Adam J. Dixon

Simultaneous photoacoustic microscopy of microvascular anatomy, oxygen saturation, and blood flow.

Capitalizing on the optical absorption of hemoglobin, photoacoustic microscopy (PAM) is uniquely capable of anatomical and functional characterization of the intact microcirculation in vivo. However, PAM of the metabolic rate of oxygen (MRO2) at the microscopic level remains an unmet challenge, mainly due to the inability to simultaneously quantify microvascular diameter, oxygen saturation of hemoglobin (sO2), and blood flow at the same spatial scale. To fill this technical gap, we have developed a multi-parametric PAM platform. By analyzing both the sO2-encoded spectral dependence and the flow-induced temporal decorrelation of photoacoustic signals generated by the raster-scanned mouse ear vasculature, we demonstrated-for the first time-simultaneous wide-field PAM of all three parameters down to the capillary level in vivo.

Enhanced Intracellular Delivery of a Model Drug Using Microbubbles Produced by a Microfluidic Device

Focal drug delivery to a vessel wall facilitated by intravascular ultrasound and microbubbles holds promise as a potential therapy for atherosclerosis. Conventional methods of microbubble administration result in rapid clearance from the bloodstream and significant drug loss. To address these limitations, we evaluated whether drug delivery could be achieved with transiently stable microbubbles produced in real time and in close proximity to the therapeutic site. Rat aortic smooth muscle cells were placed in a flow chamber designed to simulate physiological flow conditions. A flow-focusing microfluidic device produced 8 μm diameter monodisperse microbubbles within the flow chamber, and ultrasound was applied to enhance uptake of a surrogate drug (calcein). Acoustic pressures up to 300 kPa and flow rates up to 18 mL/s were investigated. Microbubbles generated by the flow-focusing microfluidic device were stabilized with a polyethylene glycol-40 stearate shell and had either a perfluorobutane (PFB) or nitrogen gas core. The gas core composition affected stability, with PFB and nitrogen microbubbles exhibiting half-lives of 40.7 and 18.2 s, respectively. Calcein uptake was observed at lower acoustic pressures with nitrogen microbubbles (100 kPa) than with PFB microbubbles (200 kPa) (p < 0.05, n > 3). In addition, delivery was observed at all flow rates, with maximal delivery (>70% of cells) occurring at a flow rate of 9 mL/s. These results demonstrate the potential of transiently stable microbubbles produced in real time and in close proximity to the intended therapeutic site for enhancing localized drug delivery.

Oscillatory Dynamics and In Vivo Photoacoustic Imaging Performance of Plasmonic Nanoparticle‐Coated Microbubbles

Microbubbles bearing plasmonic nanoparticles on their surface provide contrast enhancement for both photoacoustic and ultrasound imaging. In this work, the responses of microbubbles with surface-bound gold nanorods-termed AuMBs-to nanosecond pulsed laser excitation are studied using high-speed microscopy, photoacoustic imaging, and numerical modeling. In response to laser fluences below 5 mJ cm(-2) , AuMBs produce weak photoacoustic emissions and exhibit negligible microbubble wall motion. However, in reponse to fluences above 5 mJ cm(-2) , AuMBs undergo dramatically increased thermal expansion and emit nonlinear photoacoustic waves of over 10-fold greater amplitude than would be expected from freely dispersed gold nanorods. Numerical modeling suggests that AuMB photoacoustic responses to low laser fluences result from conductive heat transfer from the surface-bound nanorods to the microbubble gas core, whereas at higher fluences, explosive boiling may occur at the nanorod surface, producing vapor nanobubbles that contribute to rapid AuMB expansion. The results of this study indicate that AuMBs are capable of producing acoustic emissions of significantly higher amplitude than those produced by conventional sources of photoacoustic contrast. In vivo imaging performance of AuMBs in a murine kidney model suggests that AuMBs may be an effective alternative to existing contrast agents for noninvasive photoacoustic and ultrasound imaging applications.

All-optical photoacoustic microscopy based on plasmonic detection of broadband ultrasound

We report on an implementation of all-optical photoacoustic microscopy (PAM), which capitalizes on the effect of surface plasmon resonance (SPR) for optical detection of ultrasound. The SPR sensor in our all-optical PAM shows, experimentally, a linear response to the acoustic pressure from 5.2 kPa to 2.1 MPa, an ultra-flat frequency response (±0.7 dB) from 680 kHz to 126 MHz, and a noise-equivalent pressure sensitivity of 3.3 kPa. With the broadband ultrasonic detection, our SPR-PAM has achieved high spatial resolution with relatively low anisotropy (i.e., 2.0 μm laterally and 8.4 μm axially). Three-dimensional high-resolution imaging of a single melanoma cell is demonstrated.

Intravascular near-infrared fluorescence catheter with ultrasound guidance and blood attenuation correction

Abstract. Intravascular near-infrared fluorescence (NIRF) imaging offers a new approach for characterizing atherosclerotic plaque, but random catheter positioning within the vessel lumen results in variable light attenuation and can yield inaccurate measurements. We hypothesized that NIRF measurements could be corrected for variable light attenuation through blood by tracking the location of the NIRF catheter with intravascular ultrasound (IVUS). In this study, a combined NIRF-IVUS catheter was designed to acquire coregistered NIRF and IVUS data, an automated image processing algorithm was developed to measure catheter-to-vessel wall distances, and depth-dependent attenuation of the fluorescent signal was corrected by an analytical light propagation model. Performance of the catheter sensing distance correction method was evaluated in coronary artery phantoms and ex vivo arteries. The correction method produced NIRF estimates of fluorophore concentrations, in coronary artery phantoms, with an average root mean square error of 17.5%. In addition, the correction method resulted in a statistically significant improvement in correlation between spatially resolved NIRF measurements and known fluorophore spatial distributions in ex vivo arteries (from r=0.24 to 0.69, p<0.01, n=6). This work demonstrates that catheter-to-vessel wall distances, measured from IVUS images, can be employed to compensate for inaccuracies caused by variable intravascular NIRF sensing distances.

In vivo imaging of microfluidic-produced microbubbles.

Microfluidics-based production of stable microbubbles for ultrasound contrast enhancement or drug/gene delivery allows for precise control over microbubble diameter but at the cost of a low production rate. In situ microfluidic production of microbubbles directly in the vasculature may eliminate the necessity for high microbubble production rates, long stability, or small diameters. Towards this goal, we investigated whether microfluidic-produced microbubbles directly administered into a mouse tail vein could provide sufficient ultrasound contrast. Microbubbles composed of nitrogen gas and stabilized with 3 % bovine serum albumin and 10 % dextrose were injected for 10 seconds into wild type C57BL/6 mice, via a tail-vein catheter. Short-axis images of the right and left ventricle were acquired at 12.5 MHz and image intensity over time was analyzed. Microbubbles were produced on the order of 105 microbubbles/s and were observed in both the right and left ventricles. The median rise time, duration, and decay time within the right ventricle were 2.9, 21.3, and 14.3 s, respectively. All mice survived the procedure with no observable respiratory or heart rate distress despite microbubble diameters as large as 19 μm.

Microbubble-mediated intravascular ultrasound imaging and drug delivery

Intravascular ultrasound (IVUS) provides radiation-free, real-time imaging and assessment of atherosclerotic disease in terms of anatomical, functional, and molecular composition. The primary clinical applications of IVUS imaging include assessment of luminal plaque volume and real-time image guidance for stent placement. When paired with microbubble contrast agents, IVUS technology may be extended to provide nonlinear imaging, molecular imaging, and therapeutic delivery modes. In this review, we discuss the development of emerging imaging and therapeutic applications that are enabled by the combination of IVUS imaging technology and microbubble contrast agents.

Imaging Performance of a Handheld Ultrasound System With Real-time Computer-aided Detection of Lumbar Spine Anatomy: A Feasibility Study.

