This work was supported by the National Institutes of Health (R01-EB014496). The authors would like to thank Iason Apostolakis for his contribution to the experiments.

This protocol was approved by the Institutional Animal Care and Use Committee of Columbia University. All the data acquisition and processing were performed using the Matlab environment.
1. Experimental Set-up
<ol>
	<li>Degas an ex vivo canine liver sample during 90 min. Put the liver sample in a tank filled with degassed Phosphate Buffered Solution (Figure 1). Fix the liver sample on an acoustic absorber with needles at the extremities of the liver.</li>
	<li>Insert a 64-element, 0.32 mm pitch, 2.5 MHz center frequency phased array (imaging) through a circular hole located in the center of a 93-element hemispherical array HIFU transducer (therapeutic) at 4.5 MHz center frequency, 70 mm focal depth and 1.7 mm x 0.4 mm focal size11. Align both transducers co-axially and fix the imaging transducer into the therapeutic transducer with adjustment screws.
	<ol>
		<li>Cover the HIFU transducer with a volume-controlled polyurethane membrane filled with flowing degassed water to cool it down. Mount the transducer assembly on a computer-controlled 3-D positioner.</li>
	</ol>
	</li>
	<li>Connect the HIFU transducer to a function generator sending a 25 Hz amplitude modulated sine waveform with 500 mV maximum amplitude. Connect the imaging transducer to a fully programmable ultrasound system using the software Matlab. 
	Note: A software associated with the ultrasound system and using the Matlab environment has to be installed on the computer connected to the system. A 50 dB RF amplifier and a matching network should be placed between the HIFU transducer and the function generator to respectively amplify the power and match the impedance.</li>
	<li>Create a polar grid, using Matlab, starting 50 mm from the surface of the array and 40 mm deep in the radial direction with a spatial step of 9.625 &#181;m and of 90&#176; in the azimuthal direction with 128 lines and which origin is the focus of the diverging wave. Define the source of the diverging wave 10.24 mm (half the size of the aperture) behind the surface of the array and centered in the lateral direction.
	<ol>
		<li>Create a Cartesian grid, using Matlab, starting 50 mm from the surface of the array and 40 mm deep in the axial direction with a spatial step of 9.625 &#181;m and 20 mm wide in the lateral direction with 64 lines for the plane wave. Define the source of the plane wave on the surface of the array. For each grid, compute the time from the source to each point of the grid and back to each element of the array.</li>
	</ol>
	</li>
	<li>Enter &#8220;ReconMat_DW&#8221; for diverging wave imaging or &#8220;ReconMat_PW&#8221; for plane wave imaging in the Matlab command window and press &#8220;Enter&#8221; to create a reconstruction matrix associated with a standard delay-and-sum algorithm for each grid. Apply the delay-and-sum algorithm to each vector of the standard basis and retrieve the non-zeros elements of the resulting matrix11. Allocate the non-zero elements obtained from the resulting matrix to the sparse matrix at the corresponding location. Save the reconstruction matrix on the computer hard drive. 
	Note: The diverging and the plane wave methods use two distinct reconstruction matrices.
	<ol>
		<li>Cast the reconstruction matrix to a GPU matrix. Enter &#8220;SetUpP4_2Flash_4B_streaming_DW&#8221; for diverging wave imaging or &#8220;SetUpP4_2Flash_4B_streaming_PW&#8221; for plane wave imaging in the Matlab command window and press &#8220;Enter&#8221; to create a setup file for the ultrasound channel data acquisition using the script associated with the phased array and provided by the manufacturer of the ultrasound system. Name the setup file &#8220;P4-2Flash_DivergingWave.mat&#8221; for diverging wave imaging and &#8220;P4-2Flash_PlaneWave.mat&#8221; for plane wave imaging. 
		&#160;Note: A commercial software package has to be installed on the computer to cast the reconstruction sparse matrix to a GPU matrix.</li>
	</ol>
	</li>
	<li>Synchronize the ultrasound system with the function generator using an external trigger so that high frame rate ultrasound data acquisition of the liver starts at the same time as HIFU.</li>
	<li>Open Matlab. Run the setup script &#8220;SetUpP4_2Flash_4B.m&#8221; provided by the ultrasound system manufacturer to use B-mode imaging. Name the created setup file: &#8220;P4-2Flash_4B_Bmode.mat&#8221;. Use the &#8220;VSX&#8221; command and when &#8220;Name of .mat file to process:&#8221; is prompted, enter the name of the setup file &#8220;P4-2Flash_4B_Bmode.mat&#8221;. Move both transducers and use the B-mode display that appeared on the computer screen to position them in the targeted region of the liver to ablate. Target a region approximately 1 cm under the surface of the liver to avoid high ultrasound attenuation due to absorption. Save a conventional B-mode image of the liver on the computer. 
	Note: Here we performed HIFU ablations at 11 different locations in two liver specimens by moving the transducers with the 3-D positioner for each ablation.</li>
</ol>
2. Ultrasound Data Acquisition
<ol>
	<li>Open Matlab. Use the &#8220;VSX&#8221; command and when &#8220;Name of .mat file to process:&#8221; is prompted, enter the name of the setup file &#8220;P4-2Flash_DivergingWave.mat&#8221; for diverging wave imaging or &#8220;P4-2Flash_PlaneWave.mat&#8221; for plane wave imaging. Start the HIFU and apply it during 2min to the targeted region.</li>
	<li>Acquire the RF channel data at 1,000 frames per second during 2 min using diverging waves. Alternatively, acquire the RF channel data at 1,000 frames per second during 2 min using plane waves.</li>
	<li>Transfer the data to a host computer every 200 frames via a PCI express cable. Alternatively, for real-time streaming, acquire the RF channel data at 167 frames per second during 2 min using plane waves and transfer the data to a host computer every 2 frames. 
	Note: The imaging methods with set of 200 frames provides high temporal resolution within each set but create gaps between each set and is appropriate for off-line processing. The imaging method at 167 fps has a lower temporal resolution but does not create any gaps across the entire ablation time and is appropriate for real-time streaming.</li>
	<li>Cast the RF channel data matrix to a single precision GPU matrix with Matlab. Multiply the RF channel data matrix by the reconstruction matrix to obtain the reconstructed RF data11.</li>
</ol>
3. Displacement Imaging
<ol>
	<li>Create a 6th order Butterworth low pass filter at 4 MHz cutoff frequency using the DSP System Toolbox of Matlab. Apply this low pass filter to the reconstructed RF data to filter out the 4.5 MHz HIFU component.</li>
	<li>Estimate the axial displacement between consecutive frames using 1-D normalized cross-correlation with a 3.1mm-window length and 90% overlap.</li>
	<li>Create a 6th order Butterworth low pass filter at 100 Hz cutoff frequency using the DSP System Toolbox of Matlab. Apply this low pass filter to the temporal displacement data using Matlab to retrieve the 50 Hz-oscillatory frequency component.</li>
	<li>Define a region of interest (ROI) as the focal region at -6 dB (1.7 x 0.4 mm in water) and located 70 mm away from the transducer surface. Extract the displacement data in this ROI. Estimate the displacement signal-to-noise ratio (SNRd) at the focal region after 2 min of ablation as the ratio between the mean displacement and the standard deviation of the displacement in the ROI.</li>
	<li>Extract the 50 Hz temporal displacement signal at the focus from the displacement matrix data. Convert the temporal displacement signal at the focus into audible sound using Matlab.</li>
</ol>

