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2010

High-Frequency Ultrasound

Application: Features & Benefits

High-Frequency Ultrasound Table of Contents

High-Frequency Ultrasound....................................................................................................... 1
Overview .............................................................................................................................................. 1 Applications & Techniques ....................................................................................................................... 1

Cardiovascular .......................................................................................................................... 2 Cancer Biology .......................................................................................................................... 4 Neurobiology ............................................................................................................................ 6 Developmental Biology ............................................................................................................. 8 Gene Delivery ........................................................................................................................... 9
Gene Delivery and Microbubble Contrast Agent configurations..................................................................... 10 Sonoporation: Q & A............................................................................................................................. 10 Select Sonoporation References.............................................................................................................. 11

Nephrology ............................................................................................................................. 12 Ophthalmology ....................................................................................................................... 15 Reproductive Biology .............................................................................................................. 17 Rheumatology and Musculoskeletal ........................................................................................ 19 Contrast Imaging.................................................................................................................... 22
Quantification of Relative Perfusion ......................................................................................................... 23 Quantification of Biomarkers .................................................................................................................. 23

3D Visualization...................................................................................................................... 26

High-Frequency Ultrasound
Overview Imagine visualizing anatomical structures and hemodynamic functions in vivo, longitudinally in a noninvasive manner. The Vevo high-resolution in vivo micro-imaging systems provides high-resolution imaging down to 30 microns and the ability to easily export derived statistical data to any PC or Macintosh-based application for analysis. With an interface designed for multiple subject-data collation needs, the researcher is offered opportunities that until now were simply not possible. All areas of research involving phenotypic expression within the small animal model can apply the highresolution, real-time visualization benefits of the Vevo. This section describes a number of the main application areas and techniques. Applications & Techniques Overview Cardiovascular Cancer Biology Neurobiology Developmental Biology Gene Delivery Nephrology Hepatology Ophthalmology Reproductive Biology Rheumatology & Muscuskeletal Regenerative Medicine & Stem Cell Contrast Imaging Image-Guided Injection Blood Flow Analysis 3D Visualization & Measurement

High-Frequency Ultrasound Applications and Techniques

High-Frequency Ultrasound Cardiovascular


Vevo technology provides echocardiograms FOR TRANSLATIONAL RESEARCH and biomarker development in animals with a resolution down to 30 microns. Software and quantification tools are customized for small animal physiology. The high frame rate of acquisition provides the best temporal and spatial resolution specifications compared to other imaging modalities such as MRI or CT. VisualSonics has developed protocols using our dedicated animal handling platform to ensure that the small animal (mouse/rat) is maintained in the most stable state during imaging. The result is reproducible, reliable results for large cohorts being studied longitudinally. Quantitative evaluation of the cardiovascular system: Real-time evaluation and quantification of physiological function of the developing heart and the associated major vessels. Including: left ventricular function, right ventricular function, valve movement and blood flow. Evaluation of cardiovascular disease and therapies: The Vevo can also be used to assess the extent of cardiovascular disease and evaluate the effectiveness of cardiovascular therapies. The non-invasive nature of the system allows for longitudinal analysis (studying treatment effectiveness in the same animal over time). Examples of pathologies that can easily be assessed include: myocardial infarct, diastolic dysfunction, cardiac hypertrophy, pulmonary hypertension, atherosclerosis, inflammation, valvular regurgitation, vessel stenosis and diabetes. Evaluation of cardiovascular function in conscious mice: Light weight and easily handheld probes allow imaging of conscious mice for researchers interested in assessing VALVULAR DISEASE AT HIGH FRAME RATES. Extensive measurement and annotation functionality: The Vevo software provides measurement and annotation functionality that allows the cardiovascular researcher to conduct comprehensive analysis for monitoring and measuring the progression of disease and the efficacy of therapeutics. Examples of calculation packages include: quantification of ejection fraction, fractional shortening, cardiac output, strain, strain rate, pulsatility indices and resistivity indices. Furthermore all data can be exported in excel and images can be formatted in BMP, TIFF, AVI, GIF and DICOM. Monitoring of full animal physiology: The Integrated Rail and Animal Handling Table allow physiological parameters such as body temperature, ECG, heart rate and respiration to be captured and monitored throughout an imaging session. Furthermore these numbers can be integrated in calculation packages and exported for further data analysis and quantification. ECG gating is also possible. Integration of blood pressure external devices: Obtain pressure volume data by being able to integrate external blood pressure recordings with the Vevo. This is critical for pharmacologic and stress-echo type studies. Perform and visualize cardiac and vascular injections: The integrated rail system and image-guided needle injection system are easily mounted, prepared and operated. Precision adjustments are easily made to ensure that the probe, the injection system, and the mouse table are all in alignment to most effectively ensure a successful procedure. The Integrated Rail system allows the precise position of a needle/syringe to be mounted and the animal to be carefully positioned on a heated table. The injection can be performed with minimal invasiveness (just watch the needle pierce through the skin, into the target of interest). This
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convenient set-up eliminates invasive surgical procedures and consequently allows for improved animal survival. Easy to learn and obtain results: The Vevo is simple to use so that researchers can maximize their time performing post-imaging analysis. An offline workstation is available to analyze all data. In addition several on-line tutorials and protocols are available to allow researchers to learn quickly, image, analyze and obtain results.

