Displacement detection with sub-pixel accuracy and high spatial resolution using deep learning.

PURPOSE: The purpose of this study was to detect two dimensional and sub-pixel displacement with high spatial resolution using an ultrasonic diagnostic apparatus. Conventional displacement detection methods assume neighborhood uniformity and cannot achieve both high spatial resolution and sub-pixel displacement detection. METHODS: A deep-learning network that utilizes ultrasound images and output displacement distribution was developed. The network structure was constructed by modifying FlowNet2, a widely used network for optical flow estimation, and a training dataset was developed using ultrasound image simulation. Detection accuracy and spatial resolution were evaluated via simulated ultrasound images, and the clinical usefulness was evaluated with ultrasound images of the liver exposed to high-intensity-focused ultrasound (HIFU). These results were compared to the Lucas-Kanade method, a conventional sub-pixel displacement detection method. RESULTS: For a displacement within ± 40 µm (± 0.6 pixels), a pixel size of 67 µm, and signal noise of 1%, the accuracy was above 0.5 µm and 0.2 µm, the precision was above 0.4 µm and 0.3 µm, and the spatial resolution was 1.1 mm and 0.8 mm for the lateral and axial displacements, respectively. These improvements were also observed in the experimental data. Visualization of the lateral displacement distribution, which determines the edge of the treated lesion using HIFU, was also realized. CONCLUSION: Two-dimensional and sub-pixel displacement detection with high spatial resolution was realized using a deep-learning methodology. The proposed method enabled the monitoring of small and local tissue deformations induced by HIFU exposure.

Investigation of feasibility of noise suppression method for cavitation-enhanced high-intensity focused ultrasound treatment.

In high-intensity focused ultrasound (HIFU) treatment, a method that monitors tissue changes while irradiating therapeutic ultrasound is needed to detect changes in the order of milliseconds due to thermal coagulation and the presence of cavitation bubbles. The new filtering method in which only the HIFU noise was reduced while the tissue signals remained intact was proposed in the conventional HIFU exposure in our preliminary study. However, HIFU was irradiated perpendicular to the direction of the imaging ultrasound in the preliminary experiment, which was believed to be impractical. This study investigated the efficacy of the proposed method a parallel setup, in which both HIFU and imaging beams have the same axis just as in a practical application. In addition, this filtering algorithm was applied to the "Trigger HIFU" sequence in which ultrasound-induced cavitation bubbles were generated in the HIFU focal region to enhance heating. In this setup and sequence, HIFU noise level was increased and the summation or difference tone induced by the interaction of HIFU waves with the imaging pulse has the potential to affect this proposed method. Ex-vivo experiments proved that the HIFU noise was selectively eliminated by the proposed filtering method in which chaotic acoustic signals were emitted by the cavitation bubbles at the HIFU focus. These results suggest that the proposed method was practically efficient for monitoring tissue changes in HIFU-induced cavitation bubbles.

Evaluation of an octahydroisochromene scaffold used as a novel SARS 3CL protease inhibitor.

An octahydroisochromene scaffold has been introduced into a known SARS 3CL protease inhibitor as a novel hydrophobic core to interact with the S2 pocket of the protease. An alkyl or aryl substituent was also introduced at the 1-position of the octahydroisochromene scaffold and expected to introduce additional interactions with the protease. Sharpless-Katsuki asymmetric epoxidation and Sharpless asymmetric dihydroxylation were employed to construct the octahydroisochromene scaffold. The introductions of the P1 site His-al and the substituent at 1-position was achieved using successive reductive amination reactions. Our initial evaluations of the diastereo-isomeric mixtures (16a-d) revealed that the octahydroisochromene moiety functions as a core hydrophobic scaffold for the S2 pocket of the protease and the substituent at the 1-position may form additional interactions with the protease. The inhibitory activities of the diastereoisomerically-pure inhibitors (3a-d) strongly suggest that a specific stereo-isomer of the octahydroisochromene scaffold, (1S, 3S) 3b, directs the P1 site imidazole, the warhead aldehyde, and substituent at the 1-position of the fused ring to their appropriate pockets in the protease.

Ultrasound Sub-pixel Motion-tracking Method with Out-of-plane Motion Detection for Precise Vascular Imaging.

