Elucidating the mechanisms underlying cancer progression and identifying tailored therapies for patients can be enhanced by using patient-derived preclinical models. In this study, we investigated whether patient-derived xenograft (PDX) models of well-differentiated neuroendocrine tumors (NETs) could be established in the avian embryo ex ovo model. We found that fresh surgical tumor samples from well-differentiated primary small-intestine and pancreatic NETs, as well as metastatic sites, exhibited engraftment rates exceeding 80% in the avian embryo model. The NET PDXs in the avian model preserved the distinct histological features of NETs, including characteristic tumor nests and the ‘salt and pepper’ chromatin pattern in nuclei. Using immunostaining, we showed that the engrafted patient tumor fragments remained viable and maintained the proliferation rate, e.g. tumor grade, of the corresponding patient tumors. The NET PDXs continued to express characteristic neuroendocrine markers, such as the insulinoma-associated 1 (INSM1) transcription factor and chromogranin A (CgA). Importantly, they also retained the patient’s somatostatin receptor 2 (SSTR2) expression pattern in cancer cells, which is the target of radioligand therapy. Using high-frequency ultrasound imaging and immunostaining, we also demonstrated that the engrafted tumor specimens were vascularized and exhibited functional blood perfusion. Overall, this is the first study to demonstrate the feasibility and characterize PDXs of well-differentiated NETs in the avian embryo model.

Patient-derived xenograft (PDX) tumor models initiated in avian chorioallantoic membranes (CAM) are under investigation to evaluate the effectiveness of therapeutic options with the objective of personalizing treatments. CAM PDXs paired with ultra-high frequency ultrasound (UHFUS) imaging could potentially constitute prospective high throughput assays that can rapidly assess tumor volume and vascular response to therapy. To date, little work has been conducted to adapt and validate UHFUS flow imaging methods to CAM tumor models. Here we report the development and evaluation of an imaging pipeline for UHFUS detection of microvascular flow in a CAM tumor model using interframe subtraction (IS) to suppress tissue clutter. The IS pipeline included a tissue motion compensation (MC) stage prior to clutter filtering and was compared to a singular value decomposition (SVD) clutter filter. The performance was evaluated using UHFUS data acquired in phantom and in vivo sunitinib-treated renal cell carcinoma. MC substantially reduced tissue motion effects. MC + IS was comparable to MC + SVD filtering at detecting flow within tumors. The results for both IS and SVD filters were dependent on the details of implementation. The UHFUS imaging methods detected a significant decrease in blood flow metrics in treated versus control tumors. An effective imaging pipeline was developed for the assessment of the treatment response of CAM PDX models in a clinically relevant timeframe. The MC + IS approach implemented on B-scan image derived data is less computationally intensive and can be used with widely available UHFUS systems.

Intraluminal photoacoustic (PA) imaging has the potential for providing physiological and functional information in wide-ranging clinical applications. Along with endoluminal ultrasound transducers, these applications require compact light delivery devices which can deliver high-energy ns-pulsed laser to the target region. In this work, we describe the design, method of fabrication and characterization of a new compact, side-fire optical fiber that can deliver high-energy laser pulses for PA imaging. Side-fire illuminators were fabricated using UV laser ablation to create windows on the side of a 1.5 mm diameter single core, multi-mode optical fiber with a reflective silver coating and a beveled end. Devices with 10 mm, 20 mm, and 30 mm window lengths were fabricated and their beam profiles characterized. Elongated side-fire fibers with −6 dB beam size up to 30.79 mm × 5.5 mm were developed. A side-fire to total output ratio of up to 0.69 and a side fire efficiency of up to 40%, relative to a standard front-fire fiber, were achieved. We evaluated the effects of high-energy ns-pulsed light propagation on the fiber by coupling the fiber to 18 mJ or 100 MW/cm2 (at 750 nm) beam from a Q-switched laser. The PA imaging with the fiber was demonstrated by detecting India ink targets embedded in chicken breast tissue over the full length of a 20 mm illumination window and over a 100° angle and by visualizing in vivo the rat ear vasculature.