<ol>
	<li>Al-Bataineh, O., Jenne, J., Huber, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clinical+and+future+applications+of+high+intensity+focused+ultrasound+in+cancer.">Clinical and future applications of high intensity focused ultrasound in cancer.</a> Cancer Treat Rev. 38, 346-353 (2012).</li><li>Dewhirst, M. W., Viglianti, B. L., Lora-Michiels, M., Hanson, M., Hoopes, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Basic+principles+of+thermal+dosimetry+and+thermal+thresholds+for+tissue+damage+from+hyperthermia.">Basic principles of thermal dosimetry and thermal thresholds for tissue damage from hyperthermia.</a> Int J Hyperthermia. 19, 267-294 (2003).</li><li>Napoli, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MR-guided+high-intensity+focused+ultrasound:+current+status+of+an+emerging+technology.">MR-guided high-intensity focused ultrasound: current status of an emerging technology.</a> Cardiovasc Intervent Radiol. 36, 1190-1203 (2013).</li><li>Gudur, M. S., Kumon, R. E., Zhou, Y., Deng, C. 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The authors declare that they have no competing financial interests.

Real-time monitoring of HIFU lesions is important to ensure proper and efficient lesion delivery. As the lesion forms, the tissue stiffens and its motion amplitude under excitation decreases. Applying HIFU in a region of the tissue results in an acoustic radiation force that induces tissue displacement. The relative change in displacement is a surrogate of relative change in tissue stiffness. This technique offers the advantage of monitoring HIFU lesion without stopping the treatment in contrast to other ultrasound based...