Video 52: Apical 4 Chamber View in Adult Mouse

Video 14: Parasternal short axis

Image 16: Parasternal short axis

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Video 17: Infarcted mouse heart


Commonly used terminology:

heart, valve, left ventricle, right ventricle, mitral, tricuspid, atrium, atria, pulmonary, hypertension, diastolic dysfunction, diabetes, heart function, strain, strain rate, speckle tracking, aorta, vessel, pulmonary artery, pulmonary vein, blood flow, regurgitation

Cancer Biology
The Vevo is a multidisciplinary cancer research tool which allows early tumor detection, quantification of tumor size in 2D and 3D and the ability to quantify vascularity and angiogenesis. Furthermore all soft tissue tumors can be visualized whether they are subcutaneous or orthotopic. Ability to detect tumors before they are palpable: The Vevo allows for precise and accurate detection of early tumors. No need to use calipers any longer. The Vevo allows for exact measurements to be recorded and for the morphology to be precisely outlined. Monitor progression of tumor burden over time: As a non-invasive imaging system, the Vevo performs longitudinal studies that monitor the progression of disease in the same animal over time. Examples of tumor models include: TRAMP prostate, pancreatic ductul adenocarcinoma (KPC), mammary (4T1, 67NR), hepatocellular carcinoma and many others. 3D Visualization and Volumetric Measurement of Tumor Size and Shape: VisualSonics 3D visualization and segmentation tool allows high-resolution 3D rendering of an area of interests at resolution down to 30 microns. The user can also apply segmentation algorithms to determine the volume of the orthotopic tumor in vivo and follow the progression or regression of tumor volume longitudinally. The 3D volumes are obtained literally in seconds! Detection and quantification of metastasis: Ultrasound is an excellent tool for general soft tissue imaging, therefore metastasis to organs and lymph nodes can be observed. Identification and quantification of tumor blood flow: Power and Color Doppler functionality allows the identification and measurement of blood flow in and
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around tumors. Furthermore relative tumor perfusion can also be quantified using Untargeted MicroMarker contrast agents. Quantification of biomarkers: Expression of endothelial cell markers can be quantified using Target-Ready MicroMarker contrast agents. Evaluation of antiangiogenic therapies: The Vevo can directly quantify tumor neovasculature. Possibilities include quantification of VEGFR2 and Integrin expression, in vivo, by utilizing Target-Ready contrast agents. Evaluation of matrigel or collagen plugs: Determine the physiological status in these models by examining blood flow. Perform and visualize tumor injections: The integrated rail system and image-guided needle injection system are easily mounted, prepared, and operated. Precision adjustments are easily made to ensure that the probe, the injection system, and the mouse table are all in alignment to most effectively ensure a successful procedure. The Integrated Rail system allows the precise position of a needle/syringe to be mounted and the animal to be carefully positioned on a heated table. The injection can be performed without having to perform any surgically invasive procedure; eliminating invasive surgical procedures and consequently allows for improving animal survival rates. Easy to learn and obtain results: The Vevo software is simple to use so that researchers can maximize their time performing postimaging analysis. User friendly offline software is available to analyze all data. In addition, several online tutorials and protocols are available to allow researchers to learn quickly, analyze their data and obtain results.

Insert Video #4

3D tumor volume Orthotopic prostate tumor found to measure 441mm3 using the 3D visualization and segmentation tools. Tumor outlined in turquoise, bladder outlined in yellow.
Image courtesy of X. Zheng. Rutgers, The State University of New Jersey, 2009.

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Untargeted MicroMarker Contrast Agents Visualization of perfusion in a subcutaneous hepatoma. Graph shows bolus wash-in curve for tumor; quantification preformed by the Vevo software provides values of time to peak, as well as assessments of relative blood volume.
Commonly used terminology: tumor, angiogenesis, radiology, VEGF, VEGFR2, matrigel, collagen plug, hepatoma, neuroblastoma, hepatocarcinoma, mass

Neurobiology
Because of its high-resolution imaging and the ability to provide real-time visualization, the Vevo can visualize the development of early brain structures and spinal cord in utero and guide pulled glass needles for injections of cells, drugs, genetic material or metabolic agents into developing small animal embryos. The system can also be used for extraction procedures. Many neurobiological applications including the study of embryonic and neonatal brain development, cell lineage and progressive neural degenerative diseases associated with small animal models can be complimented with the use of the Vevo high-resolution imaging system. The Vevo successfully provides high-resolution images of the neonate mouse brain prior to ossification of the skull. Furthermore, structure and function of the adult brain can be visualized if a craniotomy is performed. Cerebral blood flow can also be visualized and quantified. Monitoring of small animal brain development: Due to the high-resolution images that can be derived, the neurobiologist can readily and reliably study embryonic and neonatal brain development. Furthermore, the non-invasive nature of the system allows for longitudinal study of the same animal without the need for contrast agents. Visualization and quantification of cerebral blood flow: For better assessment of disorders and angiogenesis studies. Real-time image-guided needle injections and extractions: Targeted needle injections of cells, genetic material, drugs and retroviruses can be visualized in realtime within small animals from embryonic to adult stages of development. Easy to learn and obtain results: The Vevo software is simple to use so that researchers can maximize their time performing postimaging analysis. User friendly offline software is available to analyze all data. In addition, several online tutorials and protocols are available to allow researchers to learn quickly, analyze their data and obtain results.
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Video 18: Injection into brain E12