OBJECTIVE:High-frequency ultrasound elastography (USE) can measure the mechanical properties of biomaterials and engineered tissues in vitro. Previously developed USE systems have been limited by contact acoustic radiation force (ARF) excitations and insufficient spatiotemporal resolution for sub-millimetre sub-surface mechanical property measurements. METHODS:We present a novel high-frequency USE system with a highly focused (f-number 1) 15 MHz ARF excitation transducer and a broadband (f-number 3) 40 MHz ARF tracking transducer. RESULTS:When comparing shear moduli measured via USE with shear rheometry, shear moduli of 1% and 5% agar-silica phantoms estimated by USE, were 8.8 ± 2.2 kPa and 117.0 ± 12.3 kPa (8.0 ± 0.4 kPa by rheometry, p = 0.573 for 1%; 114.4 ± 7.2 kPa, p = 0.777 for 5%) and oil-agar silica phantoms were 105.0 ± 3.4 kPa (0%) and 77.0 ± 22.1 kPa (10%) by USE (101.0 ± 4.8 kPa by rheometry; p = 0.311 for 0%; 75.8 ± 5.3 kPa; p = 0.938 for 10%). The speed of sound, acoustic impedance, and acoustic attenuation of these samples were also determined. We also used in silico analysis to mimic our experimental system and analyze the spectral content of the resulting shear waves in elastic and viscoelastic tissues with parametric changes to the ARF excitation duration, shear modulus, and viscosity. Notably, we observed a nonlinear dependency of shear wave frequency on ARF excitation duration and material properties, where shear wave frequency was most sensitive to tissue elastic modulus at longer ARF durations but more sensitive to tissue viscosity at shorter ARF durations. CONCLUSION:Our system enables noninvasive, nondestructive estimation of the mechanical properties of thin biomaterials via focused axial localization of the ARF, opening new avenues for future USE applications in engineered tissue systems.

Photoacoustic imaging adds functional information on the conventional B-mode ultrasound images. Combining photoacoustic information with micro-ultrasound has potential to aid localization and delineation of cancerous lesions to be targeted for focal therapies. The photoacoustic signal in the tumor is primarily generated by hemoglobin but exogenous photoacoustic contrast agents such as novel, biocompatible porphysome nanoparticles can be used to enhance photoacoustic signal. This work demonstrates 3D in vivo micro-ultrasound and photoacoustic imaging of porphysome nanoparticles in a subcutaneous mouse tumor model up to 48 hours after the tail-vein injection. Linear spectral unmixing was used to separate the photoacoustic signal contribution from oxygenated, deoxygenated hemoglobin and porphysomes. 3D imaging revealed the spatial distribution of nanoparticles in the tumor over time. Longitudinal imaging with porphysomes showed that the photoacoustic signal from contrast agent increased after the injection and was co-localized with oxygenated hemoglobin. The presence of nanoparticles in the tumor was also confirmed by the fluorescence imaging which showed the signal peak at 24 hours post-injection potentially due to disassociation of the particle in vivo. Fluorescence histology also confirmed the presence of nanoparticles in the tumor.

The acoustic angiography leverages the superharmonic response of microbubbles against linear tissue to generate 3-D maps of microvasculature. This contrast-enhanced ultrasound imaging approach uses dual-frequency (DF) transducers that transmit at frequencies less than 5 MHz and receive at frequencies three times or greater than the fundamental frequency to selectively detect microbubble signals. Previous iterations of the hardware were designed mainly to image preclinical models. In pilot clinical imaging studies, these transducers suffered from poor microbubble sensitivity and shallow imaging depths. Here, we investigate multiple DF transducers operating at varying transmit frequencies less than 2 MHz and center receive frequencies ranging from 7 to 18 MHz designed for deeper imaging and greater bubble sensitivity than earlier generation devices. We assess the superharmonic imaging (SpHI) performance of these transducers in vitro and in vivo by characterizing contrast sensitivity and resolution. We demonstrate improvements in sensitivity at lower transmit (<1 MHz) and receive (<10 MHz) frequencies, measuring contrast signal enhancement up to 31.8 dB. At these lower frequencies, we also achieve imaging depths up to 50-55 mm-the deepest application of acoustic angiography to date. These advances in imaging sensitivity and depth address the primary barriers to the clinical translation of acoustic angiography.

