumor penetration of liposomes was found to be time-dependent in all regions of the tumor however independent of location in the tumor. Importantly, a multi-scalar comparison of liposome distribution reveals that macro-accumulation in tissues (e.g., blood, whole tumor) may not reflect micro-accumulation levels present within specific regions of the tumor as a function of time.

A mathematical model of the enhanced permeability and retention effect for liposome transport in solid tumors.

The discovery of the enhanced permeability and retention (EPR) effect has resulted in the development of nanomedicines, including liposome-based formulations of drugs, as cancer therapies. The use of liposomes has resulted in substantial increases in accumulation of drugs in solid tumors; yet, significant improvements in therapeutic efficacy have yet to be achieved. Imaging of the tumor accumulation of liposomes has revealed that this poor or variable performance is in part due to heterogeneous inter-subject and intra-tumoral liposome accumulation, which occurs as a result of an abnormal transport microenvironment. A mathematical model that relates liposome accumulation to the underlying transport properties in solid tumors could provide insight into inter and intra-tumoral variations in the EPR effect. In this paper, we present a theoretical framework to describe liposome transport in solid tumors. The mathematical model is based on biophysical transport equations that describe pressure driven fluid flow across blood vessels and through the tumor interstitium. The model was validated by direct comparison with computed tomography measurements of tumor accumulation of liposomes in three preclinical tumor models. The mathematical model was fit to liposome accumulation curves producing predictions of transport parameters that reflect the tumor microenvironment. Notably, all fits had a high coefficient of determination and predictions of interstitial fluid pressure agreed with previously published independent measurements made in the same tumor type. Furthermore, it was demonstrated that the model attributed inter-subject heterogeneity in liposome accumulation to variations in peak interstitial fluid pressure. These findings highlight the relationship between transvascular and interstitial flow dynamics and variations in the EPR effect. In conclusion, we have presented a theoretical framework that predicts inter-subject and intra-tumoral variations in the EPR effect based on fundamental properties of the tumor microenvironment and forms the basis for transport modeling of liposome drug delivery.

Reprogramming metabolism with metformin improves tumor oxygenation and radiotherapy response.

PURPOSE: Tumor hypoxia is a negative prognostic factor in multiple cancers, due in part to its role in causing resistance to radiotherapy. Hypoxia arises in tumor regions distal to blood vessels as oxygen is consumed by more proximal tumor cells. Reducing the rate of oxygen consumption is therefore a potential strategy to reduce tumor hypoxia. We hypothesized that the anti-diabetic drug metformin, which reduces oxygen consumption through inhibition of mitochondrial complex I, would improve radiation response by increasing tumor oxygenation. EXPERIMENTAL DESIGN: Tumor hypoxia was measured in xenografts before and after metformin treatment using 2-nitroimidazole hypoxia markers quantified by immunohistochemistry (IHC), flow cytometry, and positron emission tomography (PET) imaging. Radiation response was determined by tumor growth delay and clonogenic survival in xenografts with and without administration of metformin. The impact of metformin use on outcome was assessed in 504 patients with localized prostate cancer treated with curative-intent, image-guided radiotherapy (IGRT) from 1996 to 2012. Three-year biochemical relapse-free rates were assessed using the Kaplan-Meier method. RESULTS: Metformin treatment significantly improved tumor oxygenation in two xenograft models as measured by IHC, flow cytometry, and PET imaging. Metformin also led to improved radiotherapy responses when mice were administered metformin immediately before irradiation. Clinically, metformin use was associated with an independent and significant decrease in early biochemical relapse rates (P = 0.0106). CONCLUSION: Our data demonstrate that metformin can improve tumor oxygenation and response to radiotherapy. Our study suggests that metformin may represent an effective and inexpensive means to improve radiotherapy outcome with an optimal therapeutic ratio.

Tumor perfusion imaging predicts the intra-tumoral accumulation of liposomes.

Liposomes have proven to be a viable drug delivery strategy resulting in significant increases in tumor accumulation of drugs via exploitation of the enhanced permeability and retention (EPR) effect. However, significant variability has been observed in their bulk tumor accumulation and intra-tumoral distribution. The heterogeneous accumulation of liposomes in solid tumors is largely believed to result from the chaotic morphology and physiology of tumor blood vessels. Thus, tumor perfusion imaging may provide a novel method to predict the accumulation and resulting therapeutic effect of liposome formulations. In this study, dynamic contrast enhanced computed tomography (DCE-CT) was employed to quantitatively estimate the intra-tumoral distribution of perfusion and anatomical CT was used to map the spatio-temporal accumulation of a CT-liposome contrast agent. A statistically significant positive correlation was found between quantitative and semi-quantitative measures of tumor perfusion (i.e. K(trans), vp, and AUC(iox)) and liposome accumulation (AUC(lipo) and C(peak)) in two mouse xenograft models of human cervical cancer. Specifically, it was found that regions with higher K(trans),vp, and AUC(iox) had greater liposome accumulation. These findings demonstrate that DCE-CT measurements of tumor perfusion may be an important technique for selecting patients that are likely to respond to liposome and potentially other nanoparticle-based therapies.

A spinal cord window chamber model for in vivo longitudinal multimodal optical and acoustic imaging in a murine model.