Ultrasound vascularity imaging provides important information for differential diagnosis of tumors. Peak-hold (PH) is a useful technique for precisely imaging small vessels by selecting a maximum brightness in each pixel through the frames obtained sequentially. To use PH successfully one needs motion compensation to reduce image blur, but out-of-plane motion cannot be avoided. To address this problem, we developed a sub-pixel motion-tracking method with out-of-plane motion detection (OPMD). It is a combination of the sum of the absolute differences (SAD) method and the Kanade-Lucas-Tomasi method and can be accurately applied to various motions. The value from OPMD (γ) is defined as a statistical value obtained from the distribution of residual values in the SAD procedure with the obtained frames. The value is ideally 0, and the frames having large γ are removed from the PH procedure. The accuracy of the proposed tracking method was found by a simulation study to be approximately 20 µm. We also found, through a phantom experiment, that the value of γ sensitively increased enough to detect out-of-plane motion. Most important, γ begins to increase before tracking errors occur. This suggests that OPMD can be used to predict tracking errors and effectively remove frames from the PH procedure. An in vivo experiment with a rabbit showed that the PH image obtained with motion tracking clearly revealed peripheral vessels that were blurred in the PH image obtained without motion tracking. We also found that the image quality becomes better when OPMD was used to remove frames including out-of-plane motion.

Sonodynamic Therapy With Anticancer Micelles and High-Intensity Focused Ultrasound in Treatment of Canine Cancer.

Sonodynamic therapy (SDT) is a minimally invasive anticancer therapy involving a chemical sonosensitizer and high-intensity focused ultrasound (HIFU). SDT enables the reduction of drug dose and HIFU irradiation power compared to those of conventional monotherapies. In our previous study, mouse models of colon and pancreatic cancer were used to confirm the effectiveness of SDT vs. drug-only or HIFU-only therapy. To validate its usefulness, we performed a clinical trial of SDT using an anticancer micelle (NC-6300) and our HIFU system in four pet dogs with spontaneous tumors, including chondrosarcoma, osteosarcoma, hepatocellular cancer, and prostate cancer. The fact that no adverse events were observed, suggests the usefulness of SDT.

Sonodynamic Therapy: Advances and Challenges in Clinical Translation.

Sonodynamic therapy (SDT) consists of the synergetic interaction between ultrasound and a chemical agent. In SDT, the cytotoxicity is triggered by ultrasonic stimuli, notably through cavitation. The unique features of SDT are relevant in the clinical context more than ever: the need for efficacy, accuracy, and safety while being noninvasive and preserving the patient's quality of life. However, despite the promising results of this technique, only a few clinical reports describe the use of SDT. The objective of this article is to provide an extensive overview of the clinical and preclinical research conducted in vivo on SDT, to identify the limitations, and to detail the developed strategies to overcome them.

Visualization of murine lymph vessels using photoacoustic imaging with contrast agents.

Metastasis frequently occurs even in the early stage of breast cancer. This research studied the feasibility of using photoacoustic (PA) imaging for identifying metastasis in the lymph vessels of mice. The photoacoustic efficiency of various contrast agents was investigated, and the influence of scattered light was evaluated by using a lymph vessel phantom. The lymph vessels of mice were then visualized using the selected contrast agents: indocyanine green (ICG) and gold nanorods (AuNR). The attenuation of the PA imaging was -1.90 dB/mm, whereas that of the fluorescence imaging was -4.45 dB/mm. The results indicate the potential of identifying sentinel lymph nodes by using PA imaging with these contrast agents.

Cavitation-threshold Determination and Rheological-parameters Estimation of Albumin-stabilized Nanobubbles.

Nanobubbles (NBs) are of high interest for ultrasound (US) imaging as contrast agents and therapy as cavitation nuclei. Because of their instability (Laplace pressure bubble catastrophe) and low sensitivity to US, reducing the size of commonly used microbubbles to submicron-size is not trivial. We introduce stabilized NBs in the 100-250-nm size range, manufactured by agitating human serum albumin and perfluoro-propane. These NBs were exposed to 3.34- and 5.39-MHz US, and their sensitivity to US was proven by detecting inertial cavitation. The cavitation-threshold information was used to run a numerical parametric study based on a modified Rayleigh-Plesset equation (with a Newtonian rheology model). The determined values of surface tension ranged from 0 N/m to 0.06 N/m. The corresponding values of dilatational viscosity ranged from 5.10-10 Ns/m to 1.10-9 Ns/m. These parameters were reported to be 0.6 N/m and 1.10-8 Ns/m for the reference microbubble contrast agent. This result suggests the possibility of using albumin as a stabilizer for the nanobubbles that could be maintained in circulation and presenting satisfying US sensitivity, even in the 3-5-MHz range.

