Viability variation of T-cells under ultrasound exposure according to adhesion condition with bubbles.

PURPOSE: Although cellular immunotherapy is expected as a new cancer treatment, its therapeutic efficiency is limited in solid tumors, because most cells return to the bloodstream rather than adhere to the target site. Therefore, we are motivated to develop a technique to concentrate the cells in the blood flow using active control of bubble-surrounded cells under ultrasound exposure considering both aspects of cell controllability and viability. METHODS: We prepared a lipid bubble conjugating ligand to adhere to the surface of the T-cells. First, we evaluated the cell controllability by retaining the cells on a wall of an artificial blood vessel through continuous ultrasound exposure. Next, we investigated the cell viability under ultrasound exposure in a suspension with various bubble concentrations. RESULTS: We estimated the concentration of bubbles when the adhesion to the cell surface was saturated. Then, we evaluated the cell viability with various conditions of ultrasound exposure and bubble concentrations. However, it was confirmed that cell damage occurred under conditions that achieved proper control of the cells. Therefore, we exposed the cells to burst waves to reduce the applied ultrasound intensity. Consequently, the significant increase in cell viability was confirmed to be inversely proportional to the duty ratio. CONCLUSION: To retain cells on a vessel wall, determining the appropriate ultrasound condition including sound pressure and waveform is important to maintain cell viability.

Registration between 2D and 3D Ultrasound Images to Track Liver Blood Vessel Movement.

BACKGROUND: For the accurate positioning of surgical tools, conventional intraoperative navigation systems have been developed to recognize the relationship between target positions and the tools. However, since an internal organ is deformed during the operation, registration between realtime two-dimensional (2D) ultrasound images and three-dimensional (3D) CT or MRI images is not always effective. Therefore, this study developed image registration between 2D and 3D ultrasound images considering deformation for tracking target vessel movement in the liver. METHODS: 3D ultrasound image was obtained in advance with 3D coordinates, including the target vessel. Then real-time 2D images and ultrasound probe position were simultaneously acquired using a 3D position sensor. We applied multiple image resolution registration, where rapid and fine optimizations can be expected at higher and lower levels, respectively. Meanwhile, the gradient descent method was adopted for the optimization, which determines the relative arrangements to obtain maximum similarity between 2D and 3D images. We experimentally established resolution level parameters using a phantom before applying it to track liver blood vessel movements in a normal healthy subject. RESULTS: Comparing the 2D images and the registered images, although the approach has some limitations in tracking large displacement, we confirmed that the cross-section of the target blood vessel was clearly visualized. CONCLUSION: This method has the potential for an ultrasound therapy targeting blood vessels under natural respiration conditions.

Ultrasound image-guided gene delivery using three-dimensional diagnostic ultrasound and lipid-based microbubbles.

Gene therapy is a promising technology for genetic and intractable diseases. Drug delivery carriers or systems for genes and nucleic acids have been studied to improve transfection efficiency and achieve sufficient therapeutic effects. Ultrasound (US) and microbubbles have also been combined for use in gene delivery. To establish a clinically effective gene delivery system, exposing the target tissues to US is important. The three-dimensional (3D) diagnostic probe can three-dimensionally scan the tissue with mechanical regulation, and homogenous US exposure to the targeted tissue can be expected. However, the feasibility of therapeutically applying 3D probes has not been evaluated, especially gene delivery. In this study, we evaluated the characteristics of a 3D probe and lipid-based microbubbles (LB) for gene delivery and determined whether the 3D probe in the diagnostic US device could be used for efficient gene delivery to the targeted tissue using a mouse model. The 3D probe RSP6-16 with LB delivered plasmid DNA (pDNA) to the kidney after systemic injection with luciferase activity similar to that of probes used in previously studies. No toxicity was observed after treatment and, therefore, the combined 3D probe and LB would deliver genes to targeted tissue safely and efficiently.

