@article{rojas_joiner_velasco_bautista_aji_moore_beaumont_pylayeva-gupta_dayton_gessner_et al._2022, title={Validation of a combined ultrasound and bioluminescence imaging system with magnetic resonance imaging in orthotopic pancreatic murine tumors}, volume={12}, ISSN={["2045-2322"]}, DOI={10.1038/s41598-021-03684-z}, abstractNote={AbstractPreclinical mouse solid tumor models are widely used to evaluate efficacy of novel cancer therapeutics. Recent reports have highlighted the need for utilizing orthotopic implantation to represent clinical disease more accurately, however the deep tissue location of these tumors makes longitudinal assessment challenging without the use of imaging techniques. The purpose of this study was to evaluate the performance of a new multi-modality high-throughput in vivo imaging system that combines bioluminescence imaging (BLI) with robotic, hands-free ultrasound (US) for evaluating orthotopic mouse models. Long utilized in cancer research as independent modalities, we hypothesized that the combination of BLI and US would offer complementary advantages of detection sensitivity and quantification accuracy, while mitigating individual technological weaknesses. Bioluminescent pancreatic tumor cells were injected into the pancreas tail of C57BL/6 mice and imaged weekly with the combination system and magnetic resonance imaging (MRI) to serve as a gold standard. BLI photon flux was quantified to assess tumor activity and distribution, and US and MRI datasets were manually segmented for gross tumor volume. Robotic US and MRI demonstrated a strong agreement (R2 = 0.94) for tumor volume measurement. BLI showed a weak overall agreement with MRI (R2 = 0.21), however, it offered the greatest sensitivity to detecting the presence of tumors. We conclude that combining BLI with robotic US offers an efficient screening tool for orthotopic tumor models.}, number={1}, journal={SCIENTIFIC REPORTS}, author={Rojas, Juan D. and Joiner, Jordan B. and Velasco, Brian and Bautista, Kathlyne Jayne B. and Aji, Adam M. and Moore, Christopher J. and Beaumont, Nathan J. and Pylayeva-Gupta, Yuliya and Dayton, Paul A. and Gessner, Ryan C. and et al.}, year={2022}, month={Jan} }
 @article{sendi_yazdimamaghani_hu_sultanpuram_wang_moody_mccabe_zhang_graboski_li_et al._2022, title={Nanoparticle Delivery of miR-122 Inhibits Colorectal Cancer Liver Metastasis}, volume={82}, ISSN={["1538-7445"]}, DOI={10.1158/0008-5472.CAN-21-2269}, abstractNote={Abstract
               
                  
                  Liver metastasis is a leading cause of cancer morbidity and mortality. Thus, there has been strong interest in the development of therapeutics that can effectively prevent liver metastasis. One potential strategy is to utilize molecules that have broad effects on the liver microenvironment, such as miR-122, a liver-specific miRNA that is a key regulator of diverse hepatic functions. Here we report the development of a nanoformulation miR-122 as a therapeutic agent for preventing liver metastasis. We engineered a galactose-targeted lipid calcium phosphate (Gal-LCP) nanoformulation of miR-122. This nanotherapeutic elicited no significant toxicity and delivered miR-122 into hepatocytes with specificity and high efficiency. Across multiple colorectal cancer liver metastasis models, treatment with Gal-LCP miR-122 treatment effectively prevented colorectal cancer liver metastasis and prolonged survival. Mechanistic studies revealed that delivery of miR-122 was associated with downregulation of key genes involved in metastatic and cancer inflammation pathways, including several proinflammatory factors, matrix metalloproteinases, and other extracellular matrix degradation enzymes. Moreover, Gal-LCP miR-122 treatment was associated with an increased CD8+/CD4+ T-cell ratio and decreased immunosuppressive cell infiltration, which makes the liver more conducive to antitumor immune response. Collectively, this work presents a strategy to improve cancer prevention and treatment with nanomedicine-based delivery of miRNA.
               
               
                  Significance:
                  Highly specific and efficient delivery of miRNA to hepatocytes using nanomedicine has therapeutic potential for the prevention and treatment of colorectal cancer liver metastasis.
