Image-Guided Measurement of Radiation Force Induced by Focused Ultrasound Beams.

The radiation force balance (RFB) is a widely used method for measuring acoustic power output of ultrasonic transducers. The reflecting cone target is attractive due to its simplicity and long-term stability, at a reasonable cost. However, accurate measurements using this method depend on the alignment between the ultrasound beam and cone axes, especially for highly focused beams utilized in therapeutic applications. With the advent of dual-mode ultrasound arrays (DMUAs) for imaging and therapy, image-guided measurements of acoustic output using the RFB method can be used to improve measurement accuracy. In this article, we describe an image-guided RFB measurement of focused DMUA beams using a widely used commercial instrument. DMUA imaging is used to optimize the alignment between the acoustic beam and reflecting cone axes. In addition to image-guided alignment, DMUA echo data is used to track the displacement of the cone, which provides an auxiliary measurement of acoustic power. Experimental results using a DMUA prototype with [Formula: see text] shows that 1-2 mm of misalignment can result in 5%-14% error in the measured acoustic power. In addition to the use of B-mode image guidance for improving measurement accuracy, we present preliminary results demonstrating the benefit of displacement tracking using real-time DMUA imaging during the application of (sub)therapeutic focused beams. Displacement tracking provides a direct measurement of the radiation force with high sensitivity and follows the expected dependence on changes in amplitude and duty cycle (DC) of the focused ultrasound (FUS) beam. This could lead to simpler, more reliable methods for measuring acoustic power based on the radiation force principle. Combined with appropriate computational modeling, the direct measurement of acoustic radiation force could lead to reliable dosimetry in situ in emerging applications such as transcranial FUS (tFUS) therapies.

Precision Targeted Ablation of Fine Neurovascular Structures In Vivo Using Dual-mode Ultrasound Arrays.

Carotid bodies (CBs) are chemoreceptors that monitor and register changes in the blood, including the levels of oxygen, carbon dioxide, and pH, and regulate breathing. Enhanced activity of CBs was shown to correlate with a significant elevation in the blood pressure of patients with hypertension. CB removal or denervation were previously shown to reduce hypertension. Here we demonstrate the feasibility of a dual-mode ultrasound array (DMUA) system to safely ablate the CB in vivo in a spontaneously hypertensive rat (SHR) model of hypertension. DMUA imaging was used for guiding and monitoring focused ultrasound (FUS) energy delivered to the target region. In particular, 3D imaging was used to identify the carotid bifurcation for targeting the CBs. Intermittent, high frame rate imaging during image-guided FUS (IgFUS) delivery was used for monitoring the lesion formation. DMUA imaging provided feedback for closed-loop control (CLC) of the lesion formation process to avoid overexposure. The procedure was tolerated well in over 100 SHR and normotensive rats that received unilateral and bilateral treatments. The measured mean arterial pressure (MAP) exhibited measurable deviation from baseline 2-4 weeks post IgFUS treatment. The results suggest that the direct unilateral FUS treatment of the CB might be sufficient to reduce the blood pressure in hypertensive rats and justify further investigation in large animals and eventually in human patients.

The Optimization of Transcostal Phased Array Refocusing Using the Semidefinite Relaxation Method.

Tumors in organs partially obscured by the rib cage represent a challenge for high-intensity focused ultrasound (HIFU) therapy. The ribs distort the HIFU beams in a manner that reduces the focusing gain at the target, which could result in treatment-limiting collateral damage. In fact, skin burns are a common complication during the ablation of hepatic tumors. This problem can be addressed by employing optimal refocusing algorithms that are designed to achieve a specified focusing gain at the target while controlling the exposure to the ribs in the path of the HIFU beam. However, previously proposed optimal refocusing algorithms did not allow for the controlled transmission through the ribs. In this article, we introduce a new approach for refocusing that can more efficiently steer power toward the target while limiting the power deposition on the ribs. The approach utilizes the semidefinite relaxation (SDR) technique to approximate the original (nonconvex) optimization problem. An important advantage of the SDR-based method over previously proposed optimization methods is the control of the side lobes in the focal plane. The method also allows for specifying an acceptable level of exposure to the ribs. Simulation results using a 1-MHz spherical concave phased array focused on an inhomogeneous medium are presented to demonstrate the performance of the SDR refocusing approach. A finite-difference time-domain propagation model was used to model the propagation in the inhomogeneous tissues, including the ribs. Temperature simulations based on the inhomogeneous transient bioheat transfer equation (tBHTE) demonstrate the significance of the improvements in the focusing gain when using the limited power deposition (LPD) method. The results also demonstrate that the LPD method yields well-behaved array excitation vectors, realizable by currently existing drivers.