Three-dimensional extension of blood vessel network by combining multiple ultrasound volumes from different directions.

We have previously proposed the use of acoustic radiation force in blood vessels for therapeutic application of ultrasound. For this purpose, we have developed a blood vessel network reconstruction algorithm to fuse between B-mode and Doppler-mode volumes. However, a size of ultrasound volume was insufficient to recognize the network for treatment. Therefore, using multiple ultrasound volumes, we propose a method to extend a network with neighbor networks. First, an ultrasound volume was analyzed to extract tree-structure including the nodes and the edges. Then we configured an extension method between two tree-structures, which performs the insertion of nodes in a source tree to a target tree. Next, similarity between two networks were evaluated by introducing edge lengths in the network and the edit distance. By analyzing in vivo blood vessel of porcine liver, we confirmed that the construction of the network was reliable according to the extension with the similarity of 60% compared with CT data. We confirmed that the proposed method is effective for reconstructing blood vessel network.

Effect of ultrasonic irradiation on bacterial biofilms.

PURPOSE: The purpose of this study was to clarify the effect of ultrasonic irradiation on biofilm produced by Staphylococcus epidermidis (S. epidermidis), which causes central venous catheter-related infections. MATERIALS AND METHODS: Staphylococcus epidermidis (S. epidermidis, ATCC 35984 RP 62A) was used in this study. First, biofilm was prepared from S. epidermidis on the bottom of the upper left well of a 6-well plate. Next, the biofilm was irradiated for 24 h with 1-MHz ultrasound (US) in the continuous wave mode to serve as the US irradiation group. The acoustic power irradiated below the bottom of the well was 3.8 mW. As a control (non-US irradiation group), non-irradiated biofilm on the bottom of a 6-well plate was incubated at 37 °C in an atmosphere of 5.0% CO2. After US irradiation, the bottoms of the wells were stained with 0.1% crystal violet for 60 s. To extract the crystal violet, 99.5% ethanol was added to the wells, and the extracted solutions were measured at an absorbance of 595 nm. RESULT: The absorbance of the US irradiation group was significantly less than that of the non-US irradiation group (p < 0.01). CONCLUSION: US irradiation can decrease the amount of S. epidermidis biofilm when the duration of US irradiation is sufficiently long even if the acoustic intensity is low.

Active induction of in vivo microbubbles by acoustic radiation force at the bifurcation of blood vessel and its evaluation.

Alhough the development of drug delivery system using microbubbles and ultrasound is expected, because microbubbles diffuse in bloodstream, we have so far reported our attempts for active control of the microbubbles in flow by acoustic radiation force in order to increase local concentration of the microbubbles. However, there was no evidence that in vivo microbubbles act as similar as in vitro experiments, because there were limitations for reproduction of in vivo conditions. In this study, we have elucidated the relationship between brightness variation and microbubbles concentration in the suspension to estimate the absolute concentration in an invisible condition considering in vivo experiment. Then we conducted an experiment of active induction of microbubbles in a Y-form bifurcation of artificial blood vessel, where experimental conditions were with focused ultrasound, the central frequency of 5 MHz, flow velocity of 30 mm/s, and maximum sound pressure of 300 kPa-pp, respectively. Then we applied the conditions for active induction of in vivo microbubbles to compare with in vitro experiments. We used a bifurcation of blood vessel in an ear of a rabbit because the bifurcation shape in its blood vessel is visible. As the results of the experiment, the microbubbles concentration in the induced path was almost two times higher than that in the other path, which agrees with the results from in vitro experiments.

Control of a thin catheter bending at bifurcation points in artificial blood vessel by using acoustic radiation force.

In this paper, control of a thin catheter bending by using acoustic radiation force was carried out to develop precise and noninvasive surgery in small blood vessel. First, it was elucidated that the acting force to a thin catheter made from perfluoroalkoxy (PFA) copolymer could be obtained from the cantilever equation in the effective range, where the displacement of the catheter divided by the cube of the length of the catheter was less than 1.0×10(-5) mm(-2). Next, under the above cantilever theory, acoustic radiation force acting to the catheter was measured in the condition of the continuous ultrasound radiation. Furthermore, it was observed that the force depended on the ultrasound frequency. We confirmed that the force was obtained in the practical condition by the experiment and controlled it bending in artificial blood vessel including multiple bifurcations. It was suggested that the therapy using thin catheter and ultrasound is fully promising.

Image plane positioning by pneumatic actuators for ultrasound guidance.

Image guided procedures such as percutaneous needle insertion or high intensity focused ultrasound, have become quite widespread. In images acquisition, ultrasound (US) is convenient to use in a conventional operating room, and inexpensive compared to CT and MRI. However, US requires to handle an US probe and do not have the base coordinate system. Therefore, intraoperative image position is unclear and cannot position to interested area. To address the issues, we have developed a robotic system based on US calibration and a probe scanning robot. In this study, to validate the implement system, positioning accuracy of an image plane was evaluated. Moreover, we developed an automated US guidance system with a conventional US probe. The system enables image plane positioning to visualize a therapeutic tool automatically. From the results, positioning accuracy of the image plane was 1.6 mm and 1.5 deg, maximally. In the phantom test, the error between the positions of the image plane and the mock needle was 2.5 mm and 0.9 deg. We have confirmed that the proposed system is greatly applicable for an intraoperative US guidance.

Active control of microbubbles stream in multi-bifurcated flow by using 2D phased array ultrasound transducer.

We have previously reported our attempt to propel microbbles in flow by a primary Bjerknes force, which is a physical phenomenon where an acoustic wave pushes an obstacle along its direction of propagation. However, when ultrasound was emitted from surface of the body, controlling bubbles in against flow was needed. It is unpractical to use multiple transducers to produce the same number of focal points because single element transducer cannot produce more than two focal points. In this study, we introduced a complex artificial blood vessel according to a capillary model and a 2D array transducer to produce multiple focal points for active control of microbubbles in against flow. Furthermore, we investigated bubble control in viscous fluid. As the results, we confirmed clearly path selection of MBs in viscous fluid as well as in water.

Production of acoustic field with multiple focal points to control high amount of microbubbles in flow using a 2D array transducer.

We have newly developed a 2D array transducer to control the behavior of microbubbles, which is different from that for HIFU therapy, to emit continuous wave by designing acoustic field including multiple focal points. In the experiment using a straight path model, we have confirmed that higher concentration of acoustic energy does not result more aggregation. We also have confirmed the trapped areas of microbubbles are located not in the peak of the distribution of sound pressure, but in the middle range. The dispersion of acoustic energy is important because there was a relation in the trapping performance of microbubbles and the shape of acoustic field.

An accurate calibration method of ultrasound images by center positions of a metal ball.

This paper provides a novel method for three-dimensional tracking of ultrasound images. One of the issues to determine the position of a ultrasound image plane is the thickness of the image plane. The proposed methodology address the issue by the calibration phantom using a fiducial sphere with the diameter of 5.5 mm because comet-trail artifact can be observed in the image plane through the center of the sphere. Meanwhile, to measure the sphere center accurately by a tracking device, a pointer tool with the same sphere at the tip is also proposed. To validate the feasibility of the method, simulation and phantom tests were conducted. From the results of the phantom test, the accuracy of the calibration was 0.65, 0.40, and 0.42 mm in 10, 50, 100 points calibration. The results demonstrate that the proposed method has a great potential for accurate US probe calibration.

Observation of flow variation in capillaries of artificial blood vessel by producing microbubble aggregations.

Microbubbles form their aggregations between the neighboring microbubbles by the effect of secondary Bjerknes force under ultrasound exposure. However, because of the difficulty to reproduce a capillary-mimicking artificial blood vessel, the behavior of aggregations in a capillary has not been predicted. Thus we prepared artificial blood vessels including a capillary model, which was made of poly(vinyl alcohol) (PVA) by grayscale lithography method, with minimum diameter of the path of 0.5 mm. By using this model we investigated the possibility of artificial embolization, where the microbubble aggregations might block entire vessels not to penetrate flow in downstream. Confirming that the sizes of flown aggregation were greater than the section area of the minimum path in the capillary model, we investigated the probability of path block in it. As the results we confirmed the probability increased in proportion to sound pressure and inversely to flow velocity. We are going to investigate with more kinds of parameters to enhance the possibility of artificial embolization.

Supporting reconstruction of the blood vessel network using graph theory: an abstraction method.

The blood vessel network (BVN) has a complex structure. As this structure is unique for each individual, it is not possible to establish a general model for the BVN. However, many medical applications do rely on this structure. For example, a drug delivery system would be greatly improved if it could control the drug flow towards destination. To address this BVN structure issue, several reconstruction methods have been introduced. In this paper, we describe an abstraction method supporting BVN reconstruction by using graph theory. Starting from an original BVN reconstruction, we define the so-called induced graph of that reconstruction, allowing for an efficient analysis. By applying this method, we were able to improve an original BVN reconstruction of a human kidney by pointing out probable errors inside that original reconstruction.

Development of support system to handle ultrasound probe by coordinated motion with medical robot.

We have developed a support system using our ultrasound diagnosis robot, which is able to support manual handling of ultrasound probe in echography to alleviate fatigue of examiner. This system realizes a coordinated motion according to the motion of the probe, which is hold by the robot and is moved by an examiner. We have established four kinds of situations, which are initial fixation, coordinate motions with/without contact on the body surface, and automatic chase motion of an internal organ. The system recognizes when the examiner grasps the ultrasound probe by 6-axis force sensor and touches it on body surface by processing echograms. Not only unskilled examiners but also a professional sonographer have evaluated the performance of the system after elucidating multiple parameters for compliance control and self-weight and moment compensation of the probe. As the results, this system has the potential to be able to support advanced diagnosis for conventional echography.

Dependence of aggregate formation of microbubbles upon ultrasound condition and exposure time.

We have previously reported our attempts to control microbubbles (microcapsules) behavior in flow by primary Bjerknes force to increase the local concentration of the bubbles at a diseased part. However, there was a limitation in efficiency to propel bubbles of µm-order size. Thus we consider that forming aggregates of bubbles is effective to be propelled before entering into an ultrasound field by making use of secondary Bjerknes force under continuous ultrasound exposure. In this study, we observed the phenomena of aggregates formation by confirming variation of diameter and density of aggregates under various conditions of ultrasound exposure. Then we elucidated frequency dependence of the size of aggregates of micro-bubbles.

Study to prevent the density of microcapsules from diffusing in blood vessel by local acoustic radiation force.

We have already reported our attempt to constrain direction of microcapsules in flow owing to an acoustic radiation force. However, the diameter of capsules was too large not to be applied in vivo. Furthermore, acoustic radiation force affected only in focal area because focused ultrasound was used. Thus we have improved our experiment by using microcapsules as small as blood cells and introducing a plane wave of ultrasound. We prepared an artificial blood vessel including a Y-form bifurcation established two observation areas. Then we newly defined the induction index to evaluate the difference of capsule density in two paths of downstream. As the result, optimum angle of ultrasound emission to induce to desired path was derived. And the induction index increased in proportion to the central frequency of ultrasound, which is affected by forming aggregation of capsules to receive more radiation force.

Fabrication and conductive properties of multilayered ultrathin films designed by layer-by-layer assembly of water-soluble fullerenes.

Fullerene ultrathin films were fabricated by layer-by-layer (LbL) assembly of an anionic fullerene C(61)(COO(-))(2) (FDCA) and poly(diallyldimethylammonium chloride) (PDDA) or a cationic fullerene C(60)C(2)H(4)N(CH(3))(2)(+) (FMAC) and poly(sodium 4-styrenesulfonate) (PSS). The dynamic light scattering and zeta-potential measurements revealed that both water-soluble fullerenes are stably dispersed as polyelectrolytes with a diameter of 20-70 nm for FDCA and 60-180 nm for FMAC in aqueous solutions. In spite of such large fullerene aggregates, the thickness of fullerene LbL films increased regularly by a few nanometers with each deposition, and the resultant LbL films were homogeneous. For FMAC/PSS LbL films, the monolayer thickness was evaluated to be 4 nm for FMAC and 0.5 nm for the PSS layer. In other words, the volume fraction of the fullerene moiety is as high as approximately 80 vol %. The conductivity of fullerene LbL films was comparable to that of a C(60)-dispersed polystyrene film with a similar fullerene fraction, suggesting that there exist effective percolating networks due to the high fullerene fraction in the LbL films. The electron mobility of FMAC/PSS LbL films was as high as 3 x 10(-5) cm(2) V(-1) s(-1), which is comparable to the hole mobility of poly(p-phenylenevinylene) LbL films reported previously.

Active control of microcapsules in artificial blood vessel by producing local acoustic radiation force.

Micrometer-sized microcapsules collapse upon exposure to ultrasound. Use of this phenomenon for a drug delivery system (DDS), not only for local delivery of medication but also for gene therapy, should be possible. However, enhancing the efficiency of medication is limited because capsules in suspension diffuse in the human body after injection, since the motion of capsules in blood flow cannot be controlled. To control the behavior of microcapsules, acoustic radiation force was introduced. We detected local changes in microcapsule density by producing acoustic radiation force in an artificial blood vessel. Furthermore, we theoretically estimated the conditions required for active path selection of capsules at a bifurcation point in the artificial blood vessel. We observed the difference in capsule density at both in the bifurcation point and in alternative paths downstream of the bifurcation point for different the acoustic radiation forces. Also we confirmed the microcapsules are trapped against flow with the condition when the acoustic radiation force is more than fluid resistance of the capsules. The possibility of controlling capsule flow towards a specific point in a blood vessel was demonstrated.

Development of 3D space-sharing interface using augmented reality technology for domestic tele-echography.

We propose a domestic tele-echography system linking between the patient's home and a hospital because of the demand due to increase in the number of patients by aging society and recent progress in portable echography enabled us to develop this system. In previous researches three-dimensional position of the ultrasound probe was difficult to specify because a remote doctor observe the patient through a video camera. Therefore we have developed a reproduction system of the probe position and angle using the ARToolKit and GUI interface using OpenGL. Only an USB camera and two markers for the body surface and the probe are necessary to memorize and transfer three-dimensional position of the probe. We have also designed a doctor side interface including echogram, patient scene and CG to instruct probe operation. As a result of evaluation experiments, guided position was satisfied to reproduce the echogram for diagnosis.

Field testing of a remote controlled robotic tele-echo system in an ambulance using broadband mobile communication technology.

We report the testing of a mobile Robotic Tele-echo system that was placed in an ambulance and successfully transmitted clear real time echo imaging of a patient's abdomen to the destination hospital from where this device was being remotely operated. Two-way communication between the paramedics in this vehicle and a doctor standing by at the hospital was undertaken. The robot was equipped with an ultrasound probe which was remotely controlled by the clinician at the hospital and ultrasound images of the patient were transmitted wirelessly. The quality of the ultrasound images that were transmitted over the public mobile telephone networks and those transmitted over the Multimedia Wireless Access Network (a private networks) were compared. The transmission rate over the public networks and the private networks was approximately 256 Kbps, 3 Mbps respectively. Our results indicate that ultrasound images of far higher definition could be obtained through the private networks.