               }, number={1}, journal={CANCER RESEARCH}, author={Sendi, Hossein and Yazdimamaghani, Mostafa and Hu, Mengying and Sultanpuram, Nikhila and Wang, Jie and Moody, Amber S. and McCabe, Ellie and Zhang, Jiajie and Graboski, Amanda and Li, Liantao and et al.}, year={2022}, month={Jan}, pages={105–113} }
 @article{deruiter_markley_rojas_pinton_dayton_2020, title={Transient acoustic vaporization signatures unique to low boiling point phase change contrast agents enable super-resolution ultrasound imaging without spatiotemporal filtering}, volume={10}, ISSN={["2158-3226"]}, DOI={10.1063/5.0029207}, abstractNote={The unique activation signal of phase-change contrast agents (PCCAs or droplets) can be separated from the tissue signal and localized to generate super-resolution (SR) ultrasound (US) images. Lipid-shelled, perfluorocarbon PCCAs can be stochastically vaporized (activated) by a plane wave US transmission thereby enabling them to be used as separable targets for ultrasound localization microscopy. The unique signature of droplet vaporization imaging and the transient inherent nature of this signature increases signal contrast and therefore localization confidence, while the poor resolution of the low-frequency vaporization signal is overcome by the super-resolution result. Furthermore, our proposed PCCA SR technique does not require the use of user-dependent and flow-dependent spatio-temporal filtering via singular-value decomposition. Rather, matched filters selected by Fourier-domain analysis are able to identify and localize PCCA activations. Droplet SR was demonstrated in a crossed-microtube water phantom by localizing the activation signals of octafluoropropane nanodroplets (OFP, C3F8, −37 °C boiling point) to resolve 100 µm diameter fluorinated ethylene propylene tubes, which are ordinarily 35% smaller than the native diffraction-limited resolution of the imaging system utilized.}, number={10}, journal={AIP ADVANCES}, author={DeRuiter, R. M. and Markley, E. N. and Rojas, J. D. and Pinton, G. F. and Dayton, P. A.}, year={2020}, month={Oct} }
 @article{czernuszewicz_papadopoulou_rojas_rajamahendiran_perdomo_butler_harlacher_o'connell_zukic_dayton_et al._2019, title={A preclinical ultrasound platform for widefield 3D imaging of rodent tumors}, volume={79}, ISSN={["1538-7445"]}, DOI={10.1158/1538-7445.AM2019-1955}, abstractNote={Abstract
               Background: Preclinical ultrasound (US) and contrast-enhanced ultrasound (CEUS) imaging have long been used in oncology to noninvasively measure tumor volume and vascularity. While the value of preclinical US has been repeatedly demonstrated, these modalities are not without several key limitations that make them unattractive to cancer researchers, including: high user-variability, low throughput, and limited imaging field-of-view (FOV). Herein, we present a novel robotic preclinical US/CEUS system that addresses these limitations and demonstrates its use in evaluating tumors in 3D in a rodent model.
               Methods: The imaging system was designed to allow seamless whole-body 3D imaging, which requires rodents to be imaged without physical contact between the US transducer and the animal. To achieve this, a custom dual-element transducer was mounted on a robotic carriage, submerged in a hydrocarbon fluid, and the reservoir sealed with an acoustically transmissive top platform. Eight NOD/scid/gamma (NSG) female mice were injected subcutaneously in the flank with 8×109 786-O human clear-cell renal cell carcinoma (ccRCC) cells. Weekly imaging commenced after tumors reached a size of 150 mm3 and continued until tumors reached a maximum size of 1 cm3 (∼4-5 weeks). An additional six nude athymic female mice were injected subcutaneously in the flank with 7 × 105 SVR angiosarcoma cells to perform an inter-operator variability study. Imaging consisted of 3D B-mode (conventional ultrasound) of the whole abdomen (< 1 min), as well as contrast-enhanced acoustic angiography (< 10 min) to measure blood vessel density (BVD). Tumors were manually segmented in 3D (< 2 min) and inter-operator and inter-reader reliability was assessed with Krippendorff’s alpha.