Dancing Styles of Collective Cell Migration: Image-Based Computational Analysis of JRAB/MICAL-L2.

Collective cell migration is observed during morphogenesis, angiogenesis, and wound healing, and this type of cell migration also contributes to efficient metastasis in some kinds of cancers. Because collectively migrating cells are much better organized than a random assemblage of individual cells, there seems to be a kind of order in migrating clusters. Extensive research has identified a large number of molecules involved in collective cell migration, and these factors have been analyzed using dramatic advances in imaging technology. To date, however, it remains unclear how myriad cells are integrated as a single unit. Recently, we observed unbalanced collective cell migrations that can be likened to either precision dancing or awa-odori, Japanese traditional dancing similar to the style at Rio Carnival, caused by the impairment of the conformational change of JRAB/MICAL-L2. This review begins with a brief history of image-based computational analyses on cell migration, explains why quantitative analysis of the stylization of collective cell behavior is difficult, and finally introduces our recent work on JRAB/MICAL-L2 as a successful example of the multidisciplinary approach combining cell biology, live imaging, and computational biology. In combination, these methods have enabled quantitative evaluations of the "dancing style" of collective cell migration.

Sonodynamic Therapy Based on Combined Use of Low Dose Administration of Epirubicin-Incorporating Drug Delivery System and Focused Ultrasound.

Sonodynamic therapy (SDT) is currently considered as one of the promising minimally invasive treatment options for solid cancers. SDT is based on the combined use of a sonosensitizer drug and high-intensity focused ultrasound (HIFU) to produce cytotoxic reactive oxygen species (ROS) in and around neoplastic cells. Anthracycline drugs, including epirubicin (EPI), have been well known as effective sonosensitizers after interaction with focused ultrasound. Recently a new anticancer drug delivery system (DDS), NC-6300, has been developed that comprises EPI through an acid-labile hydrazone bond. In previous in vivo studies, NC-6300 showed basic drug safety and an excellent concentration property of EPI, and recently has been tested in clinical trials. For realizing minimally invasive cancer treatment, the present study demonstrated the effectiveness and feasibility of DDS-based SDT, which combined a small dose of NC-6300 and low energy of HIFU in mouse models of colon cancer and pancreatic cancer.

Development of Real-Time 3-D Photoacoustic Imaging System Employing Spherically Curved Array Transducer.

Photoacoustic (PA) imaging is a promising imaging modality to visualize specific living tissues based on the light absorption coefficients without dyeing. In this paper, a real-time PA imaging system with a tunable laser was newly developed with an originally designed spherically curved array transducer. Five different series of experiments were conducted to validate the PA measurement system. The peak frequency of the transducer response was 17.7 MHz, and a volume-imaging rate of 3-D volume imaging was 10-20 volumes per second. The spatial resolution of imaging was 90- [Formula: see text] along both the axial and lateral directions. The developed imaging system could measure a difference on an absorption coefficient of gold nanorods. Additionally, the PA imaging could visualize the in vivo microvasculatures of a human hand. This PA imaging system with higher spatial-temporal resolution and the tunable laser further should enhance our understanding of not only basic properties of the photo acoustics but also clinical applications.

Conformational plasticity of JRAB/MICAL-L2 provides "law and order" in collective cell migration.

In fundamental biological processes, cells often move in groups, a process termed collective cell migration. Collectively migrating cells are much better organized than a random assemblage of individual cells. Many molecules have been identified as factors involved in collective cell migration, and no one molecule is adequate to explain the whole picture. Here we show that JRAB/MICAL-L2, an effector protein of Rab13 GTPase, provides the "law and order" allowing myriad cells to behave as a single unit just by changing its conformation. First, we generated a structural model of JRAB/MICAL-L2 by a combination of bioinformatic and biochemical analyses and showed how JRAB/MICAL-L2 interacts with Rab13 and how its conformational change occurs. We combined cell biology, live imaging, computational biology, and biomechanics to show that impairment of conformational plasticity in JRAB/MICAL-L2 causes excessive rigidity and loss of directionality, leading to imbalance in cell group behavior. This multidisciplinary approach supports the concept that the conformational plasticity of a single molecule provides "law and order" in collective cell migration.

Detection of tissue coagulation by decorrelation of ultrasonic echo signals in cavitation-enhanced high-intensity focused ultrasound treatment.