Objectives The aim of this study was to evaluate the imaging performance of a handheld ultrasound system and the accuracy of an automated lumbar spine computer-aided detection (CAD) algorithm in the spines of human subjects. Materials and Methods This study was approved by the institutional review board of the University of Virginia. The authors designed a handheld ultrasound system with enhanced bone image quality and fully automated CAD of lumbar spine anatomy. The imaging performance was evaluated by imaging the lumbar spines of 68 volunteers with body mass index between 18.5 and 48 kg/m2. The accuracy, sensitivity, and specificity of the lumbar spine CAD algorithm were assessed by comparing the algorithms results to ground-truth segmentations of neuraxial anatomy provided by radiologists. Results The lumbar spine CAD algorithm detected the epidural space with a sensitivity of 94.2% (95% confidence interval [CI], 85.1%–98.1%) and a specificity of 85.5% (95% CI, 81.7%–88.6%) and measured its depth with an error of approximately ±0.5 cm compared with measurements obtained manually from the 2-dimensional ultrasound images. The spine midline was detected with a sensitivity of 93.9% (95% CI, 85.8%–97.7%) and specificity of 91.3% (95% CI, 83.6%–96.9%), and its lateral position within the ultrasound image was measured with an error of approximately ±0.3 cm. The bone enhancement imaging mode produced images with 5.1- to 10-fold enhanced bone contrast when compared with a comparable handheld ultrasound imaging system. Conclusions The results of this study demonstrate the feasibility of CAD for assisting with real-time interpretation of ultrasound images of the lumbar spine at the bedside.

Synthesis of albumin microbubbles using a microfluidic device for real-time imaging and therapeutics

A method for producing monodisperse albumin stabilized microbubbles (MBs) using a flow-focusing microfluidic device (FFMD) is introduced. This method allows for localized delivery of short life-time microbubbles with a biocompatible shell, thereby potentially improving patient safety. In this study, microbubbles stabilized with bovine serum albumin (BSA) are characterized for microbubble coalescence, size, and production rate using a high speed camera. Microbubbles were produced with diameters between 10-20 μm and production rates up to 6×105 MB/s were achieved. Microbubble diameter was observed to decrease linearly (R2 > 0.99) with liquid flow rate. Microbubble stability was evaluated acoustically using a clinical ultrasound scanner. The image intensity of a well-mixed solution containing 13 μm microbubbles returned to baseline from peak intensity within 30s. Delivery of the membrane impermeable fluorophore calcein was demonstrated using microbubbles produced in situ within an in vitro flow chamber. 11 μm diameter microbubbles insonated at a peak negative pressure (PNP) of 200 kPA resulted in 58.0% of cells to uptake calcein. To demonstrate the ability to stabilize microbubbles directly with blood, plasma was separated from whole bovine blood, input into the FFMD, and used to produce microubbbles. These plasma microbubbles were imaged under flow in a gelatin flow phantom and demonstrated a 6.5 dB increase in lumen contrast.

Large diameter microbubbles produced by a catheter-sized microfluidic device for sonothrombolysis applications

Therapeutic approaches that enhance thrombolysis by combining tissue plasminogen activator (tPA), ultrasound (US), and/or microbubbles (MBs) are known generally as sonothrombolysis techniques. To date, sonothrombolysis clinical trials and experimental investigations have primarily utilized commercially available MB formulations (or derivatives thereof) with MB diameters generally in the range 1 - 4 μm. The restriction on MB diameter is due to a risk of gas emboli formation, which has left MBs outside of this diameter range virtually unexplored for sonothrombolysis applications. However, it is broadly understood that large MBs confer larger bioeffects when excited acoustically, as has been shown in sonoporation, blood brain barrier disruption, and sonothrombolysis applications. In support of the hypothesis that large MBs confer enhanced therapeutic effects, we demonstrate that MBs with diameters between 10 - 20 μm achieve a 4.5-fold increase in in vitro sonothrombolysis rates compared to MBs with diameters between 1 - 4 μm. In addition, we present the development of a catheter (1.5 mm diameter) containing a flow-focusing microfluidic device (FFMD) capable of producing large-diameter MBs suitable for catheter-directed sonothrombolysis applications. The microfluidically-produced MBs are comprised of N2 gas and a weak albumin/dextrose shell, which confers short MB half-lives and reduces the risk of gas emboli formation. Finally, we present the results of administering microfluidically produced MBs directly into the rat brain to demonstrate that large MBs with short lifetimes are safe for in vivo deployment.