               Results: Wide-field US images reconstructed from 3D volumetric data showed superior FOV over conventional US. Several anatomical landmarks could be identified within each image surrounding the tumor, including the liver, small intestines, bladder, and inguinal lymph nodes. Tumor boundaries were clearly delineated in both B-mode and BVD images, with BVD images showing heterogeneous microvessel density at later timepoints suggesting tumor necrosis. Excellent agreement was measured for both inter-reader and inter-operator experiments, with alpha coefficients of 0.914 (95% CI: 0.824-0.948) and 0.959 (0.911-0.981), respectively.
               Conclusion: We have demonstrated a novel preclinical US imaging system that can accurately and consistently evaluate tumors in rodent models. The system leverages cost-effective robotic technology, and a new scanning paradigm that allows for easy and reproducible data acquisition to enable wide-field, 3D, multi-parametric ultrasound imaging.
               Note: This abstract was not presented at the meeting.
               Citation Format: Tomasz Czernuszewicz, Virginie Papadopoulou, Juan D. Rojas, Rajalekha Rajamahendiran, Jonathan Perdomo, James Butler, Max Harlacher, Graeme O'Connell, Dzenan Zukic, Paul A. Dayton, Stephen Aylward, Ryan C. Gessner. A preclinical ultrasound platform for widefield 3D imaging of rodent tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1955.}, number={13}, journal={CANCER RESEARCH}, author={Czernuszewicz, Tomasz and Papadopoulou, Virginie and Rojas, Juan D. and Rajamahendiran, Rajalekha and Perdomo, Jonathan and Butler, James and Harlacher, Max and O'Connell, Graeme and Zukic, Dzenan and Dayton, Paul A. and et al.}, year={2019}, month={Jul} }
 @article{rojas_borden_dayton_2019, title={EFFECT OF HYDROSTATIC PRESSURE, BOUNDARY CONSTRAINTS AND VISCOSITY ON THE VAPORIZATION THRESHOLD OF LOW-BOILING-POINT PHASE-CHANGE CONTRAST AGENTS}, volume={45}, ISSN={["1879-291X"]}, DOI={10.1016/j.ultrasmedbio.2018.11.006}, abstractNote={The vaporization of low-boiling-point phase-change contrast agents (PCCAs) using ultrasound has been explored in vitro and in vivo. However, it has been reported that the pressure required for activation is higher in vivo, even after attenuation is accounted for. In this study, the effect of boundary constraints, hydrostatic pressure and viscosity on PCCA vaporization pressure threshold are evaluated to explore possible mechanisms for variations in in vivo vaporization behavior. Vaporization was measured in microtubes of varying inner diameter and a pressurized chamber under different hydrostatic pressures using a range of ultrasound pressures. Furthermore, the activation threshold was evaluated in the kidneys of rats. The results confirm that the vaporization threshold is higher in vivo and reveal an increasing activation threshold inversely proportional to constraining tube size and inversely proportional to surrounding viscosity in constrained environments. Counterintuitively, increased hydrostatic pressure had no significant effect experimentally on the PCCA vaporization threshold, although it was confirmed that this result was supported by homogeneous nucleation theory for liquid perfluorocarbon vaporization. These factors suggest that constraints caused by the surrounding tissue and capillary walls, as well as increased viscosity in vivo, contribute to the increased vaporization threshold compared with in vitro experiments, although more work is required to confirm all relevant factors.}, number={4}, journal={ULTRASOUND IN MEDICINE AND BIOLOGY}, author={Rojas, Juan D. and Borden, Mark A. and Dayton, Paul A.}, year={2019}, month={Apr}, pages={968–979} }
 @article{rojas_rajamahendiran_czernuszewicz_velasco_perdomo_harlacher_o'connell_butler_damania_dayton_et al._2019, title={Tracking angiogenesis induced microvascular changes in a lymphoma model via a new high throughput non-invasive dual modality imaging platform}, volume={79}, ISSN={["1538-7445"]}, DOI={10.1158/1538-7445.AM2019-1935}, abstractNote={Abstract
               This study presents a novel dual-modality imaging system for assessing cancer progression in rodents. The system incorporates bioluminescence imaging (BLI), used to assess tumor growth, and contrast-enhanced ultrasound (CEUS), used to assess anatomical information and map microvasculature. The combination of the two modalities has previously been shown to reduce inter-user variability of BLI quantification, and in this work, we demonstrate that a dual BLI/US system can provide a more holistic assessment of disease.