BACKGROUND: A noninvasive technique to monitor thermal lesion formation is necessary to ensure the accuracy and safety of high-intensity focused ultrasound (HIFU) treatment. The purpose of this study is to ultrasonically detect the tissue change due to thermal coagulation in the HIFU treatment enhanced by cavitation microbubbles. METHODS: An ultrasound imaging probe transmitted plane waves at a center frequency of 4.5 MHz. Ultrasonic radio-frequency (RF) echo signals during HIFU exposure at a frequency of 1.2 MHz were acquired. Cross-correlation coefficients were calculated between in-phase and quadrature (IQ) data of two B-mode images with an interval time of 50 and 500 ms for the estimation of the region of cavitation and coagulation, respectively. Pathological examination of the coagulated tissue was also performed to compare with the corresponding ultrasonically detected coagulation region. RESULTS: The distribution of minimum hold cross-correlation coefficient between two sets of IQ data with 50-ms intervals was compared with a pulse inversion (PI) image. The regions with low cross-correlation coefficients approximately corresponded to those with high brightness in the PI image. The regions with low cross-correlation coefficients in 500-ms intervals showed a good agreement with those with significant change in histology. CONCLUSIONS: The results show that the regions of coagulation and cavitation could be ultrasonically detected as those with low cross-correlation coefficients between RF frames with certain intervals. This method will contribute to improve the safety and accuracy of the HIFU treatment enhanced by cavitation microbubbles.

Focused Ultrasound and Lithotripsy.

Shock wave lithotripsy has generally been a first choice for kidney stone removal. The shock wave lithotripter uses an order of microsecond pulse durations and up to a 100 MPa pressure spike triggered at approximately 0.5-2 Hz to fragment kidney stones through mechanical mechanisms. One important mechanism is cavitation. We proposed an alternative type of lithotripsy method that maximizes cavitation activity to disintegrate kidney stones using high-intensity focused ultrasound (HIFU). Here we outline the method according to the previously published literature (Matsumoto et al., Dynamics of bubble cloud in focused ultrasound. Proceedings of the second international symposium on therapeutic ultrasound, pp 290-299, 2002; Ikeda et al., Ultrasound Med Biol 32:1383-1397, 2006; Yoshizawa et al., Med Biol Eng Comput 47:851-860, 2009; Koizumi et al., A control framework for the non-invasive ultrasound the ragnostic system. Proceedings of 2009 IEEE/RSJ International Conference on Intelligent Robotics and Systems (IROS), pp 4511-4516, 2009; Koizumi et al., IEEE Trans Robot 25:522-538, 2009). Cavitation activity is highly unpredictable; thus, a precise control system is needed. The proposed method comprises three steps of control in kidney stone treatment. The first step is control of localized high pressure fluctuation on the stone. The second step is monitoring of cavitation activity and giving feedback on the optimized ultrasound conditions. The third step is stone tracking and precise ultrasound focusing on the stone. For the high pressure control we designed a two-frequency wave (cavitation control (C-C) waveform); a high frequency ultrasound pulse (1-4 MHz) to create a cavitation cloud, and a low frequency trailing pulse (0.5 MHz) following the high frequency pulse to force the cloud into collapse. High speed photography showed cavitation collapse on a kidney stone and shock wave emission from the cloud. We also conducted in-vitro erosion tests of model and natural kidney stones. For the model stones, the erosion rate of the C-C waveform showed a distinct advantage with the combined high and low frequency waves over either wave alone. For optimization of the high frequency ultrasound intensity, we investigated the relationship between subharmonic emission from cavitation bubbles and stone erosion volume. For stone tracking we have also developed a non-invasive ultrasound theragnostic system (NIUTS) that compensates for kidney motion. Natural stones were eroded and most of the resulting fragments were less than 1 mm in diameter. The small fragments were small enough to pass through the urethra. The results demonstrate that, with the precise control of cavitation activity, focused ultrasound has the potential to be used to develop a less invasive and more controllable lithotripsy system.

Measurement of regional pulse-wave velocity using spatial compound imaging of the common carotid artery in vivo.

Pulse-wave velocity (PWV) is an important index for diagnosing cardiovascular diseases. The pulse wave is volumetric change induced by heartbeat or inflowing blood, and significantly depends on the propagating path and stiffness of the artery. In this study, PWV of the propagating wave was visualized using spatial compound imaging with high temporal resolution. The frame rate was 1000 Hz, or a time interval of 1 ms. Subjects were four young healthy males and one young healthy female (n=5, age: 23.8±1.17 years old), and the measurement area was the right common carotid artery. PWVs in four phases (the four phases of heart valve opening and closing) were investigated during a cardiac cycle. In phase I, the heart pulsates. In phase II, the tricuspid and mitral valves close, and the aortic and pulmonic valves open. In phase III, the tricuspid and mitral valves open, and the aortic and pulmonic valves close. In phase IV, the propagating wave is reflected. PWVs in phases II and III were easily observed. PWVs were 3.52±1.11 m/s in phase I, 5.62±0.30 m/s in phase II, 7.94±0.85 m/s in phase III, and -4.60±0.99 m/s for the reflective wave. PWV was measured using Spatial Compound Imaging with high temporal resolution, and the PWV in each phase may be used as the index for diagnosing stages of arteriosclerosis progression.