The study of vascular diseases such as stroke can be advanced by characterizing hemodynamic changes in the microvasculature. While contrast-enhanced ultrasound has been effective for imaging rat brain vasculature, its sensitivity is limited for slow flow and the smallest vessels. Superharmonic contrast imaging, which exploits the nonlinear response of microbubbles to ultrasound waves, has the potential to improve the detection in the microvasculature by providing a higher contrast-to-tissue ratio. In this work, we present the fabrication and in vitro characterization of the 4 & 40 MHz dual-frequency probe designed for rat brain imaging. The probe integrates a high-frequency linear array (40 MHz center frequency) and two flanking low-frequency elements (4 MHz center frequency) in the elevation direction. Acoustic pressure measurements verified the alignment of the low-frequency transmit focus with the high-frequency receive beam. To assess the sensitivity and imaging performance under realistic conditions, a flow phantom incorporating a rat skull was developed. Results showed that the probe successfully detected microbubble superharmonic signals while suppressing bone-related artifacts. Compared to conventional B-mode and plane-wave imaging, superharmonic contrast imaging provided a CTR improvement of 22 dB at 7 mm depth.

Compact high-frequency arrays are of interest for clinical and preclinical applications in which a small-footprint or endoscopic device is needed to reach the target anatomy. However, the fabrication of compact arrays entails the connection of several dozens of small elements to the imaging system through a combination of flexible printed circuit boards at the array end and micro-coaxial cabling to the imaging system. The methods currently used, such as wire bonding, conductive adhesives, or a dry connection to a flexible circuit, considerably increase the array footprint. Here, we propose an interconnection method that uses vacuum-deposited metals, laser patterning, and electroplating to achieve a right-angle, compact, reliable connection between array elements and flexible-circuit traces. The array elements are thickened at the edges using patterned copper traces, which increases their cross-sectional area and facilitates the connection. We fabricated a 2.3 mm by 1.7 mm, 64-element linear array with elements at a 36 μm pitch connected to a 4 cm long flexible circuit, where the interconnect adds only 100 μm to each side of the array. Pulse-echo measurements yielded an average center frequency of 55 MHz and a −6 dB bandwidth of 41%. We measured an imaging resolution of 35 μm in the axial direction and 114 μm in the lateral direction and demonstrated the ex vivo imaging of porcine esophageal tissue and the in vivo imaging of avian embryonic vasculature.

Superharmonic imaging (SpHI) using dual-frequency probes enables high-contrast microvasculature imaging by taking advantage of higher order harmonics of the broadband nonlinear response from microbubble (MB) contrast agents. We previously introduced a DF probe with a low-frequency (LF, 2 MHz; 32 elements) array behind a high-frequency (HF, 21 MHz; 256 elements) array and demonstrated SpHI with conventional walking-aperture approaches which limit acquisition rates. In this work, ultrafast imaging is investigated to overcome this challenge. We demonstrate SpHI with plane waves and coherent compounding in vitro and in vivo while evaluating acquisition frame rates. LF plane waves were implemented on VevoF2 systems (FUJIFILM Visualsonics, Toronto) with beam steering enabled by element-specific delays (9 angles between ±10°, step: 2.5°). All SpHI images showed almost complete suppression of tissue clutter to the background noise level. A 2.5 dB contrast improvement was found in vitro with coherent compounding . Tumor perfusion and fine vascular structures were visualized in vivo. SpHI acquisition frame rate reached 3.5 kHz at 0° and 396 Hz with 9 angles, ∼40 times that of walking-aperture approaches. These results demonstrate plane wave imaging approaches can increase SpHI frame rates while maintaining a high image contrast for visualizing vasculature, enabling SpHI for fast flow imaging.