High Intensity Focused Ultrasound (HIFU) is a technique that generates temperature elevation at the focal region and can be used to ablate cancerous tissue 1. Temperature elevation at the focus causes thermal lesions in the tissue 2. In order to avoid overtreating a region and to reduce treatment duration, it is imperative to reliably monitor the ablation. Magnetic resonance-guided focused ultrasound (MRgFUS) is the main technique used in clinic to guide and monitor HIFU treatment 3. MRI provides high spatial resolution images of the treated region with tissue displacement or thermal dose but has a frame rate of 0.1-1 Hz and is costly. Several ultrasound-based techniques such as B-mode imaging 4, passive acoustic mapping 5, shear wave imaging 6 and acoustic radiation force impulse 7 have been developed to guide and monitor thermal ablation. However, B-mode imaging and passive acoustic mapping do not provide imaging of mechanical properties of the ablated region which is useful to the operator to improve lesion delivery. 
Shear wave imaging and acoustic radiation force impulse can both characterize the elasticity of the tissue by measuring acoustic radiation force-induced displacements 7,8. However, in both methods, the HIFU treatment is typically interrupted to monitor the ablation. Our group has developed a technique called Harmonic Motion Imaging for Focus Ultrasound (HMIFU) which can monitor the HIFU treatment with ultrasound without stopping the ablation9,10. Briefly, a HIFU transducer sends an amplitude-modulated wave to the region to ablate while simultaneously generating an oscillatory motion in the focal region. A co-axially aligned ultrasound transducer is used to image this oscillation. The magnitude of the induced motion is related to the stiffness of the tissue.
To ensure proper lesion delivery, the temporal resolution of real-time monitoring is of key interest in ablation guidance. Recently, our group has shown real-time streaming of displacement at a frame rate up to 15 Hz, imaged with diverging waves in a narrow field of view and using a fast image reconstruction method 11. Several beamforming techniques can be used to image the displacements. A large field of view can be obtained with diverging wave imaging by changing the delay profile but the axial direction is not aligned with the HIFU beam on the lateral regions and the wave is attenuated due to geometric spreading in the lateral direction, which can affect the quality of the displacement estimation. In contrast, the lateral field of view for plane wave is upper bounded by the active aperture but the axial direction is aligned with the HIFU beam at the focus and there is no geometric spreading in the lateral direction. Depending on the type of application, one or the other imaging method can be selected. The objectives of this protocol are to show how plane wave imaging can provide real-time streaming of displacements images using HMIFU during ablation and to compare the quality of the motion estimation between diverging and plane wave imaging.

<table><tbody><tr><td>P4-2 Phased array</td><td>ATL</td><td></td><td></td></tr><tr><td>H-178 HIFU transducer</td><td>Sonic Concepts</td><td></td><td></td></tr><tr><td>3-D positioner</td><td>Velmex Inc.</td><td></td><td></td></tr><tr><td>AT33522A function generator</td><td>Agilent Technologies</td><td></td><td></td></tr><tr><td>V-1 ultrasound system</td><td>Verasonics</td><td></td><td></td></tr><tr><td>3100L RF amplifier</td><td>ENI</td><td></td><td></td></tr><tr><td>Matching network</td><td>Sonic Concepts</td><td></td><td></td></tr><tr><td>Degasing system</td><td>Sonic Concepts</td><td></td><td></td></tr><tr><td>Programming software</td><td>Matlab</td><td></td><td></td></tr><tr><td>Jacket software package</td><td>Accelereyes</td><td></td><td></td></tr></tbody></table>

real-time monitoring of high intensity focused ultrasound (hifu) ablation of in vitro canine livers using harmonic motion imaging for focused ultrasound (hmifu)