               NSG (NOD/scid/gamma) female mice were implanted with luc-tagged lymphoma cells (BCBL-1, RRID: CVCL_0165, 1x105 cells, intraperitoneal (IP) injection, N = 8 mice) and imaged using the US and BLI hybrid modality system (SonoVol, Inc.), and a BLI-alone system (Perkin Elmer, Inc.) for comparison to a widely available commercial BLI system. BLI sensitivity was evaluated using a weakly luminescent tritium phantom to find the shortest exposure required to detect signal. In vivo studies consisted of an IP injection of D-luciferin (250 µL at 15 mg/mL) and serial captures of images with exposure times of 60 s every 3 min. Acoustic Angiography (AA), a high-resolution CEUS technique, was used to acquire 3D volumes in the abdomen surrounding the tumor site to assess angiogenesis-induced vascular remodeling associated with tumor growth.
               In vitro BLI sensitivity experiments showed that the dual-modality system required an exposure of 3 sec to detect signal (p < 0.05) and the BLI-alone system required an exposure of 1 sec (p< 0.05). For in vivo studies, the change in luminescence occurring between week 2 and 3 post-cell implantation was calculated (a surrogate measurement for tumor growth), and the difference in signal was 17.15 ± 10.1 photons/sec and 16.04 ± 7.6 for the dual-modality and BLI-alone systems, respectively. Images of the vascular remodeling arising during the first two weeks of tumor growth were captured with AA and demonstrated an increase in perfusion in the vicinity of BLI signal by a factor of 1.4 ± 0.38, with vascular remodeling being evident even at the periphery of BLI signal.
               This work demonstrates that non-invasive measurements of in vivo microvascular remodeling can be precisely mapped to changes in tumor growth with a hybrid modality system. The system has comparable sensitivity to a BLI-alone system and provides similar assessments of longitudinal tumor growth. Adding quantitative metrics for vascular remodeling to the widely used luminescent imaging could provide a more comprehensive assessment for tumor functional status than either modality could individually. This should prove valuable when using antiangiogenic therapies because changes in vasculature will precede cell death, and the ability to monitor both the cells and their blood supply might help to elucidate underlying biological processes.
               Citation Format: Juan D. Rojas, Rajalekha Rajamahendiran, Tomasz J. Czernuszewicz, Brian Velasco, Jonathan Perdomo, Max Harlacher, Graeme O'Connell, James Butler, Blossom Damania, Paul A. Dayton, Ryan C. Gessner. Tracking angiogenesis induced microvascular changes in a lymphoma model via a new high throughput non-invasive dual modality imaging platform [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1935.}, number={13}, journal={CANCER RESEARCH}, author={Rojas, Juan D. and Rajamahendiran, Rajalekha and Czernuszewicz, Tomasz J. and Velasco, Brian and Perdomo, Jonathan and Harlacher, Max and O'Connell, Graeme and Butler, James and Damania, Blossom and Dayton, Paul A. and et al.}, year={2019}, month={Jul} }
 @article{rojas_dayton_2019, title={IN VIVO MOLECULAR IMAGING USING LOW-BOILING-POINT PHASE-CHANGE CONTRAST AGENTS: A PROOF OF CONCEPT STUDY}, volume={45}, ISSN={["1879-291X"]}, DOI={10.1016/j.ultrasmedbio.2018.08.004}, abstractNote={Sub-micron phase-change contrast agents (PCCAs) have been proposed as a tool for ultrasound molecular imaging based on their potential to extravasate and target extravascular markers and also because of the potential to image these contrast agents with a high contrast-to-tissue ratio. We compare in vivo ultrasound molecular imaging with targeted low-boiling-point PCCAs and targeted microbubble contrast agents. Both agents were targeted to the intravascular (endothelial) integrin αvß3via a cyclic RGD peptide (cyclo-Arg-Gly-Asp-D-Tyr-Cys) mechanism and imaged in vivo in a rodent fibrosarcoma model, which exhibits angiogenic microvasculature. Signal intensity was measured using two different techniques, conventional contrast-specific imaging (amplitude/phase modulation) and a droplet vaporization imaging sequence, which detects the unique signature of vaporizing PCCAs. