Measurement of flow volume in the presence of reverse flow with ultrasound speckle decorrelation

Direct measurement of volumetric flow rate in the cardiovascular system with ultrasound is valuable but has been a challenge because most current 2-D flow imaging techniques are only able to estimate the flow velocity in the scanning plane (in-plane). Our recent study demonstrated that high frame rate contrast ultrasound and speckle decorrelation (SDC) can be used to accurately measure the speed of flow going through the scanning plane (through-plane). The volumetric flow could then be calculated by integrating over the luminal area, when the blood vessel was scanned from the transverse view. However, a key disadvantage of this SDC method is that it cannot distinguish the direction of the through-plane flow, which limited its applications to blood vessels with unidirectional flow. Physiologic flow in the cardiovascular system could be bidirectional due to its pulsatility, geometric features, or under pathologic situations. In this study, we proposed a method to distinguish the through-plane flow direction by inspecting the flow within the scanning plane from a tilted transverse view. This method was tested on computer simulations and experimental flow phantoms. It was found that the proposed method could detect flow direction and improved the estimation of the flow volume, reducing the overestimation from over 100% to less than 15% when there was flow reversal. This method showed significant improvement over the current SDC method in volume flow estimation and can be applied to a wider range of clinical applications where bidirectional flow exists

Multi-Frame Rate Plane Wave Contrast-Enhance Ultrasound Imaging for Tumour Vasculature Imaging and Perfusion Quantification

A multi-frame rate plane wave imaging strategy is developed to simultaneously image tumor vasculature and quantify tumor perfusion. Customised imaging sequences interleaving a short but high frame rate (HFR) plane wave imaging sequence with a long but low frame rate imaging (LFR) sequence were implemented using a programmable ultrasound research platform. The results from a spatio-temporal coherence processing technique of ours demonstrated a significant improvement in the SNR and vasculature contrast when compared with the existing ultrafast Power Doppler (PD) using the same data. Initial perfusion quantification using LFR imaging was also demonstrated. Mean time intensity curve and some parametric measures were generated. Combining both structural and functional perfusion imaging using the multiframe rate sequences, a better evaluation of the tumour angiogenesis can be assessed

Quantification of vaporized targeted nanodroplets using high-frame-rate ultrasound and optics

Owing to their ability to efficiently deliver biological cargo and sense the intracellular milieu, vertical arrays of high aspect ratio nanostructures, known as nanoneedles,are being developed as minimally invasive tools for cell manipulation. However, little is known of the mechanisms of cargo transfer across the cell membrane-nanoneedle interface. Particularly,the contributions of membrane piercing, modulation of membrane permeability and endocytosis to cargo transfer remain largelyunexplored. Here, combining state-of-the-art electron and scanning ion conductance microscopy with molecular biology techniques, we show that porous silicon nanoneedle arrays concurrently stimulate independent endocytic pathways which contribute to enhanced biomolecule delivery into human mesenchymal stem cells. Electron microscopy of the cell membrane at nanoneedle sites shows an intact lipid bilayer, accompanied by an accumulation of clathrin-coated pits and caveolae. Nanoneedles enhance the internalisation of biomolecular markers of endocytosis, highlighting the concurrent activation of caveolae-and clathrin-mediated endocytosis, alongside macropinocytosis. These events contribute to the nanoneedle-mediated delivery (nanoinjection) of nucleic acids into human stem cells, which distribute across the cytosol and the endolysosomal system. This data extends the understanding of how nanoneedles modulate biological processes to mediate interaction with the intracellular space, providing indications for the rational design of improved cell-manipulation technologies

Acoustic wave sparsely activated localization microscopy (AWSALM): super-resolution ultrasound imaging using acoustic activation and deactivation of nanodroplets

Photo-activated localization microscopy (PALM) has revolutionized the field of fluorescence microscopy by breaking the diffraction limit in spatial resolution. In this study, “acoustic wave sparsely activated localization microscopy (AWSALM),” an acoustic counterpart of PALM, is developed to super-resolve structures which cannot be resolved by conventional B-mode imaging. AWSALM utilizes acoustic waves to sparsely and stochastically activate decafluorobutane nanodroplets by acoustic vaporization and to simultaneously deactivate the existing vaporized nanodroplets via acoustic destruction. In this method, activation, imaging, and deactivation are all performed using acoustic waves. Experimental results show that sub-wavelength micro-structures not resolvable by standard B-mode ultrasound images can be separated by AWSALM. This technique is flow independent and does not require a low concentration of contrast agents, as is required by current ultrasound super resolution techniques. Acoustic activation and deactivation can be controlled by adjusting the acoustic pressure, which remains well within the FDA approved safety range. In conclusion, this study shows the promise of a flow and contrast agent concentration independent super-resolution ultrasound technique which has potential to be faster and go beyond vascular imaging