In vivo and direct imaging of the murine spinal cord and its vasculature using multimodal (optical and acoustic) imaging techniques could significantly advance preclinical studies of the spinal cord. Such intrinsically high resolution and complementary imaging technologies could provide a powerful means of quantitatively monitoring changes in anatomy, structure, physiology and function of the living cord over time after traumatic injury, onset of disease, or therapeutic intervention. However, longitudinal in vivo imaging of the intact spinal cord in rodent models has been challenging, requiring repeated surgeries to expose the cord for imaging or sacrifice of animals at various time points for ex vivo tissue analysis. To address these limitations, we have developed an implantable spinal cord window chamber (SCWC) device and procedures in mice for repeated multimodal intravital microscopic imaging of the cord and its vasculature in situ. We present methodology for using our SCWC to achieve spatially co-registered optical-acoustic imaging performed serially for up to four weeks, without damaging the cord or induction of locomotor deficits in implanted animals. To demonstrate the feasibility, we used the SCWC model to study the response of the normal spinal cord vasculature to ionizing radiation over time using white light and fluorescence microscopy combined with optical coherence tomography (OCT) in vivo. In vivo power Doppler ultrasound and photoacoustics were used to directly visualize the cord and vascular structures and to measure hemoglobin oxygen saturation through the complete spinal cord, respectively. The model was also used for intravital imaging of spinal micrometastases resulting from primary brain tumor using fluorescence and bioluminescence imaging. Our SCWC model overcomes previous in vivo imaging challenges, and our data provide evidence of the broader utility of hybridized optical-acoustic imaging methods for obtaining multiparametric and rich imaging data sets, including over extended periods, for preclinical in vivo spinal cord research.

3D image-guided robotic needle positioning system for small animal interventions.

PURPOSE: This paper presents the design of a micro-CT guided small animal robotic needle positioning system. In order to simplify the robotic design and maintain a small targeting error, a novel implementation of the remote center of motion is used in the system. The system has been developed with the objective of achieving a mean targeting error of <200 µm while maintaining a high degree of user friendliness. METHODS: The robot is compact enough to operate within a 25 cm diameter micro-CT bore. Small animals can be imaged and an intervention performed without the need to transport the animal from one workspace to another. Not requiring transport of the animal reduces opportunities for targets to shift from their localized position in the image and simplifies the workflow of interventions. An improved method of needle calibration is presented that better characterizes the calibration using the position of the needle tip in photographs rather than the needle axis. A calibration fixture was also introduced, which dramatically reduces the time requirements of calibration while maintaining calibration accuracy. Two registration modes have been developed to correspond the robot coordinate system with the coordinate system of the micro-CT scanner. The two registration modes offer a balance between the time required to complete a registration and the overall registration accuracy. The development of slow high accuracy and fast low accuracy registration modes provides users with a degree of flexibility in selecting a registration mode best suited for their application. RESULTS: The target registration error (TRE) of the higher accuracy primary registration was TRE(primary) = 31 ± 12 µm. The error in the lower accuracy combined registration was TRE(combined) = 139 ± 63 µm. Both registration modes are therefore suitable for small-animal needle interventions. The targeting accuracy of the robotic system was characterized using targeting experiments in tissue-mimicking gelatin phantoms. The results of the targeting experiments were combined with the known calibration and needle deflection errors to provide a more meaningful measure of the needle positioning accuracy of the system. The combined targeting errors of the system were 149 ± 41 µm and 218 ± 38 µm using the primary and combined registrations, respectively. Finally, pilot in vivo experiments were successfully completed to demonstrate the performance of the system in a biomedical application. CONCLUSIONS: The device was able to achieve the desired performance with an error of <200 µm and improved repeatability when compared to other designs. The device expands the capabilities of image-guided interventions for preclinical biomedical applications.

Acoustic and kinetic behaviour of definity in mice exposed to high frequency ultrasound.

Microbubble contrast agents have shown clinical potential for characterising blood flow using 1 to 10 MHz ultrasound; however, scaling their use for similar applications in the mouse with high frequency ultrasound (20 to 60 MHz) has not been addressed. The goal was to determine the utility of microbubbles for mouse imaging with 30 MHz ultrasound by investigating their attenuation and backscatter characteristics as a function of concentration in vitro and dose response in vivo. The agent was exposed to a 30 MHz, 20% bandwidth pulse with a peak negative pressure of 244 kPa. In vitro results showed that the attenuation and backscatter increased linearly for concentrations between 2.8 x 10(6) and 28 x 10(6) bubbles per mL of deionized water. In vivo experiments where performed in the jugular vein of CD-1 mice and time intensity curves were acquired for doses between 10 and 100 microL kg(-1). These doses corresponded to the range of concentrations used in vitro. In vivo results showed that the peak enhancement of the agent increased linearly for doses between 10 and 60 microL kg(-1), the duration of enhancement varied between 200 to 300 s and the integrated enhancement (area under the curve) increased linearly up to 100 microL kg(-1). A maximum enhancement of 13 dB over the blood pool was observed for a dose of 100 microL kg(-1). The intra- and inter-mouse variabilities were 10% to 40% and indicate that further optimisations are required. These results suggest that quantitative contrast flow studies in the mouse using high frequency ultrasound are possible for doses between 10 and 60 microL kg(-1).