Nonlinear Imaging of Microbubble Contrast Agent Using the Volterra Filter: In Vivo Results.

A nonlinear filtering approach to imaging the dynamics of microbubble ultrasound contrast agents (UCAs) in microvessels is presented. The approach is based on the adaptive third-order Volterra filter (TVF), which separates the linear, quadratic, and cubic components from beamformed pulse-echo ultrasound data. The TVF captures polynomial nonlinearities utilizing the full spectral components of the echo data and not from prespecified bands, e.g., second or third harmonics. This allows for imaging using broadband pulse transmission to preserve the axial resolution and the SNR. In this paper, we present the results from imaging the UCA activity in a 200- [Formula: see text] cellulose tube embedded in a tissue-mimicking phantom using a linear array diagnostic probe. The contrast enhancement was quantified by computing the contrast-to-tissue ratio (CTR) for the different imaging components, i.e., B-mode, pulse inversion (PI), and the TVF components. The temporal mean and standard deviation of the CTR values were computed for all frames in a given data set. Quadratic and cubic images, referred to as QB-mode and CB-mode, produced higher mean CTR values than B-mode, which showed improved sensitivity. Compared with PI, they produced similar or higher mean CTR values with greater spatial specificity. We also report in vivo results from imaging UCA activity in an implanted LNCaP tumor with heterogeneous perfusion. The temporal means and standard deviations of the echogenicity were evaluated in small regions with different perfusion levels in the presence and absence of UCA. The in vivo measurements behaved consistently with the corresponding calculations obtained under microflow conditions in vitro. Specifically, the nonlinear VF components produced larger increases in the temporal mean and standard deviation values compared with B-mode in regions with low to relatively high perfusion. These results showed that polynomial filters such as the TVF can provide an important tool for imaging UCA activity in regions with heterogeneous perfusion as is the case in some tumors and ischemic tissues.

In Vivo application and localization of transcranial focused ultrasound using dual-mode ultrasound arrays.

Focused ultrasound (FUS) has been proposed for a variety of transcranial applications, including neuromodulation, tumor ablation, and blood-brain barrier opening. A flurry of activity in recent years has generated encouraging results demonstrating its feasibility in these and other applications. To date, monitoring of FUS beams has been primarily accomplished using MR guidance, where both MR thermography and elastography have been used. The recent introduction of real-time dual-mode ultrasound array (DMUA) systems offers a new paradigm in transcranial focusing. In this paper, we present first experimental results of ultrasound-guided transcranial FUS (tFUS) application in a rodent brain, both ex vivo and in vivo. DMUA imaging is used for visualization of the treatment region for placement of the focal spot within the brain. This includes the detection and localization of pulsating blood vessels at or near the target point(s). In addition, DMUA imaging is used to monitor and localize the FUS-tissue interactions in real time. In particular, a concave (40 mm radius of curvature), 32-element, 3.5-MHz DMUA prototype was used for imaging and tFUS application in ex vivo and in vivo rat models. The ex vivo experiments were used to evaluate the point spread function of the transcranial DMUA imaging at various points within the brain. In addition, DMUA-based transcranial ultrasound thermography measurements were compared with thermocouple measurements of subtherapeutic tFUS heating in rat brain ex vivo. The ex vivo setting was also used to demonstrate the capability of DMUA to produce localized thermal lesions. The in vivo experiments were designed to demonstrate the ability of the DMUA to apply, monitor, and localize subtherapeutic tFUS patterns that could be beneficial in transient blood-brain barrier opening. The results show that although the DMUA focus is degraded due to the propagation through the skull, it still produces localized heating effects within a sub-millimeter volume. In addition, DMUA transcranial echo data from brain tissue allow for reliable estimation of temperature change.

Anatomical-based model for simulation of HIFU-induced lesions in atherosclerotic plaques.