Video 19: Motor Cortex Injury Model

Image 20: Motor Cortex Injury Model

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Image 21: Motor Cortex Injury Model


Commonly used terminology: brain, spinal cord, white matter, grey matter, cortex, hippocampus

Developmental Biology
With the mouse being the preferred model for mammalian development and its applications for phenotypic analysis, the high-resolution Vevo imaging system makes it an invaluable tool for providing real-time visualization of anatomical structures and hemodynamic function in vivo in utero of small animals. The Vevo allows detailed in vivo visualization as early as embryonic day 5.5 through neonate to adulthood. The non-invasive nature of the system therefore facilitates longitudinal studies of the same mouse, providing added utility to phenotypic analysis. In addition to the mouse, the Vevo is well-suited for other small animal models including the rat, chick embryo, and zebra fish. Furthermore 3D images and quantification can also be generated. Ability to detect early pregnancy/implantation: It allows screening of early embryonic structures as well as characterization and visualization of specific gestational stages. Furthermore, the Vevo allows you to observe embryonic resorption and to count the number of embryos present. Visualize and quantify rodent embryonic development: The ability to generate real-time images from embryonic day 4.5 in vivo provides the developmental biologist with a powerful research tool for assessment and measurement of target anatomy or physiology through to adulthood. Extensive Measurement and Annotation Functionality: The Vevo software provides measurement and annotation functionality that allows the developmental biologist to conduct comprehensive analysis for monitoring and measuring the progression of disease and the efficacy of therapeutics. Examples of calculation packages include: quantification of ejection fraction, fractional shortening, cardiac output, blood flow measurements, crown rump length. Furthermore all data can be exported in excel and images can be formatted in BMP, TIFF, AVI, GIF and DICOM. Real-time visualization of image-guided needle injections and extractions: Targeted needle injections of cells, genetic material, drugs and retroviruses can be visualized in realtime in embryos
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Easy to learn and obtain results: The Vevo software is simple to use so that researchers can maximize their time performing postimaging analysis. User friendly offline software is available to analyze all data. In addition, several online tutorials and protocols are available to allow researchers to learn quickly, analyze their data and obtain results.

Video 22: Mouse Embryo Profile E12

Video 23: Placental Flow E12


Commonly used terminology: embryo, fetus, teratogenesis, embryogenesis, implantation, amnion, amniotic cavity, coelemic cavity, ectoplacental cavity, resorption, placenta, placental, OBGYN, obstetrics

Gene Delivery
Ultrasound-based gene delivery methods are becoming more widely used based on the critical attributes of small animal research. These attributes include, but are not limited to, the important benefits of a noninvasive, non-immunogenic, longitudinal and reproducible approach of delivering genes, drugs or proteins at the cellular level. The introduction of the Vevo SoniGene and microbubble contrast agents provides a powerful and promising approach for gene delivery applications. More importantly, the combination of these products with the Vevo High-Resolution Imaging System provides a complete solution for small animal gene delivery applications. Gene and/or Drug Delivery are now possible with the combinations of the following products:
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Vevo Micro-Imaging System Vevo SoniGene

The combination of these products, along with microbubbles, provides an effective mechanism for the targeted delivery of genes or drugs at the cellular level. The Vevo system is used to identify the region of interest and provide longitudinal assessment of the effect of the gene or drug delivered. Image-Guided Needle Injection can also be used for regional delivery of the gene/drug solution. The Vevo SoniGene is a low frequency ultrasound device that is integrated with the Vevo system. The SoniGene delivers a low frequency/high-powered ultrasound pulse sequence. When used in conjunction with the contrast agents, SoniGene will cause a sonoporation of the targeted cells and allow the gene or drug to transfect to the cellular level. Contrast agents can also act as a carrier agent for the gene or drug to the targeted site. Gene Delivery and Microbubble Contrast Agent configurations

{Use current idea/image from website} (http://www.visualsonics.com/applications/applications_genedelivery.htm) Sonoporation: Q & A What is Sonoporation? Sonoporation refers to the effect of low frequency (1 to 3MHz) ultrasound on living cells. The application of ultrasound in the presence of cavitation nuclei (microbubbles) can create transient pores in a cell membrane, allowing drug molecules, proteins, or foreign DNA to enter the cell. What are microbubbles? A microbubble is a microscopic (1 to 3 micron) bubble containing an inert gas. The shell of the microbubble is composed of lipids.