Notwithstanding recanalization treatments in the acute stage of stroke, many survivors suffer long-term impairments. Physical rehabilitation is the only widely available strategy for chronic-stage recovery, but its optimization is hindered by limited understanding of its effects on brain structure and function. Using micro-ultrasound, behavioral testing, and electrophysiology, we investigated the impact of skilled reaching rehabilitation on cerebral hemodynamics, motor function, and neuronal activity in a rat model of focal ischemic stroke. A 50 MHz micro-ultrasound transducer and intracortical electrophysiology were utilized to characterize neurovascular changes three weeks following focal ischemia elicited by endothelin-1 injection into the sensorimotor cortex. Sprague-Dawley rats were rehabilitated through tray reaching, and their fine skilled reaching was assessed via the Montoya staircase. Focal ischemia led to a sustained deficit in forelimb reaching; and increased tortuosity of the penetrating vessels in the perilesional cortex; with no lateralization of spontaneous neuronal activity. Rehabilitation improved skilled reaching; decreased cortical vascularity; was associated with elevated peri- vs. contralesional hypercapnia-induced flow homogenization and increased perilesional spontaneous cortical neuronal activity. Our study demonstrated neurovascular plasticity accompanying rehabilitation-elicited functional recovery in the subacute stage following stroke, and multiple micro-ultrasound-based markers of cerebrovascular structure and function modified in recovery from ischemia and upon rehabilitation.

Catheter based procedures are typically guided by X-Ray, which suffers from low soft tissue contrast and only provides 2D projection images of a 3D volume. Intravascular ultrasound (IVUS) can serve as a complementary imaging technique. Forward viewing catheters are useful for visualizing obstructions along the path of the catheter. The CathEye system mechanically steers a single-element transducer to generate a forward-looking surface reconstruction from an irregularly spaced 2-D scan pattern. The steerable catheter leverages an expandable frame with cables to manipulate the distal end independently of vessel tortuosity. The tip position is estimated by measuring the cable displacements and used to create surface reconstructions of the imaging workspace with the single-element transducer. CathEye's imaging capabilities were tested with an agar phantom and an ex vivo chronic total occlusion (CTO) sample while the catheter was confined to various tortuous paths. The CathEye maintained similar scan patterns regardless of path tortuosity and was able to recreate major features of the imaging targets, such as holes and extrusions. The feasibility of forward-looking IVUS with the CathEye is demonstrated in this study. The CathEye mechanism can be applied to other imaging modalities with field-of-view (FOV) limitations and represents the basis for an interventional device fully integrated with image guidance.

Objective: High-frequency, high-resolution transrectal micro-ultrasound (micro-US: >= 15 MHz) imaging of the prostate is emerging as a beneficial tool for scoring disease risk and accurately targeting biopsies. Adding photoacoustic (PA) imaging to visualize abnormal vascularization and accumulation of contrast agents in tumors has potential for guiding focal therapies. In this work, we describe a new imaging platform that combines a transrectal micro-US system with transurethral light delivery for PA imaging. Methods: A clinical transrectal micro-US system was adapted to acquire PA images synchronous to a tunable laser pulse. A transurethral side-firing optical fiber was developed for light delivery. A polyvinyl chloride (PVC)-plasti- sol phantom was developed and characterized to image PA contrast agents in wall-less channels. After resolution measurement in water, PA imaging was demonstrated in phantom channels with dyes and biodegradable nanoparticle contrast agents called porphysomes. In vivo imaging of a tumor model was performed, with porphysomes administered intravenously. Results: Photoacoustic imaging data were acquired at 5 Hz, and image reconstruction was performed offline. PA image resolution at a 14-mm depth was 74 and 261 mu m in the axial and lateral directions, respectively. The speed of sound in PVC-plastisol was 1383 m/s, and the attenuation was 4 dB/mm at 20 MHz. PA signal from porphysomes was spectrally unmixed from blood signals in the tumor, and a signal increase was observed 3 h after porphysome injection. Conclusion: A combined transrectal micro-US and PA imaging system was developed and characterized, and in vivo imaging demonstrated. High-resolution PA imaging may provide valuable additional information for diagnostic and therapeutic applications in the prostate.

Objective: Endobronchial ultrasound (EBUS) is commonly used to guide transbronchial needle biopsies for the staging of lymph nodes in non-small cell lung cancer patients. Although contrast-enhanced ultrasound (CEUS) and microbubbles (MBs) can improve the diagnostic accuracy in tumors, the ability of contrast-enhanced EBUS (CE-EBUS) to image MBs has not yet been comprehensively evaluated. In this study, we assessed the ability of a CE-EBUS system (Olympus EU-ME2 PREMIER and BF-UC180F bronchoscope) to detect laboratory-synthesized MBs in comparison to clinical (Toshiba Aplio SSA-790A) and pre-clinical (VisualSonics Vevo 2100) CEUS systems in vitro and in vivo, respectively.Methods: Agar flow phantoms and reference tissue were used to assess CE-EBUS MB imaging in vitro, and A549 tumor-bearing athymic nude and AE17-OVA tumor-bearing C57BL/6 mice were used to assess MB detectability and perfusion in vivo, respectively. Results: Results revealed that despite the lower sensitivity of CE-EBUS to MB concentration in comparison to clini-cal CEUS, CE-EBUS yielded a similar contrast-to-tissue ratio (CTR) in vitro of 28.9 +/- 4.5 dB for CE-EBUS, compared with 29.7 +/- 2.6 dB for clinical CEUS (p < 0.05). In vivo, CE-EBUS generated a perfusion curve highly correlated with that obtained with the pre-clinical CEUS system (Pearson correlation coefficient = 0.927, p < 0.05). More-over, CE-EBUS yielded a CTR 2.7 times higher than that obtained with the pre-clinical ultrasound system.Conclusion: These findings together suggest that CE-EBUS can perform contrast imaging comparable to that pro-duced by commercial pre-clinical and clinical ultrasound systems, with potential for clinical characterization of mediastinal lymph nodes in lung cancer patients.

This dataset includes raw acquired ultrasound data, processing scripts, and statistical data for acoustic property characterization of cell-free and cell-seeded fibrin hydrogels.

Superharmonic imaging (SpHI) using dual-frequency (DF) transducers enables high-contrast microvasculature imaging while suppressing tissue clutter. It takes advantage of the high-frequency components within the nonlinear response of microbubble contrast agents when excited with a low-frequency pulse. SpHI has been demonstrated with array-based DF transducers with traditional line-by-line imaging schemes where the acquisition frame rate is limited by the number of image lines needed over the imaging field-of-view. In this work, we implement plane wave imaging approaches on programmable systems and investigate the image acquisition frame rate and image quality of SpHI in vitro and in vivo, using an integrated DF probe comprising a 21 MHz (high-frequency; 256 elements) array stacked on a 2 MHz (low-frequency; 32 elements) array. Micro-ultrasound imaging with high-frequency plane wave transmission at 25 steering angles and coherent compounding on receive was demonstrated in vivo, which showed good image contrast and resolution compared to traditional line-by-line imaging, while achieving an acquisition frame rate of 158 Hz. The 5-angle coherently compounded in vitro SpHI images showed ~4 dB improvement in tissue clutter suppression compared to images reconstructed from 0° steering. With 5-angle compounding, the acquisition frame rate in SpHI reached 588 Hz for a 30 mm imaging depth, moving SpHI towards 2D real-time imaging.