PURPOSE: The aim of this study was to simulate the effect of high intensity focused ultrasound (HIFU) in non-homogenous medium for targeting atherosclerotic plaques in vivo. MATERIALS AND METHODS: A finite-difference time-domain heterogeneous model for acoustic and thermal tissue response in the treatment region was derived from ultrasound images of the treatment region. A 3.5 MHz dual mode ultrasound array suitable for targeting peripheral vessels was used. The array has a lateral and elevation focus at 40 mm with fenestration in its centre through which a 7.5 MHz diagnostic transducer can be placed. Two cases were simulated where seven adjacent HIFU shots (∼5000 W/cm2, 2-s exposure time) were targeted on the plaque tissue within the femoral artery. The transient bioheat equation with a convective term to account for blood flow was used to predict the thermal dose. The results of the simulation model were then validated against the histology data. RESULTS: The simulation model predicted the HIFU-induced damage for both cases, and correlated well with the histology data. For the first case thermal damage was detected within the targeted plaque, while for the second case thermal damage was detected in the pre-focal region. CONCLUSION: The results suggest that a realistic, image-based acoustic and thermal model of the treatment region is capable of predicting the extent of thermal damage to target plaque tissue. The model considered the effect of the wall thickness of large arteries and the heat-sink effect of flowing blood. The model is used for predicting the size and pattern of HIFU damage in vivo.

Ultrasound-guided therapeutic focused ultrasound: current status and future directions.

This paper reviews ultrasound imaging methods for the guidance of therapeutic focused ultrasound (USgFUS), with emphasis on real-time preclinical methods. Guidance is interpreted in the broadest sense to include pretreatment planning, siting of the FUS focus, real-time monitoring of FUS-tissue interactions, and real-time control of exposure and damage assessment. The paper begins with an overview and brief historical background of the early methods used for monitoring FUS-tissue interactions. Current imaging methods are described, and discussed in terms of sensitivity and specificity of the localisation of the FUS effects in both therapeutic and sub-therapeutic modes. Thermal and non-thermal effects are considered. These include cavitation-enhanced heating, tissue water boiling and cavitation. Where appropriate, USgFUS methods are compared with similar methods implemented using other guidance modalities, e.g. magnetic resonance imaging. Conclusions are drawn regarding the clinical potential of the various guidance methods, and the feasibility and current status of real-time implementation.

High-intensity focused ultrasound for potential treatment of polycystic ovary syndrome: toward a noninvasive surgery.

OBJECTIVE: To investigate the feasibility of using high-intensity focused ultrasound (HIFU), under dual-mode ultrasound arrays (DMUAs) guidance, to induce localized thermal damage inside ovaries without damage to the ovarian surface. DESIGN: Laboratory feasibility study. SETTING: University-based laboratory. ANIMAL(S): Ex vivo canine and bovine ovaries. INTERVENTION(S): DMUA-guided HIFU. MAIN OUTCOME MEASURE(S): Detection of ovarian damage by ultrasound imaging, gross pathology, and histology. RESULT(S): It is feasible to induce localized thermal damage inside ovaries without damage to the ovarian surface. DMUA provided sensitive imaging feedback regarding the anatomy of the treated ovaries and the ablation process. Different ablation protocols were tested, and thermal damage within the treated ovaries was histologically characterized. CONCLUSION(S): The absence of damage to the ovarian surface may eliminate many of the complications linked to current laparoscopic ovarian drilling (LOD) techniques. HIFU may be used as a less traumatic tool to perform LOD.

Feasibility of targeting atherosclerotic plaques by high-intensity-focused ultrasound: an in vivo study.

PURPOSE: To investigate the feasibility and acute safety of targeting atherosclerotic plaques by high-intensity-focused ultrasound (US) in vivo through a noninvasive extracorporeal approach. MATERIALS AND METHODS: Four swine were included in this prospective study, three of which were familial hypercholesterolemic swine. The procedure was done under general anesthesia. After US identification of atherosclerotic plaques within the femoral arteries, plaques were targeted by high-intensity focused US with an integrated dual-mode US array system. Different ablation protocols were used to meet the study objectives, and animals were then euthanized at different time points. Targeted arterial segments were stained by hematoxylin and eosin for histopathologic examination. Numeric values are presented as means ± standard deviation. RESULTS: All swine tolerated the procedure well, with no arterial dissection, perforation, or rupture. Discrete lesions were detected in the first two swine, measuring 0.54 mm ± 0.10 and 0.25 mm ± 0.03 in cross-sectional dimensions in the first and 0.50 mm ± 0.12 and 0.24 mm ± 0.15 in the second. Confluent ablation zones were identified in the last two swine, measuring 6.92 mm and 0.93 mm in the third and 2.97 mm and 2.52 mm in the fourth. Lesions showed necrotic cores and peripheral reactive inflammatory infiltration. The endothelium overlying targeted arterial segments remained intact. CONCLUSIONS: The results demonstrate the feasibility and acute safety of targeting atherosclerotic plaques by high-intensity-focused US in vivo. Further long-term studies are needed to assess how induction of these lesions can modify the progression of atherosclerotic plaques.

Adaptive transthoracic refocusing of dual-mode ultrasound arrays.

We present experimental validation results of an adaptive, image-based refocusing algorithm of dual-mode ultrasound arrays (DMUAs) in the presence of strongly scattering objects. This study is motivated by the need to develop noninvasive techniques for therapeutic targeting of tumors seated in organs where the therapeutic beam is partially obstructed by the ribcage, e.g., liver and kidney. We have developed an algorithm that takes advantage of the imaging capabilities of DMUAs to identify the ribs and the intercostals within the path of the therapeutic beam to produce a specified power deposition at the target while minimizing the exposure at the rib locations. This image-based refocusing algorithm takes advantage of the inherent registration between the imaging and therapeutic coordinate systems of DMUAs in the estimation of array directivity vectors at the target and rib locations. These directivity vectors are then used in solving a constrained optimization problem allowing for adaptive refocusing, directing the acoustical energy through the intercostals, and avoiding the rib locations. The experimental validation study utilized a 1-MHz, 64-element DMUA in focusing through a block of tissue-mimicking phantom [0.5 dB/(cm .MHz)] with embedded Plexiglas ribs. Single transmit focus (STF) images obtained with the DMUA were used for image-guided selection of the critical and target points to be used for adaptive refocusing. Experimental results show that the echogenicity of the ribs in STF images provide feedback on the reduction of power deposition at rib locations. This was confirmed by direct comparison of measured temperature rise and integrated backscatter at the rib locations. Direct temperature measurements also confirm the improved power deposition at the target and the reduction in power deposition at the rib locations. Finally, we have compared the quality of the image-based adaptive refocusing algorithm with a phase-conjugation solution obtained by direct measurement of the complex pressures at the target location. It is shown that our adaptive refocusing algorithm achieves similar improvements in power deposition at the target while achieving larger reduction of power deposition at the rib locations.

Monitoring and guidance of minimally-invasive thermal therapy using diagnostic ultrasound.

We present specialized ultrasound imaging modes for monitoring and guidance of noninvasive and minimally-invasive thermal therapy. One mode is based on two-dimensional imaging of temperature change using diagnostic ultrasound. We have validated this method both in vivo and in vitro in monitoring the heating patterns produced by noninvasive HIFU source and minimally-invasive RF ablation device, respectively. In addition, a nonlinear method for imaging the quadratic echo components from HIFU-induced lesions has also been developed and tested in vivo. Illustrative results from both modes of imaging are presented. These results demonstrate the unique advantages of ultrasound as an image-guidance modality. Specifically, the high spatial and temporal resolutions that allow for imaging highly-localized short-duration therapeutic and sub-therapeutic HIFU beams. With the advent of highperformance computing hardware, these imaging modes are now implementable in real-time. This will lead to active realtime monitoring and control of a range of thermal therapies in the very near future.

Real-time two-dimensional temperature imaging using ultrasound.

We present a system for real-time 2D imaging of temperature change in tissue media using pulse-echo ultrasound. The frontend of the system is a SonixRP ultrasound scanner with a research interface giving us the capability of controlling the beam sequence and accessing radio frequency (RF) data in real-time. The beamformed RF data is streamlined to the backend of the system, where the data is processed using a two-dimensional temperature estimation algorithm running in the graphics processing unit (GPU). The estimated temperature is displayed in real-time providing feedback that can be used for real-time control of the heating source. Currently we have verified our system with elastography tissue mimicking phantom and in vitro porcine heart tissue, excellent repeatability and sensitivity were demonstrated.

Monitoring and guidance of HIFU beams with dual-mode ultrasound arrays.

We present experimental results illustrating the unique advantages of dual-mode array (DMUA) systems in monitoring and guidance of high intensity focused ultrasound (HIFU) lesion formation. DMUAs offer a unique paradigm in image-guided surgery; one in which images obtained using the same therapeutic transducer provide feedback for: 1) refocusing the array in the presence of strongly scattering objects, e.g. the ribs, 2) temperature change at the intended location of the HIFU focus, and 3) changes in the echogenicity of the tissue in response to therapeutic HIFU. These forms of feedback have been demonstrated in vitro in preparation for the design and implementation of a real-time system for imaging and therapy with DMUAs. The results clearly demonstrate that DMUA image feedback is spatially accurate and provide sufficient spatial and contrast resolution for identification of high contrast objects like the ribs and significant blood vessels in the path of the HIFU beam.

A post-beamforming 2-D pseudoinverse filter for coarsely sampled ultrasound arrays.

Beamforming artifacts due to coarse discretization of imaging apertures represent a significant barrier against the use of array probes in high-frequency applications. Nyquist sampling of array apertures dictates center-to-center spacing of lambda/2 for elimination of grating lobes in the array pattern. However, this requirement is hard to achieve using current transducer technologies, even at the lower end of high-frequency ultrasonic imaging (in the range 25-35 MHz). In this paper, we present a new design approach for 2-D regularized pseudoinverse (PIO) filters suitable for restoring imaging contrast in systems employing coarsely sampled arrays. The approach is based on a discretized 2-D imaging model for linear arrays assuming scattering from a Cartesian grid in the imaging field of view (FOV). We show that the discretized imaging operator can be represented with a block Toeplitz matrix with the blocks themselves being Toeplitz. With sufficiently large grid size in the axial and lateral directions, it is possible to replace this Toeplitz-block block Toeplitz (TBBT) operator with its circulant-block block circulant (CBBC) equivalent. This leads to a computationally efficient implementation of the regularized pseudoinverse filtering approach using the 2-D fast Fourier transform (FFT). The derivation of the filtering equation is shown in detail and the regularization procedure is fully described. Using FIELD, we present simulation data to show the 2-D point-spread functions (PSFs) for imaging systems employing linear arrays with fine and coarse sampling of the imaging aperture. PSFs are also computed for a coarsely sampled array with different levels of regularization to demonstrate the tradeoff between contrast and spatial resolution. These results demonstrate the well-behaved nature of the PSF with the variation in a single regularization parameter. Specifically, the 6 dB axial and lateral dimensions of the PSF increase gradually with increasing value of the regularization parameter. On the other hand, the peak grating lobe level decreases gradually with increasing value of the regularization parameter. The results are supported by image reconstructions from a simulated cyst phantom obtained using finely and coarsely sampled apertures with and without the application of the regularized 2-D PIO.

Viscoelastic characterization of thin tissues using acoustic radiation force and model-based inversion.

By means of the viscoelastodynamic model for a two-layer solid-fluid system and a detailed account of the locally induced acoustic radiation force, a rational analytical and computational framework is established for the viscoelastic characterization of thin tissues from high-frequency ultrasound (HFUS) measurements. For practical applications, the back-analysis is set up to interpret the frequency response function, signifying the tissue's axial displacement (captured by the imaging transducer) per squared voltage driving the 'pushing' transducer, as experimental input. On parametrizing the tissue's viscoelastic behavior in terms of the standard linear model, the proposed methodology is applied to a set of measurements performed on tissue-mimicking phantom constructs with thicknesses ranging from 0.5 to 4 mm. The results demonstrate that the model-based inversion, which carefully mimics the local boundary conditions and applied ultrasound excitation, yields viscoelastic properties for the phantom that are virtually invariant over the range of specimen thicknesses tested. Beyond its immediate application to in vitro viscoelastic characterization of thin excised tissues and tissue constructs, the proposed methodology may also find use in the characterization of skin or skin lesions over bone in vivo.

Imaging with concave large-aperture therapeutic ultrasound arrays using conventional synthetic-aperture beamforming.

Several dual-mode ultrasound array (DMUA) systems are being investigated for potential use in image- guided surgery. In therapeutic mode, DMUAs generate pulsed or continuous-wave (CW) high-intensity focused ultrasound (HIFU) beams capable of generating localized therapeutic effects within the focal volume. In imaging mode, pulse-echo data can be collected from the DMUA elements to obtain B-mode images or other forms of feedback on the state of the target tissue before, during, and after the application of the therapeutic HIFU beam. Therapeutic and technological constraints give rise to special characteristics of therapeutic arrays. Specifically, DMUAs have concave apertures with low f-number values and are typically coarsely sampled using directive elements. These characteristics necessitate pre- and post-beamforming signal processing of echo data to improve the spatial and contrast resolution and maximize the image uniformity within the imaging field of view (IxFOV). We have recently developed and experimentally validated beamforming algorithms for concave large-aperture DMUAs with directive elements. Experimental validation was performed using a 1 MHz, 64-element, concave spherical aperture with 100 mm radius of curvature. The aperture was sampled in the lateral direction using elongated elements 1-lambda x 33.3-lambda with 1.333-lambda center-to-center spacing (lambda is the wavelength). This resulted in f-number values of 0.8 and 2 in the azimuth and elevation directions, respectively. In this paper, we present a new DMUA design approach based on different sampling of the shared concave aperture to improve image quality while maintaining therapeutic performance. A pulse-wave (PW) simulation model using a modified version of the Field II program is used in this study. The model is used in generating pulse-echo data for synthetic-aperture (SA) beamforming for forming images of a variety of targets, e.g., wire arrays and speckle-generating cyst phantoms. To provide validation for the simulation model and illustrate the improvements in image quality, we show SA images of similar targets using pulse-echo data acquired experimentally using our existing 64-element prototype. The PW simulation model is used to investigate the effect of transducer bandwidth as well as finer sampling of the concave DMUA aperture on the image quality. The results show that modest increases in the sampling density and transducer bandwidth result in significant improvement in spatial and contrast resolutions in addition to extending the DMUA IxFOV.

Dual-mode ultrasound phased arrays for image-guided surgery.

A 64-element, 1 MHz prototype dual-mode array (DMUA) with therapeutic and imaging capabilities is described. Simulation and experimental results for the characterization of the therapeutic operating field (ThxOF) and imaging field-of-view (IxFOV) for a DMUA are given. In addition, some of the special considerations for imaging with DMUAs are given and illustrated experimentally using wire-target arrays and commercial, quality-assurance phantoms. These results demonstrate what is potentially the most powerful advantage of the use of DMUAs in image-guided surgery; namely, inherent registration between the imaging and therapeutic coordinate systems. We also present imaging results before and after discrete and volumetric HIFU-induced lesions in freshly-excised tissues. DMUA images consistently show changes in echogenicity after lesion formation with shape and extent reflecting the actual shape of the lesion. While changes in echogenicity cannot be used as an indicator of irreversible HIFU-induced tissue damage, they provide important feedback on the location and extent of the expected lesion. Thus, together with the self-registration property of DMUAs, lesion images can be expected to provide immediate and spatially-accurate feedback on the tissue response to the therapeutic HIFU beams. Based on the results provided here, the imaging capabilities of DMUAs can add unique features to other forms of image guidance, e.g. MRI, CT and diagnostic ultrasound.

Enhancement of tumor thermal therapy using gold nanoparticle-assisted tumor necrosis factor-alpha delivery.

Tumor necrosis factor-alpha (TNF-alpha) is a potent cytokine with anticancer efficacy that can significantly enhance hyperthermic injury. However, TNF-alpha is systemically toxic, thereby creating a need for its selective tumor delivery. We used a newly developed nanoparticle delivery system consisting of 33-nm polyethylene glycol-coated colloidal gold nanoparticles (PT-cAu-TNF-alpha) with incorporated TNF-alpha payload (several hundred TNF-alpha molecules per nanoparticle) to maximize tumor damage and minimize systemic exposure to TNF-alpha. SCK mammary carcinomas grown in A/J mice were treated with 125 or 250 microg/kg PT-cAu-TNF-alpha alone or followed by local heating at 42.5 degrees C using a water bath for 60 minutes, 4 hours after nanoparticle injection. Increases in tumor growth delay were observed for both PT-cAu-TNF-alpha alone and heat alone, although the most dramatic effect was found in the combination treatment. Tumor blood flow was significantly suppressed 4 hours after an i.v. injection of free TNF-alpha or PT-cAu-TNF-alpha. Tumor perfusion, imaged by contrast enhanced ultrasonography, on days 1 and 5 after treatment revealed perfusion defects after the injection of PT-cAu-TNF-alpha alone and, in many regions, complete flow inhibition in tumors treated with combination treatment. The combination treatment of SCK tumors in vivo reduced the in vivo/in vitro tumor cell survival to 0.05% immediately following heating and to 0.005% at 18 hours after heating, suggesting vascular damage-mediated tumor cell killing. Thermally induced tumor growth delay was enhanced by pretreatment with TNF-alpha-coated gold nanoparticles when given i.v. at the proper dosage and timing.