Is it necessary that I use microbubbles? No, but the application of ultrasound in the presence of microbubbles and naked DNA has been found to be one of the most simple and effective methods of gene transfer available. If your laboratory already has a protocol in place using a lipid based transfection reagent you can use ultrasound in conjunction with your existing procedures. The application of ultrasound in conjunction with lipid based transfection reagents has been shown to increase gene expression (1), as well as to possibly allow a reduction in the amount of reagent used.
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How does sonoporation compare to other transfection methods in terms of cell viability? When all parameters are optimized, sonoporation causes little irreversible cell damage in most cell lines. Cell membrane recovery time has been shown to be less than 10 seconds (2). The factors that must be controlled to prevent irreversible cell damage and death are: 1. Concentrations of transfection reagent or microbubbles (3) 2. Ultrasound power output 3. Ultrasound application time 4. Ultrasound duty cycle In the published literature, everyone seems to be using different frequencies, which is best? Much experimentation was/is done using diagnostic ultrasound systems, since many medical research labs already have these instruments on hand. Because these systems vary in frequency based on their intended application, the result is a wide range of frequencies in the literature. Recent research has found a frequency of 1MHz to be most effective when used in conjunction with microbubbles. Application of 1MHz ultrasound results in the greatest relative expansion of the microbubbles prior to bursting (4), apparently causing the greatest effect. The Vevo SoniGene is pre-configured for use at 1MHz. How much output power do I need? Though not an easy question, the answer is the minimum needed to do the job. Experimenters who have tried various ranges of power output and application times have found there is a point where additional time and power no longer result in increased gene expression. Increasing power and exposure time beyond this point will only result in a decrease in the number of viable cells. Typically, in vitro sonoporation uses 0.5 to 1W/cm2 while in vivo sonoporation works well with 2W/cm2 or more. The Vevo SoniGene can provide power outputs from 0.1 to 5.0W/cm2 and duty cycles of 10, 20, 50, and 100% are standard but custom duty cycles and power outputs are available. How do I apply ultrasound to my experiment? Since 1MHz ultrasound does not travel through air, it is necessary for the probe to contact the medium directly or indirectly using a coupling gel. The Vevo SoniGene probe may be placed directly into the culture medium. Alternately, an ultrasound coupling gel may be placed between the probe and the bottom of the culture dish. A third method involves floating a well plate in a water bath with the ultrasound transducer in the water below the plate. Ideally, the Vevo SoniGene transducer should be mounted in plane with the Vevo ultrasound transducer to ensure the correct target is being affected. How does it work? Research has yet to yield a conclusive theory on the mechanism of transfer and so the debate continues. The current theory for microbubble-enhanced transfection is the destruction of each microbubble causes a chain reaction of cavitation events (2). This cavitation opens transient pores in cell membranes, allowing the entry of foreign DNA into the cell. Select Sonoporation References Combined use of Ultrasound and Acoustic Cationic Liposomes Results in Improved Gene Delivery into Smooth Muscle Cells. Shaoling Huang, et al., American Society of Gene Therapy, Annual Meeting. June 2002. Gene Transfer with Echo-enhanced Contrast Agents: Comparison between Albunex, Optison and Levovist in Mice-Initial Results. Tiell Li, PhD et al., Radiology November 2003.

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Threshold of fragmentaion for ultrasonic contrast agents. James Chomas, Paul Dayton, Donovan May, Kathy Ferrara., Journal of Biomedical Optics. (2001), 6, 141-150. Ultrasound-Induced Membrane Porosity. Cheri X. Deng, Fred Seiling, Hua Pan, and Jianmin Cui. Ultrasound in Medicine and Biology (2004) 30, 519-526. Selective clinical ultrasound signals mediate differential gene transfer and expression in two human prostate cancer cell lines: LnCap and PC-3, Biochem. Tata, D. B. et.al. (1997). Biophys. Res. Comm. 234 (1), 64 67.

Commonly used terminology: transfection

Nephrology
The Vevo high-resolution micro-ultrasound system is an indispensible tool for nephrology research in mice, rats and other small animal models. The high spatial resolution of this system allows for excellent delineation of the renal tissue. Furthermore, the numerous features of Vevo ultrasound system and its software extend the researchers ability to quantify and analyze kidney function. High-spatial resolution up to 30 m allows for excellent delineation of renal and renal-related structures such as medulla, cortex, renal vein and artery, ureter, urethra, and bladder in 2D and 3D. Many renal pathologies such as renal failure, polycystic kidney disease, kidney stone and renal cancer can cause changes in renal tissue texture, brightness, shape and size. All of these changes can be detected and quantified with the Vevo high-resolution system. Quantification of renal function and perfusion. Renal flow can be detected and quantified at all vascular sizes. Pulsed-wave Doppler can obtain detailed velocity profiles in the renal vein and artery, providing measurements of Pulsatility Index, Resistive Index and Renal Artery Peak Velocity. Color Flow and Power Doppler techniques detect and quantify flow in the smaller vessels in the 100 m and greater range. Contrast imaging with infusion of contrast agents can quantify flow down to the capillary level. Collectively these can provide detailed assessment of renal function. Quantification of bladder and urethra size can be obtained as changes occur in pathological states. Bladder volume and wall thickness can be measured as well as tumor size and volume. Image-guided injection and biopsy. The integrated rail system and the image-guided needle injection system enables needle injections to, and biopsies from, the kidney and/or surrounding tissue without the need of a surgical abdominal incision. Examples of material injected can be cancer cells, stem cells or therapeutic compounds. Detection and quantification of biomarkers. Biomarker expressions can be detected and quantified using Target-ready MicroMarker contrast agents. For example, P-selectin-coupled MicroMarker can be used as an inflammatory marker in the kidney. Gene transfection and enhanced drug delivery. The Vevo SoniGene is a device used in conjunction with Vevo 2100 or Vevo 770 system along with injection of MicroMarker microbubbles. The SoniGene sends a pulse that disrupts microbubbles in the organ of interest. Within the process, sonoporation is induced. If gene products or drugs are mixed with the MicroMarker infusions, enhanced gene transfection and drug delivery can be induced in the renal cells.
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Video 24: Transverse View of the Kidney

Video 27: Color Doppler

Image 28: Color Doppler


Commonly used terminology: kidney, renal, cortex, medulla, adrenal gland

Hepatology
The excellent spatial resolution and expanded functionality of the Vevo system greatly facilitates hepatology researchers to visualize and assess function in the liver, gall bladder and pancreas. High-spatial resolution up to 30 m allows for excellent delineation of anatomy associated with hepatology research in 2D and 3D, such as the liver, gall bladder and pancreas. Various associated pathologies such as hepatitis B or C, pancreatitis, gall stone and liver and pancreatic cancer can
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cause abnormal tissue changes such as liver cirrhosis, growth of tumors and presence of calcification. All of these examples can be detected and quantified by the Vevo system and tracked longitudinally in the same animal when therapy is administered. Quantification of organ perfusion. Blood flow in the liver, pancreas and other associated organs can be detected and quantified at all vascular sizes. Pulsed-wave Doppler can obtain detailed velocity profiles in veins and arteries. Color and Power Doppler can detect and quantify flow in the smaller vessels in the 100 m and greater range. Contrast imaging with infusion of contrast agents can quantify flow down to the capillary level. Collectively these can provide a detailed hemodynamic assessment in the organs associated with hepatology. Image-guided injection and biopsy. The integrated rail system and the image-guided needle injection system enables needle injections to, and biopsies from, the liver, pancreas and/or surrounding tissue without the need of a surgical abdominal incision reducing recovery time and increasing throughput in large cohort studies. Examples of material injected can be cancer cells, stem cells or therapeutic compounds. Detection and quantification of biomarkers. Biomarker expressions can be detected and quantified using Target-ready MicroMarker contrast agents. For example, VEGF-R2-coupled MicroMarker can be used as an angiogenesis marker in hepatoma in the same animal as its own control in longitudinal studies. Gene transfection and enhanced drug delivery. The Vevo SoniGene is a device used in conjunction with Vevo 2100 or Vevo 770 system along with injection of MicroMarker microbubbles. The SoniGene sends a pulse that disrupts the microbubbles in the organ of interest. Within the process, sonoporation is induced. If gene products or drugs are mixed with the MicroMarker infusions, enhanced gene transfection and drug delivery can be induced in the hepatic cells.

Image 29: Mouse Hepatic Artery and Portal Vein: Color Doppler

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Video 30: Ultrasound-Guided Injection Into Mouse Portal Vein

Image 31: Visualization of VEGFR2 Expression in PAP2 Fibroblastoma Liver Metastasis Tumor Model
Commonly used terminology: liver, gallbladder, lobe, portal, hepatic, hepatocarcinoma, portal vein, hepatic artery, hepatic vein

Ophthalmology
The excellent spatial resolution and expanded functionality of the Vevo system greatly facilitates ophthalmology researchers to visualize and assess the anatomy and function in the eye. High-spatial resolution up to 30 m and depth of imaging penetration up to 30mm allows for excellent delineation of the eye anatomy in small and large animal models, ranging from small mouse developing embryos to large full-sized adult pigs. Eye anatomical structures such as (but not limited to) cornea, lens, optic nerve, pupil, iris and ciliary body can be visualized in 2D and 3D. Furthermore, any changes in anatomy and/or tissue texture and brightness in these structures can be detected and quantified. Quantification of blood flow in the eye. Blood flow in the eye can be detected and quantified at all vascular sizes. For example, Pulsed-wave, Color and Power Doppler can obtain velocity profiles in retinal vein and artery and other vessels in the 100 m and greater range. To assess and quantify flow in the tissue/capillary levels (e.g. in the retina and iris), contrast imaging with infusion of contrast agents can be performed. Collectively these can provide a detailed hemodynamic assessment in the eye.
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Image-guided injection and biopsy. The integrated rail system and the image-guided needle injection system enables needle injections to and biopsies from the eye. Examples of injected material include stem cells and therapeutic agents that elicit response that can be tracked longitudinally. Detection and quantification of biomarkers. Biomarker expressions can be detected and quantified using Target-ready MicroMarker contrast agents. For example, VEGFR2-coupled MicroMarker can be used as an angiogenesis marker in retinoblastomas. Gene transfection and enhanced drug delivery. The Vevo SoniGene is a device used in conjunction with Vevo 2100 or Vevo 770 system along with injection of MicroMarker microbubbles. The SoniGene sends a pulse that disrupts microbubbles in the organ of interest. Within the process, sonoporation is induced. If gene products or drugs are mixed with the MicroMarker infusions, enhanced gene transfection and drug delivery can be induced in cells within the eye.

Image 33: Anterior and Lateral structures

Video 34: Visualization of hemodynamics: Color Doppler

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Image 35 & 36: Hemodynamic quantification MicroMarker in the iris


Commonly used terminology: eye, retina, cornea, optic nerve, retinal, macular degeneration, diabetes, lens

Reproductive Biology
The excellent spatial resolution and expanded functionality of the Vevo system greatly facilitates reproductive biology researchers to visualize structures in male and female reproductive systems in small animal models ranging in size from zebra fish to rabbits. High-spatial resolution up to 30 m allows for excellent delineation of anatomy associated with reproductive biology research in 2D and 3D. Examples of reproductive organs that can be visualized in mice are: penis, prostate, testicle, epididymus, seminal vesicles, uterus, ovary, endometrium, placenta and mammary fat pad. Changes in any of these tissues caused by conditions such as cancer or pregnancy can be detected and quantified with the Vevo system. Quantification of organ perfusion. Blood flow in the various male and reproductive organs can be detected and quantified at all vascular sizes. Pulsed-wave Doppler can obtain detailed velocity profiles in vessels such as testicular and ovarian artery. Color and Power Doppler can detect and quantify flow in the smaller vessels in the 100 m and greater range. Contrast imaging with infusion of contrast agents can quantify flow down to the capillary level. Collectively these can provide a detailed hemodynamic assessment in the reproductive organs which can be followed longitudinally. Image-guided injection and biopsy. The integrated rail system and the image-guided needle injection system enables needle injections to, and biopsies from, the prostate, ovary, or other reproductive organs without the need of a surgical incision. Examples of material injected can be cancer cells, stem cells or therapeutic compounds Detection and quantification of biomarkers. Biomarker expressions can be detected and quantified using Target-ready MicroMarker contrast agents. For example, VEGF-R2-coupled MicroMarker can be used as an angiogenesis marker in ovarian and prostate cancer.

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Gene transfection and enhanced drug delivery. The Vevo SoniGene is a device used in conjunction with Vevo 2100 or Vevo 770 system along with injection of MicroMarker microbubbles. The SoniGene sends a pulse that disrupts microbubbles in the organ of interest. Within the process, sonoporation is induced. If gene products or drugs are mixed with the MicroMarker infusions, enhanced gene transfection and drug delivery can be induced in the cells of the reproductive organs.

Image 37: Sagittal Cervix and Endometrium

Image 38: Uterine Bifurcation


Commonly used terminology: uterus, ovary, mammary, testicle, endometriosis, placenta, OBGYN, obstetrics, breast, sperm, prostate

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The excellent spatial resolution and expanded functionality of the Vevo system greatly facilitates rheumatology and musculoskeletal researchers to visualize and assess function in joints, muscles, and other soft and connective tissue in small animal models such as mice to large models such as humans. High-spatial resolution up to 30 m allows for excellent delineation of anatomy associated with rheumatology and musculoskeletal research. For example, muscles, tendons, cartilage and ligaments can be visualized in various anatomical regions such as hind limb muscle, shoulder, knee, finger and elbow. Furthermore, bone and articular cartilage surface smoothness can also be visualized and inflammatory responses quantified. Various pathologies such as skeletal muscle ischemia, arthritis, tendinitis and certain autoimmune diseases can cause changes in these structures in terms of tissue texture, tissue brightness and smoothness of bone and cartilage surfaces. The Vevo can detect and quantify changes in joint space size, detect synovitis and quantify volumetric changes as a response to treatment. Quantification of joint perfusion. Various pathologies can affect blood flow in the joint regions. Blood flow in vessels greater than 100 m can be visualized and quantified using pulsed-wave, color and Power Doppler. Contrast imaging with infusion of contrast agents enables blood flow assessment at the tissue/capillary levels. Therapeutic intervention and cellular delivery to joints and the response can be measured and tracked longitudinally. Image-guided injection and biopsy. The integrated rail system and the image-guided needle injection system enables needle injections to and biopsies/aspiration from soft tissue in the joint regions. Examples of material injected can be cancer cells, stem cells or therapeutic compounds Detection and quantification of biomarkers. Biomarker expressions can be detected and quantified using Target-ready MicroMarker contrast agents. For example, P-selectin-coupled MicroMarker can be used as an inflammatory marker in various tissues in the joint region. Gene transfection and enhanced drug delivery. The Vevo SoniGene is a device used in conjunction with Vevo 2100 or Vevo 770 system along with injection of MicroMarker microbubbles. The SoniGene sends a pulse that disrupts microbubbles in the organ of interest. Within the process, sonoporation is induced. If gene products or drugs are mixed with the MicroMarker infusions, enhanced gene transfection and drug delivery can be induced in the skeletal cells.

