Colon cancer is the fourth most common cancer worldwide. Despite its high incidence and mortality, many cases could be prevented through accurate early detection techniques that enable timely treatment. Studies using animal models of colon disease are essential to test new diagnostic approaches, including novel imaging instrumentation. We have previously used a 3-French ultrasound mini-probe to image the layered structure of the mouse colon, detect polyps, and visualize their invasion through the colon wall. In the present work, we investigate the use of an optical fiber (OF) with a machined tip that could be incorporated into endoluminal microultrasound biomicroscopy (e mu UBM). In the manufactured OF was perfomeda profile characterization and a microphotoacoustic images (mu PAI) in a mouse colon phantom. This integration may allow the future acquisition of mu PAI from a mouse model of colon tumor, providing a novel approach for imaging tumor progression in vivo.

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.

Design strategies that lead to a more focused in vivo delivery of functionalized nanoparticles (NPs) and their cargo can potentially maximize their therapeutic efficiency while reducing systemic effects, broadening their clinical applications. Here, we report the development of a noncovalent labeling approach where immunoglobulin G (IgG)-decorated NPs can be directed to a cancer cell using a simple, linear bispecific protein adaptor, termed MFE23-ZZ. MFE23-ZZ was created by fusing a single-chain fragment variable domain, termed MFE23, recognizing carcinoembryonic antigen (CEA) expressed on tumor cells, to a small protein ZZ module, which binds to the Fc fragment of IgG. As a proof of concept, monoclonal antibodies (mAbs) were generated against a NP coat protein, namely, gas vesicle protein A (GvpA) of Halobacterium salinarum gas vesicles (GVs). The surface of each GV was therapeutically derivatized with the photoreactive agent chlorin e6 (Ce6GVs) and anti-GvpA mAbs were subsequently bound to GvpA on the surface of each Ce6GV. The bispecific ligand MFE23-ZZ was then bound to mAb-decorated Ce6GVs via their Fc domain, resulting in a noncovalent tripartite complex, namely, MFE23.ZZ-2B10-Ce6GV. This complex enhanced the intracellular uptake of Ce6GVs into human CEA-expressing murine MC38 colon carcinoma cells (MC38.CEA) relative to the CEA-negative parental cell line MC38 in vitro, making them more sensitive to light-induced cell killing. These results suggest that the surface of NP can be rapidly and noncovalently functionalized to target tumor-associated antigen-expressing tumor cells using simple bispecific linkers and any IgG-labeled cargo. This noncovalent approach is readily applicable to other types of functionalized NPs.

The nonlinear behavior of microbubbles (MBs) is dependent on both the excitation pressure and the MB properties, which rely on the composition of the gas core and shell, as well as the size. Polydisperse MBs have a broad size distribution and only a subpopulation of them may be contributing to the higher order harmonics that are used for superharmonic imaging. High MB concentrations have been used for superharmonic imaging to produce nonlinear responses from the polydisperse MB solution for good image contrast. In this work, we investigate in vitro the contrast signal intensity and longivity with in-house polydisperse MBs and size-selected MBs of similar to 1.5, 2.2, and 4.3 mu m in diameter, comparing to commonly used MicroMarker MB solutions. We found the in-house 2.2 mu m MBs showed comparable mean contrast intensities and signal decay to MicroMarker. We evaluated these two populations in vivo on mouse kidneys over an approximately 18-minute imaging duration, and assessed quantitatively the contrast intensities and longevity of superharmonic signals. We found MicroMarker showed greater superharmonic contrast over the entire acquisition in vivo, with a half-life almost twice to that of the in-house 2.2 mu m MBs.

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.

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.

Representative cine-loop obtained using ultrasound biomicroscopic (UBM) imaging corresponding to MDA-MB-231 tumor xenograft treated with PBS (control) for a 3-week period.

Description of additional methods and procedures used in the study.

Bioluminescence imaging of mice bearing primary LM2-4 tumors (S1); Assessment of MVD, HIF-1α, and tumoral drug accumulation in PDX primary tumors (S2); Immunohistochemistry staining for CD31, CAIX, caspase 3, Ki67, and HIF-1α, as well as in vitro VEGF quantification (S3); CEUS imaging assessment of HCI-002 primary tumors (S4); Assessment of vessels with open lumen and tumor cell density in PDX tumors (S5); Time-intensity curves (TICs) of CEUS in primary MDA-MB-231/LM2-4 tumors (S6).

Construction of the composite histological images.

Legend for Supplemental Figure S1.

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.

It has been previously shown that high frequency ultrasound (20-100 MHz) can be used to detect cellular structure changes in tissues and cell ensembles. However, the changes seen in the backscattered ultrasound intensity and frequency spectrum are not fully understood. In this paper we attempt to better understand the nature of these changes by examination of the backscatter power spectra from cell ensembles (in pellet form) that have undergone two different types of cell death: by exposure to the chemotherapeutic cisplatin and by withdrawal of nutrients (decay). Three different ultrasound transducers were used, centered at 20MHz and 40MHz. In both death pathways, an increase of the midband fit of 10-12dB was measured, and there were significant changes in the spectral slopes. Furthermore, our initial analysis of the backscatter from single cells and polystyrene microspheres demonstrates the potential of the technique to assess scatterer size.