Harmonic Motion Imaging for Focused Ultrasound (HMIFU) is a technique that can perform and monitor high-intensity focused ultrasound (HIFU) ablation. An oscillatory motion is generated at the focus of a 93-element and 4.5 MHz center frequency HIFU transducer by applying a 25 Hz amplitude-modulated signal using a function generator. A 64-element and 2.5 MHz imaging transducer with 68kPa peak pressure is confocally placed at the center of the HIFU transducer to acquire the radio-frequency (RF) channel data. In this protocol, real-time monitoring of thermal ablation using HIFU with an acoustic power of 7 W on canine livers in vitro is described. HIFU treatment is applied on the tissue during 2 min and the ablated region is imaged in real-time using diverging or plane wave imaging up to 1,000 frames/second. The matrix of RF channel data is multiplied by a sparse matrix for image reconstruction. The reconstructed field of view is of 90&#176; for diverging wave and 20 mm for plane wave imaging and the data are sampled at 80 MHz. The reconstruction is performed on a Graphical Processing Unit (GPU) in order to image in real-time at a 4.5 display frame rate. 1-D normalized cross-correlation of the reconstructed RF data is used to estimate axial displacements in the focal region. The magnitude of the peak-to-peak displacement at the focal depth decreases during the thermal ablation which denotes stiffening of the tissue due to the formation of a lesion. The displacement signal-to-noise ratio (SNRd) at the focal area for plane wave was 1.4 times higher than for diverging wave showing that plane wave imaging appears to produce better displacement maps quality for HMIFU than diverging wave imaging.

Real-time streaming of HMI displacement during HIFU ablation can be obtained using diverging and plane wave imaging. Figure 2 is a video screen capture showing real-time display of acoustic radiation force induced displacement using plane wave imaging in in vitro canine livers during HIFU ablation. The displacements are streamed in real-time on the computer screen at a display frame rate of 4.5 Hz. Positive displacements are shown in red and negative displacements in blue. Lesions were successfu...

Watch this Scientific Journal Video about Real-time Monitoring of High Intensity Focused Ultrasound HIFU Ablation of In Vitro Canine Livers Using Harmonic Motion Imaging for Focused Ultrasound HMIFU a

Real-time Monitoring of High Intensity Focused Ultrasound HIFU Ablation of In Vitro Canine Livers Using Harmonic Motion Imaging for Focused Ultrasound HMIFU

This article describes real-time monitoring of HIFU ablation in canine liver with high frame rate ultrasound imaging using diverging and plane wave imaging. Harmonic Motion Imaging for Focused Ultrasound is used to image the decrease of acoustic radiation force induced displacement in the ablated region.

Real-time Monitoring of High Intensity Focused Ultrasound (HIFU) Ablation of In Vitro Canine Livers Using Harmonic Motion Imaging for Focused Ultrasound (HMIFU)

Harmonic Motion Imaging for Focused Ultrasound (HMIFU) is a technique that can perform and monitor high-intensity focused ultrasound (HIFU) ablation. An ...

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Research

JoVE Journal

Bioengineering

9.9K Views.  Columbia University. This article describes real-time monitoring of HIFU ablation in canine liver with high frame rate ultrasound imaging using diverging and plane wave imaging. Harmonic Motion Imaging for Focused Ultrasound is used to image the decrease of acoustic radiation force induced displacement in the ablated region.

Video: Real-time Monitoring of High Intensity Focused Ultrasound HIFU Ablation of In Vitro Canine Livers Using Harmonic Motion Imaging for Focused Ultrasound HMIFU

Evaluation of the Feasibility, Safety, and Accuracy of an Intraoperative High-intensity Focused Ultrasound Device for Treating Liver Metastases

Today, the only potentially curative option in patients with colorectal liver metastases is surgery. However, liver resection is feasible in less than 20% of patients. Surgery has been widely used in association with radiofrequency, cryotherapy, or microwaves to expand the number of treatments performed with a curative intent. Nevertheless, several limitations have been documented when using these techniques (i.e., a traumatic puncture of the parenchyma, a limited size of lesions, and an inability to monitor the treatment in real-time).High-intensity focused ultrasound (HIFU) technology can achieve precise ablations of biological tissues without incisions or radiation. Current HIFU devices are based on an extracorporeal approach with limited access to the liver. We have developed a HIFU device designed for intraoperative use. The use of a toroidal transducer enables an ablation rate (10 cm3&#183;min-1) higher than any other treatment and is independent of perfusion.
The feasibility, safety, and accuracy of intraoperative HIFU ablation were evaluated during an ablate-and-resect prospective study. This clinical phase I and IIa study was performed in patients undergoing hepatectomy for liver metastases. The HIFU treatment was performed on healthy tissue scheduled for resection.Liver metastases measuring less than 20 mm will be targeted during phase IIb (currently ongoing). This set-up allows the real-time evaluation of HIFU ablation while protecting participating patients from any adverse effects related to this new technique.
Fifteen patients were included in phase I&#8211;IIa and 30 HIFU ablations were safely created within 40 s and with a precision of 1&#8211;2 mm. The average dimensions of the HIFU ablations were 27.5 x 21.0 mm2, corresponding to a volume of approximately 7.5 cm3. The aim of the ongoing phase IIb is to ablate metastases of less than 20 mm in diameter with a 5 mm margin.