Intrinsic elastography and its dependence on arterial flow volume.

We propose a new estimation method of tissues stiffness from deformation induced by arterial pulsation. The new method was named Intrinsic Elastography (IE). Under two assumptions that the deformation induced by pulsation propagates throughout the body, and the propagation velocity (PV) is closely related to the mechanical properties of tissues, the IE can evaluate the tissue stiffness based on the PV. However, this method has an issue that the observed PVs change with the changes of amounts of the flow. In this paper, we propose the new estimation method in order to estimate true shear elasticity in spite of the changes. The results was compared to the results of SWI. The results was almost identical to the result of SWI, or showed the similar results.

High-speed observation of bubble cloud generation near a rigid wall by second-harmonic superimposed ultrasound.

Cavitation bubbles are known to accelerate therapeutic effects of ultrasound. Although negative acoustic pressure is the principle factor of cavitation, positive acoustic pressure has a role for bubble cloud formation at a high intensity of focused ultrasound when cavitation bubbles provide pressure release surfaces converting the pressure from highly positive to negative. In this study, the second-harmonic was superimposed onto the fundamental acoustic pressure to emphasize either peak positive or negative pressure. The peak negative and positive pressure emphasized waves were focused on a surface of an aluminum block. Cavitation bubbles induced near the block were observed with a high-speed camera by backlight and the size of the cavitation generation region was measured from the high-speed images. The negative pressure emphasized waves showed an advantage in cavitation inception over the positive pressure emphasized waves. In the sequence of the negative pressure emphasized waves immediately followed by the positive pressure emphasized waves, cavitation bubbles were generated on the block by the former waves and the cavitation region were expanded toward the transducer in the latter waves with high reproducibility. The sequence demonstrated its potential usefulness in enhancing the effects of therapeutic ultrasound at a high acoustic intensity.

Changes in backscatter of liver tissue due to thermal coagulation induced by focused ultrasound.

Ultrasonic imaging has advantages in its self-consistency in guiding and monitoring ultrasonic treatment such as high-intensity focused ultrasound (HIFU) treatment. Changes in ultrasonic backscatter of tissues due to HIFU treatment have been observed, but their mechanism is still under discussion. In this paper, ultrasonic backscatter of excised and degassed porcine liver tissue was observed before and after HIFU exposure using a diagnostic scanner, and its acoustic impedance was mapped using an ultrasonic microscope. The histology of its pathological specimen was also observed using an optical microscope. The observed decrease in backscatter intensity due to HIFU exposure was consistent with a spatial Fourier analysis of the histology, which also showed changes due to the exposure. The observed increase in acoustic impedance due to the exposure was also consistent with the histological change assuming that the increase was primarily caused by the increase in the concentration of hepatic cells.

Sonodynamic therapy with 5-aminolevulinic acid and focused ultrasound for deep-seated intracranial glioma in rat.

BACKGROUND: 5-Aminolevulinic acid (5-ALA) has already been applied clinically as a photosensitizer. In this study, sonodynamically induced selective antitumour effect of 5-ALA for deep-seated lesions was evaluated. MATERIALS AND METHODS: First, normal rat brains were sonicated via a transducer placed on the dural surface to confirm safe acoustic conditions for normal rat brains. One week after inoculation of brains with C6 rat glioma cells, brains with/without administration of 5-ALA (100 mg/kg body weight) were sonicated. RESULTS: Sonodynamic therapy (SDT) with 5-ALA and focused ultrasound (10 W/cm(2), 1.04 MHz, 5 min) achieved selective antitumour effect against deep-seated experimental glioma. Mean tumour sizes in the largest coronal section in sham-operated rats and rats receiving ultrasound with/without 5-ALA were 29.94±10.39, 18.32±5.69 and 30.81±9.65 mm(2), respectively. Tumour size was significantly smaller in the SDT group than in other groups (p<0.05). CONCLUSION: This experimental rat model showed that SDT appears to be useful in the treatment of deep-seated malignant glioma.