Photoacoustic imaging using a micro-ultrasound system adds physiological information to high-resolution ultrasound images. This work demonstrates the in vivo imaging capability of a combined micro-ultrasound photoacoustic imaging system. A photoacoustic adapted, 22.5 MHz, 512-element transrectal ultrasound system was used to image human prostate cancer (PC3) grown on the hind limb of a nude mouse. Both endogenous chromophores i.e., oxygenated, and deoxygenated hemoglobin and an exogenous biocompatible photoacoustic contrast agent called porphysome nanoparticles were imaged in vivo. Spectral unmixing was used to separate the photoacoustic signals from oxygenated, deoxygenated hemoglobin and porphysomes. Longitudinal imaging with porphysomes revealed that the photoacoustic signal from contrast agent peaks at 6 hours after the injection and degrades gradually potentially due to disassociation of the particle in vivo.

Ultrasound molecular imaging (USMI) is a technique used to noninvasively estimate the distribution of molecular markers in vivo by imaging microbubble contrast agents (MCAs) that have been modified to target receptors of interest on the vascular endothelium. USMI is especially relevant for preclinical and clinical cancer research and has been used to predict tumor malignancy and response to treatment. In the last decade, methods that improve the resolution of contrast-enhanced ultrasound by an order of magnitude and allow researchers to noninvasively image individual capillaries have emerged. However, these approaches do not translate directly to molecular imaging. In this work, we demonstrate super-resolution visualization of biomarker expression in vivo using superharmonic ultrasound imaging (SpHI) with dual-frequency transducers, targeted contrast agents, and localization microscopy processing. We validate and optimize the proposed method in vitro using concurrent optical and ultrasound microscopy and a microvessel phantom. With the same technique, we perform a proof-of-concept experiment in vivo in a rat fibrosarcoma model and create maps of biomarker expression co-registered with images of microvasculature. From these images, we measure a resolution of 23 μm, a nearly fivefold improvement in resolution compared to previous diffraction-limited molecular imaging studies.

Endoscopic micro-ultrasound can potentially im-prove imaging of diseased tissue structure and function if the probe can be positioned near the tissue of interest. We previously reported the fabrication of miniaturized linear arrays using thin, high-density flexible printed circuit boards (FPCBs) connected to array elements with laser patterned deposited metals. Here we extend previous work to limit the area used for electrical connection to array elements and place element signal electrodes on the back surface of the array so the ground electrode can be at the front face and we use extended electroplated signal electrodes to create bond-pads at the edges to achieve an endoscopic form factor. We demonstrate fabrication with a forward-viewing, 50 MHz, 64-element linear array that fits within a 3-mm-diameter lumen. The array was 2.5 mm (azimuth) by 1.6 mm (elevation), had a mean resonant frequency of 48.2 MHz and a mean kt of 0.41. Laser Doppler vibrometry measurements allow further investigation of the device performance. Phantom images are shown to demonstrate array capability.

There has been growing interest in nanobubbles (NBs) for vascular and extravascular ultrasound contrast imaging and therapeutic applications. Studies to date have generally utilized low frequencies (<12 MHz), high concentrations (>109 mL-1), and uncalibrated B-mode or contrast-mode on commercial systems without reporting investigations on NB signatures upon which the imaging protocols should be based. We recently demonstrated that low concentrations (106 mL-1) of porphyrin-lipid-encapsulated NBs scatter nonlinearly at low (2.5, 8 MHz) and high (12.5, 25, 30 MHz) frequencies in a pressure threshold-dependent manner that is advantageous for amplitude modulation (AM) imaging. Here, we implement pressure-calibrated AM at high frequency on a commercial preclinical array system to enhance sensitivity to nonlinear scattering of three phospholipid-based NB formulations. With this approach, improvements in contrast to tissue ratio relative to B-mode between 12.4 and 22.8 dB are demonstrated in a tissue-mimicking phantom, and between 6.7 and 14.8 dB in vivo.