Multi-frame rate plane wave contrast-enhanced ultrasound imaging for tumour vascular imaging and perfusion quantification

Angiogenesis and blood flow dynamics play an important role in the development of malignant tumours and their response to treatment. While contrast enhanced ultrasound (CEUS) imaging with microbubble contrast agents as a tool for imaging angiogenesis and flow dynamics has shown great potential [1], recent development of plane wave high frame-rate (HFR) CEUS has offered new opportunities in such applications. In this study, we demonstrate an interleaved multi-frame rate plane wave CEUS imaging to quantify perfusion and to image vascular structure with improved resolution and contrast

Surface Charge Measurement of SonoVue, Definity and Optison: A Comparison of Laser Doppler Electrophoresis and Micro-Electrophoresis

Microbubble (MB) contrast-enhanced ultrasonography is a promising tool for targeted molecular imaging. It is important to determine the MB surface charge accurately as it affects the MB interactions with cell membranes. In this article, we report the surface charge measurement of SonoVue, Definity and Optison. We compare the performance of the widely used laser Doppler electrophoresis with an in-house micro-electrophoresis system. By optically tracking MB electrophoretic velocity in a microchannel, we determined the zeta potentials of MB samples. Using micro-electrophoresis, we obtained zeta potential values for SonoVue, Definity and Optison of −28.3, −4.2 and −9.5 mV, with relative standard deviations of 5%, 48% and 8%, respectively. In comparison, laser Doppler electrophoresis gave −8.7, +0.7 and +15.8 mV with relative standard deviations of 330%, 29,000% and 130%, respectively. We found that the reliability of laser Doppler electrophoresis is compromised by MB buoyancy. Micro-electrophoresis determined zeta potential values with a 10-fold improvement in relative standard deviation

Investigating CXCR4 expression of tumor cells and the vascular compartment: A multimodal approach

The C-X-C chemokine receptor 4 (CXCR4) is G protein-coupled receptor that upon binding to its cognate ligand, can lead to tumor progression. Several CXCR4-targeted therapies are currently under investigation, and with it comes the need for imaging agents capable of accurate depiction of CXCR4 for therapeutic stratification and monitoring. PET agents enjoy the most success, but more cost-effective and radiation-free approaches such as ultrasound (US) imaging could represent an attractive alternative. In this work, we developed a targeted microbubble (MB) for imaging of vascular CXCR4 expression in cancer. A CXCR4-targeted MB was developed through incorporation of the T140 peptide into the MB shell. Binding properties of the T140-MB and control, non-targeted MB (NT-MB) were evaluated in MDA-MB-231 cells where CXCR4 expression was knocked-down (via shRNA) through optical imaging, and in the lymphoma tumor models U2932 and SuDHL8 (high and low CXCR4 expression, respectively) by US imaging. PET imaging of [18F]MCFB, a tumor-penetrating CXCR4-targeted small molecule, was used to provide whole-tumor CXCR4 readouts. CXCR4 expression and microvessel density were performed by immunohistochemistry analysis and western blot. T140-MB were formed with similar properties to NT-MB and accumulated sensitively and specifically in cells according to their CXCR4 expression. In NOD SCID mice, T140-MB persisted longer in tumors than NT-MB, indicative of target interaction, but showed no difference between U2932 and SuDHL8. In contrast, PET imaging with [18F]MCFB showed a marked difference in tumor uptake at 40–60 min post-injection between the two tumor models (p<0.05). Ex vivo analysis revealed that the large differences in CXCR4 expression between the two models are not reflected in the vascular compartment, where the MB are restricted; in fact, microvessel density and CXCR4 expression in the vasculature was comparable between U2932 and SuDHL8 tumors. In conclusion, we successfully developed a T140-MB that can be used for imaging CXCR4 expression in the tumor vasculature

Anti-angiogenic alternatives to VEGF blockade

Angiogenesis is a major requirement for tumour formation and development. Anti-angiogenic treatments aim to starve the tumour of nutrients and oxygen and also guard against metastasis. The main anti-angiogenic agents to date have focused on blocking the pro-angiogenic vascular endothelial growth factors (VEGFs). While this approach has seen some success and has provided a proof of principle that such anti-angiogenic agents can be used as treatment, the overall outcome of VEGF blockade has been somewhat disappointing. There is a current need for new strategies in inhibiting tumour angiogenesis; this article will review current and historical examples in blocking various membrane receptors and components of the extracellular matrix important in angiogenesis. Targeting these newly discovered pro-angiogenic proteins could provide novel strategies for cancer therapy