Image 39: Rat Shoulder at 40 MHz


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Video 40: Contrast Imaging Showing Perfusion Image 41: Contrast Quantification of Rat Shoulder Anatomy
Commonly used terminology: muscle, arthritis, inflammation, osteoarthritis, diabetes

Regenerative Medicine and Stem Cell Research


The excellent spatial resolution and expanded functionality of the Vevo system greatly facilitates research in regenerative medicine by enabling researchers to visualize and assess tissue regeneration in areas such as nerve damage and repair and stem cell applications in myocardial infarction. High-spatial resolution up to 30 m allows for excellent delineation of numerous anatomical structures such as nerves. Nerve damage and healing can induce changes in nerve texture and brightness under ultrasound imaging and can be detected and quantified measuring pixel intensity with histograms by the Vevo system. Quantification of tissue perfusion. Various pathologies can affect blood flow such as myocardial infarction. Contrast imaging with contrast agent injections enables blood flow assessment in the infarct border regions. Image-guided injection and biopsy. The integrated rail system and the image-guided needle injection system enables needle injections to and biopsies/aspiration from soft tissue in the tissues of interest. For example, stem cells can be injected into the infarct border regions of the heart. Detection and quantification of biomarkers. Biomarker expressions can be detected and quantified using Target-ready MicroMarker contrast agents. For example, P-selectin-coupled MicroMarker can be used as an inflammatory marker in various tissues.

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Video 42: Adult Cardiac Injection

Video 43: Target-Ready MicroMarker Hind Limb Inflammation Model P-Selectin

Video 44: Target-Ready MicroMarker Hind Limb Inflammation Model P-Selectin


Commonly used terminology: Transplantation, stem cell, tissue regeneration, diabetes

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High-Frequency Ultrasound Contrast Imaging


The VisualSonics Contrast Imaging Program has developed and created MicroMarker Contrast Agents and application-specific protocols that will allow proprietary untargeted and targeted contrast agents to be used with the Vevo high-resolution in vivo micro-imaging systems. The contrast agents and protocols have been optimized specifically for high-frequency micro-ultrasound and for preclinical applications. Ultrasound-based contrast agents are typically small micron sized micro-bubbles that may be air or gas filled. Tissue typically reacts linearly to ultrasound energy while micro-bubbles react in a non-linear fashion to the same energy. Using *proprietary filtering the Vevo removes virtually all the linear response of tissue signal from the quantification process, allowing researchers to quantify only the micro-bubble response. As these microbubbles are intravascular, they are easily introduced intravenously and pass through the vascular stream mimicking red blood cell movement. *Available on the Vevo 2100 Untargeted MicroMarker Contrast Agents provide image enhancement of the blood pool such as imaging and quantification of tumor and organ perfusion (including myocardial perfusion).

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Visualization of perfusion in the abdomen, showing flow in the kidney, pancreas, and spleen. The bottom image shows just the non linear response of the microbubbles, virtually all tissue signal has been removed, allowing the researcher to visualize and quantify capillary blood flow. The graph shows a bolus wash-in curve for the medulla and cortex of the kidney; quantification preformed by the Vevo software provides values of time to peak, as well as assessments of relative blood volume. (Untargeted MicroMarker Contrast Agents) Target-Ready MicroMarker Contrast Agents, with the appropriate ligand, are capable of binding to a variety endothelial cell surface expressions, provides quantification of the target biomarker. Insert Image #3

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High-Frequency Ultrasound
Visualization of VEGFR2 expression in subcutaneous hepatoma tumor model. Quantification from software provides values for relative expression of VEGFR2, taken as the ratio between the contrast intensity before and after the destructive pulse (red vertical band) (Target-Ready MicroMarker Contrast Agents)

Quantification of Relative Perfusion The study and quantification of relative perfusion in vivo is an important metric for both cardiovascular studies (i.e. myocardial perfusion) as well as cancer research and tumorigenesis study (i.e. tumor perfusion) and general organ perfusion. The Vevo can be used to visualize and quantify perfusion in small animal models in vivo in both 2D and 3D. Vascular architecture and structures can be visualized in tumor models and relative tumor perfusion can be quantified. For cardiovascular studies, imaging myocardial perfusion is possible along with the Vevos capabilities to perform complete cardiovascular assessments. Quantification of Biomarkers (for all types of endothelial cell markers) The expression of endothelial cell surface markers can be visualized and quantified in both 2D and 3D. For Angiogenesis Using the microbubbles conjugated to ligands targeting VEGFR2, the Vevo is able to quantify angiogenesis in vivo in small animal models. The microbubbles, and thus the angiogenesis are quantified relative to the tumor. Since micro-ultrasound is a real-time modality, molecular imaging and quantification of angiogenesis and a variety of other biological targets can be performed in minutes. The microbubble is intravascular and clears the blood stream quickly, allowing multiple molecular experiments to be performed in the same animal repeatedly allowing for longitudinal studies. Inflammation Biomarkers Inflammation can be visualized and quantified in small animal models by introducing a targeted microbubble with ligands targeting P-Selectin as a biomarker. The microbubbles bound to the inflammatory marker can be quantified. Longitudinal studies can be performed in the same animal and therapeutic efficacy can be monitored in anti-inflammation studies.