Here, we present an ablate-and-resect prospective study to evaluate the feasibility, safety, and accuracy of intraoperative high-intensity focused ultrasound ablation in patients undergoing hepatectomy for liver metastases.

Today, the only potentially curative option in patients with colorectal liver metastases is surgery. However, liver resection is feasible in less than 20% ...

Cancer Research

Fetal Mouse Cardiovascular Imaging Using a High-frequency Ultrasound (30/45MHZ) System

Congenital heart defects (CHDs) are the most common cause of childhood morbidity and early mortality. Prenatal detection&#160;of the underlying molecular mechanisms of CHDs is&#160;crucial for inventing new preventive and therapeutic strategies. Mutant mouse models are powerful tools to discover new&#160;mechanisms&#160;and environmental stress modifiers that drive cardiac development and their potential&#160;alteration in CHDs. However, efforts to establish the causality of these putative contributors have been limited to histological&#160;and molecular studies&#160;in non-survival animal experiments, in which&#160;monitoring the key physiological and hemodynamic parameters is&#160;often absent. Live imaging technology has become an essential tool to establish the&#160;etiology of CHDs. In particular,&#160;ultrasound imaging can be used prenatally without surgically exposing the fetuses,&#160;allowing maintaining&#160;their baseline physiology while monitoring the impact of environmental stress&#160;on the hemodynamic and structural aspects of cardiac chamber development. Herein, we use the High-Frequency Ultrasound (30/45)&#160;system to examine the cardiovascular system in fetal mice at E18.5 in utero at the baseline and in response to prenatal hypoxia exposure. We demonstrate the feasibility of the system to measure cardiac chamber size, morphology, ventricular function, fetal heart rate, and umbilical artery flow indices, and their alterations in fetal mice exposed to systemic chronic hypoxia in utero in real time.

Fetal Mouse Cardiovascular Imaging Using a High-frequency Ultrasound 30/45MHZ System

High-frequency ultrasound imaging of the fetal mouse has improved imaging resolution and can provide precise non-invasive characterization of cardiac development and structural defects. The protocol outlined herein is designed to perform real-time fetal mice echocardiography in vivo.

Congenital heart defects (CHDs) are the most common cause of childhood morbidity and early mortality. Prenatal detection&#160;of the underlying molecular ...

Developmental Biology

Controllable Nucleation of Cavitation from Plasmonic Gold Nanoparticles for Enhancing High Intensity Focused Ultrasound Applications

In this study, plasmonic gold nanoparticles were simultaneously exposed to pulsed near-infrared laser light and high intensity focused ultrasound (HIFU) for the controllable nucleation of cavitation in tissue-mimicking gel phantoms. This in vitro protocol was developed to demonstrate the feasibility of this approach, for both enhancement of imaging and therapeutic applications for cancer. The same apparatus can be used for both imaging and therapeutic applications by varying the exposure duration of the HIFU system. For short duration exposures (10 &#181;s), broadband acoustic emissions were generated through the controlled nucleation of inertial cavitation around the gold nanoparticles. These emissions provide direct localization of nanoparticles. For future applications, these particles may be functionalized with molecular-targeting antibodies (e.g. anti-HER2 for breast cancer) and can provide precise localization of cancerous regions, complementing routine diagnostic ultrasound imaging. For continuous wave (CW) exposures, the cavitation activity was used to increase the localized heating from the HIFU exposures resulting in larger thermal damage in the gel phantoms. The acoustic emissions generated from inertial cavitation activity during these CW exposures was monitored using a passive cavitation detection (PCD) system to provide feedback of cavitation activity. Increased localized heating was only achieved through the unique combination of nanoparticles, laser light and HIFU. Further validation of this technique in pre-clinical models of cancer is necessary.

This protocol demonstrates the controllable nucleation of cavitation in gel phantoms, through simultaneous exposure to both near-infrared pulsed laser light and high intensity focused ultrasound (HIFU). The cavitation activity can then be used for enhancing imaging and/or therapeutic uses of HIFU.

In this study, plasmonic gold nanoparticles were simultaneously exposed to pulsed near-infrared laser light and high intensity focused ultrasound (HIFU) ...

Engineering

Multiplexing Focused Ultrasound Stimulation with Fluorescence Microscopy

By focusing low-intensity ultrasound pulses that penetrate soft tissues, LIPUS represents a promising biomedical technology to remotely and safely manipulate neural firing, hormonal secretion and genetically-reprogrammed cells. However, the translation of this technology for medical applications is currently hampered by a lack of biophysical mechanisms by which targeted tissues sense and respond to LIPUS. A suitable approach to identify these mechanisms would be to use optical biosensors in combination with LIPUS to determine underlying signaling pathways. However, implementing LIPUS to a fluorescence microscope may introduce undesired mechanical artefacts due to the presence of physical interfaces that reflect, absorb and refract acoustic waves. This article presents a step-by-step procedure to incorporate LIPUS to commercially-available upright epi-fluorescence microscopes while minimizing the influence of physical interfaces along the acoustic path. A simple procedure is described to operate a single-element ultrasound transducer and to bring the focal zone of the transducer into the objective focal point. The use of LIPUS is illustrated with an example of LIPUS-induced calcium transients in cultured human glioblastoma cells measured using calcium imaging.

Low-Intensity Pulsed Ultrasound Stimulation (LIPUS) is a modality for non-invasive mechanical stimulation of endogenous or engineered cells with high spatial and temporal resolution. This article describes how to implement LIPUS to an epi-fluorescence microscope and how to minimize acoustic impedance mismatch along the ultrasound path to prevent unwanted mechanical artefacts.

By focusing low-intensity ultrasound pulses that penetrate soft tissues, LIPUS represents a promising biomedical technology to remotely and safely ...

Evaluating Targeting Accuracy in the Focal Plane for an Ultrasound-guided High-intensity Focused Ultrasound Phased-array System

Phased arrays are increasingly used as high-intensity focused ultrasound (HIFU) transducers in the existing extracorporeal ultrasound-guided HIFU (USgHIFU) systems. The HIFU transducers in such systems are usually spherical in shape with a central hole where a US imaging probe is mounted and can be rotated. The image on the plane of treatment can be reconstructed through the image sequence acquired during the rotation of the probe. Therefore, the treatment plan can be made on the reconstructed images. In order to evaluate the targeting accuracy in the focal plane of such systems, the protocol of a method using a bovine muscle and marker-embedded phantom is described. In the phantom, four solid balls at the corners of a square resin model serve as the reference markers in the reconstructed image. The target should be moved so that both its center and the center of the square model can coincide according to their relative positions in the reconstructed image. Swine muscle with a thickness of about 30 mm is placed above the phantom to mimic the beam path in clinical settings. After sonication, the treatment plane in the phantom is scanned and the boundary of the associated lesion is extracted from the scanned image. The targeting accuracy can be evaluated by measuring the distance between the centers of target and lesion, as well as three derivative parameters. This method cannot only evaluate the targeting accuracy of the target consisting of multiple focal spots rather than a single focal spot in a clinically relevant beam path of the USgHIFU phased-array system, but it can be also used in the preclinical evaluation or regular maintenance of USgHIFU systems configured with phased-array or self-focused HIFU transducer.

This study describes a protocol to evaluate the targeting accuracy in the focal plane of an ultrasound-guided high-intensity focused ultrasound phased-array system.

Phased arrays are increasingly used as high-intensity focused ultrasound (HIFU) transducers in the existing extracorporeal ultrasound-guided HIFU ...

Magnetic Resonance-Guided High Intensity Focused Ultrasound Generated Hyperthermia: A Feasible Treatment Method in a Murine Rhabdomyosarcoma Model

Magnetic resonance-guided high intensity focused ultrasound (MRgHIFU) is an established method for producing localized hyperthermia. Given the real-time imaging and acoustic energy modulation, this modality enables precise temperature control within a defined area. Many thermal applications are being explored with this noninvasive, nonionizing technology, such as hyperthermia generation, to release drugs from thermosensitive liposomal carriers. These drugs can include chemotherapies such as doxorubicin, for which targeted release is desired due to the dose-limiting systemic side effects, namely cardiotoxicity. Doxorubicin is a mainstay for treating a variety of malignant tumors and is commonly used in relapsed or recurrent rhabdomyosarcoma (RMS). RMS is the most common solid soft tissue extracranial tumor in children and young adults. Despite aggressive, multimodal therapy, RMS survival rates have remained the same for the past 30 years. To explore a solution for addressing this unmet need, an experimental protocol was developed to evaluate the release of thermosensitive liposomal doxorubicin (TLD) in an immunocompetent, syngeneic RMS mouse model using MRgHIFU as the source of hyperthermia for drug release.

Presented here is a protocol to use controlled hyperthermia, generated by magnetic resonance-guided high intensity focused ultrasound, to trigger drug release from temperature-sensitive liposomes in a rhabdomyosarcoma mouse model.

Magnetic resonance-guided high intensity focused ultrasound (MRgHIFU) is an established method for producing localized hyperthermia. Given the real-time ...

Real-Time Intravital Multiphoton Microscopy to Visualize Focused Ultrasound and Microbubble Treatments to Increase Blood-Brain Barrier Permeability

The blood-brain barrier (BBB) is a key challenge for the successful delivery of drugs to the brain. Ultrasound exposure in the presence of microbubbles has emerged as an effective method to transiently and locally increase the permeability of the BBB, facilitating para- and transcellular transport of drugs across the BBB. Imaging the vasculature during ultrasound-microbubble treatment will provide valuable and novel insights on the mechanisms and dynamics of ultrasound-microbubble treatments in the brain.
Here, we present an experimental procedure for intravital multiphoton microscopy using a cranial window aligned with a ring transducer and a 20x objective lens. This set-up enables high spatial and temporal resolution imaging of the brain during ultrasound-microbubble treatments. Optical access to the brain is obtained via an open-skull cranial window. Briefly, a 3-4 mm diameter piece of the skull is removed, and the exposed area of the brain is sealed with a glass coverslip. A 0.82 MHz ring transducer, which is attached to a second glass coverslip, is mounted on top. Agarose (1% w/v) is used between the coverslip of the transducer and the coverslip covering the cranial window to prevent air bubbles, which impede ultrasound propagation. When sterile surgery procedures and anti-inflammatory measures are taken, ultrasound-microbubble treatments and imaging sessions can be performed repeatedly over several weeks. Fluorescent dextran conjugates are injected intravenously to visualize the vasculature and quantify ultrasound-microbubble induced effects (e.g., leakage kinetics, vascular changes). This paper describes the cranial window placement, ring transducer placement, imaging procedure, common troubleshooting steps, as well as advantages and limitations of the method.

This protocol describes the surgical and technical procedures that enable real-time in vivo multiphoton fluorescence imaging of the rodent brain during focused ultrasound and microbubble treatments to increase blood-brain barrier permeability.

The blood-brain barrier (BBB) is a key challenge for the successful delivery of drugs to the brain. Ultrasound exposure in the presence of microbubbles ...

A High-Throughput Image-Guided Stereotactic Neuronavigation and Focused Ultrasound System for Blood-Brain Barrier Opening in Rodents

The blood-brain barrier (BBB) has been a major hurdle for the treatment of various brain diseases. Endothelial cells, connected by tight junctions, form a physiological barrier preventing large molecules (&#62;500 Da) from entering the brain tissue. Microbubble-mediated focused ultrasound (FUS) can be used to induce a transient local BBB opening, allowing larger drugs to enter the brain parenchyma.
In addition to large-scale clinical devices for clinical translation, preclinical research for therapy response assessment of drug candidates requires dedicated small animal ultrasound setups for targeted BBB opening. Preferably, these systems allow high-throughput workflows with both high-spatial precision as well as integrated cavitation monitoring, while still being cost effective in both initial investment and running costs.
Here, we present a bioluminescence and X-ray guided stereotactic small animal FUS system that is based on commercially available components and fulfills the aforementioned requirements. A particular emphasis has been placed on a high degree of automation facilitating the challenges typically encountered in high-volume preclinical drug evaluation studies. Examples of these challenges are the need for standardization in order to ensure data reproducibility, reduce intra-group variability, reduce sample size and thus comply with ethical requirements and decrease unnecessary workload. The proposed BBB system has been validated in the scope of BBB opening facilitated drug delivery trials on patient-derived xenograft models of glioblastoma multiforme and diffuse midline glioma.

The blood-brain barrier (BBB) can be temporarily disrupted with microbubble-mediated focused ultrasound (FUS). Here, we describe a step-by-step protocol for high-throughput BBB opening in vivo using a modular FUS system accessible for non-ultrasound experts.

The blood-brain barrier (BBB) has been a major hurdle for the treatment of various brain diseases. Endothelial cells, connected by tight junctions, form a ...

Neuroscience

Integrated Protocol for FUN and Fiber Photometry in Free-Moving Mice

Simultaneous Focused Ultrasound Neuromodulation and Fiber Photometry Recording in Free-Moving Mouse

Focused ultrasound neuromodulation (FUN) represents a promising approach for non-invasive perturbation of neuronal circuits at deep brain regions. It is compatible with most of the existing modalities for monitoring brain functions in vivo. Integration with brain function recording modalities not only enables us to address orders and disorders of specific brain functions with closed-loop feedback but also provides us with mechanistic insights about FUN itself. Here, we provide a modified, simple, dependable, and robust protocol for the simultaneous application of FUN and fiber photometry GCaMP6s fluorescence recording in free-moving mice. This involves the fabrication of a well-sized single transducer and its temporary placement on the mice, along with the secure fixation of a fiber optical implant to facilitate the smooth passage of the transducer. The combination of FUN and fiber photometry provides for the optical recording of neural circuitry responses upon FUN in real-time in deep brain regions. To demonstrate the efficiency of this protocol, Thy1-GCaMP6s mice were used as an example to record the neuroactivity in the anterior thalamic nucleus during FUN while the mice are freely moving. We believe that this protocol can promote the widespread use of FUN in both the neuroscience field and the biomedical ultrasound field.

Author Spotlight: Integration of Fiber Photometry and Focused Ultrasound Neuromodulation for Investigating Neural Modulation in Freely Moving Mice

The protocol includes transducer manufacturing, parameters reporting, surgical procedure, and signal recording for the entire operational workflow of concurrent focused ultrasound neuromodulation and fiber photometry recording in free-moving mice.

Focused ultrasound neuromodulation (FUN) represents a promising approach for non-invasive perturbation of neuronal circuits at deep brain regions. It is ...

Ultrafast Doppler Ultrasound for Hepatic Microvasculature Analysis in Mice

Imaging and Quantification of the Hepatic Vasculature of Mice Using Ultrafast Doppler Ultrasound

Non-invasive in vivo imaging of the vasculature is a powerful tool for studying disease mechanisms in rodents. To achieve high sensitivity imaging of the microvasculature using Doppler ultrasound methods, imaging modalities employing the concept of ultrafast imaging are preferred. By increasing the frame rate of the ultrasound scanner to thousands of frames per second, it becomes possible to improve the sensitivity of the blood flow down to 2 mm/s and to obtain functional information about the microcirculation in comparison to a sensitivity of around 1 cm/s in conventional Doppler modes. While Ultrafast Doppler ultrasound (UFUS) imaging has become adopted in neuroscience, where it can capture brain activity through neurovascular coupling, it presents greater challenges when imaging the vasculature of abdominal organs due to larger motions linked to breathing. The liver, positioned anatomically under the diaphragm, is particularly susceptible to out-of-plane movement and oscillating respiratory motion. These artifacts not only adversely affect Doppler imaging but also complicate the anatomical analysis of vascular structures and the computation of vascular parameters. Here, we present a qualitative and quantitative imaging analysis of the hepatic vasculature in mice by UFUS. We identify major anatomical vascular structures and provide graphical illustrations of the hepatic macroscopical anatomy, comparing it to an in-depth anatomical assessment of the hepatic vasculature based on Doppler readouts. Additionally, we have developed a quantification protocol for robust measurements of hepatic blood volume of the microvasculature over time. To contemplate further research, qualitative vascular analysis provides a comprehensive overview and suggests a standardized terminology for researchers working with mouse models of liver disease. Furthermore, it offers the opportunity to apply ultrasound as a non-invasive complementary method to inspect hepatic vascular defects in vivo and measure functional microvascular alterations deep within the organ before unraveling blood vessel anomalies at the micron scale levels using ex vivo staining on tissue sections.

Ultrafast Doppler ultrasound (UFUS) with high spatial resolution (100 &#956;m) and sensitivity represents a suitable noninvasive imaging modality for obtaining a comprehensive qualitative overview of the hepatic vasculature. UFUS also facilitates quantitative measurements of the microvasculature, aiming to enhance our understanding of vascular disease mechanisms.

Non-invasive in vivo imaging of the vasculature is a powerful tool for studying disease mechanisms in rodents. To achieve high sensitivity imaging of the ...

Real-time Monitoring of High Intensity Focused Ultrasound (HIFU) Ablation of In Vitro Canine Livers Using Harmonic Motion Imaging for Focused Ultrasound (HMIFU)

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