MicroMarker Kits are designed and manufactured exclusively for VisualSonics by the Bracco Group, a leading manufacturer of diagnostic contrast agents at a state-of-the art GLP/GMP facility (Geneva, Switzerland) ensuring the highest standards of manufacturing quality and reagent consistency. Link to Product section on Molecular Imaging (insert Bracco Logo and http://www.bracco.com/) see original page: http://www.visualsonics.com/products/products_micromarker.htm
Commonly used terminology: Bubble, perfusion, targeted imaging, VEGFR2, VEGF, vascular marker, endothelial cell marker, microbubble, definity, optison, micromarker, nonlinear, integrins, p-selectin, VCAM, PCAM

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High-Frequency Ultrasound Image Guided Injection


Because of its high-resolution imaging and the ability to provide real-time visualization, the Vevo can guide injections of cells, drugs, genetic material or metabolic agents into developing small animal embryos as early as embryonic day 5. Also, the system allows visualization and needle guidance into adult murine models to include but not be limited to: Cardiovascular system (myocardium or ventricles), Liver, Kidney, Placenta and the Spleen. The system can also be used for extraction procedures. Insert Video #7

Image Guided Injection - Injection into the fourth ventricle of an externalized E12 embryo using a pulled glass capillary Visualize placement of needle tip without surgical techniques: Due to the real-time capabilities of the system, interventional procedures can be performed and monitored without surgical intervention allowing for increased survival rates for longitudinal studies and decreasing risk of infection (in newborn and adult rodents) Modify embryonic phenotypes by precisely injecting into anatomy: In addition, the phenotype can be observed and quantified in vivo longitudinally Easy to learn and obtain results: The Vevo software is simple to use so that researchers can maximize their time performing postimaging analysis. User friendly offline software is available to analyze all data. In addition, several online tutorials and protocols are available to allow researchers to learn quickly, analyze their data and obtain results.

Blood Flow Analysis


The Vevo allows researchers to image arteries and veins and quantify the blood flow through them. The possibilities for this application are numerous. Imagine being able to quantify the flow in vessels such as the coronary artery of a mouse or the abdominal aorta in a rat. With our resolution you can see smaller vessels, such as the Circle of Willis in a mouse embryo. In addition, the vascularity of organs/tissues can be assessed and quantified. Pulsed Wave Doppler ultrasound is a special application of ultrasound. It measures the direction, intensity and speed of blood cells as they move through vessels. The movement of blood cells causes a change in pitch of the reflected sound waves (called the Doppler effect). The signals generated are displayed in graphs or color pictures and represent the flow of blood through the blood vessels or tissues/organs and tumors. Additionally with the Vevo 2100, we have introduced Color Doppler. This allows us to identify arteries and veins with ease and accuracy. It shows us in color, flow disturbances, so we can easily sample in the correct region, to obtain accurate quantification of the vessel you are interrogating.
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The system provides both real-time images and Doppler waveforms for measurement and analysis as well as the audio output of the flow dynamics. All can be captured as static images, audio .wav files and animated CineLoops for offline review and analysis. In addition, the measurement and statistical data can be exported to any PC or Macintosh-compatible spreadsheet or database applications for further analysis. Image in real-time the blood flow profiles of all major vessels (arteries and veins) including: renals, hepatics, splenics, carotids and aorta. Diagnose and assess the extent of vascular abnormalities such as: stenosis, thrombus, atherosclerosis and ischemia. Evaluate tumor and organ flow: Quantification tools are available in 2D and 3D.

Video 45: Aortic Outflow Tract

Video 46: Aortic Outflow Tract

Image 47: Aortic Outflow Tract

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Image 48 & 49: High Aortic Velocity and Regurgitation

Image 50 & 51: High Aortic Velocity and Regurgitation

3D Visualization
3D measurement and visualization of datasets is available in an optional 3D package from VisualSonics. When visualizing irregular structures, 3D measurement is especially valuable because it reduces the chance of ambiguous or erroneous results. The 3D package allows users to view a series of 2D images as a 3D volume and apply various techniques. In addition, the user can view and analyze any arbitrary plane and perform 3D volume measurements, such as determining volumes for orthotopic tumors in vivo.

Insert Video #8

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3D Tumor Volume Transgenic prostate tumor measuring 6.9mm3 is outlined in red, with the surrounding prostate tissue in blue, and the bladder in yellow.
Image courtesy of Robarts Research Institute, 2008.

Insert Image #9

3D Power Doppler imaging Rendered 3D Power Doppler image of an adult mouse testicle, this image shows the vascular network within this organ. Generate 3D volumes of organs/tissues: For a more complete picture of the anatomy and morphology Obtain 3D images of vessels: Evaluate vessel architecture, including stenosis Generate 3D blood flow data using Power Doppler or MicroMarker contrast agents allowing detailed vascular network to be visualized within an organ or tumor High resolution 3D imaging of the heart (using ECG gating)

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VisualSonics Inc. T.1.416.484.5000 Toll Free (North America) 1.866.416.4636 Toll Free (Europe) +800.0751.2020 E. info@visualsonics.com www.visualsonics.com

VisualSonics, Vevo, MicroMarkerTM, VevoStrainTM, DEPO, SoniGeneTM, RMVTM, EKV and Insight through In Vivo ImagingTM are trademarks of VisualSonics Inc.

High-Frequency Ultrasound Applications and Techniques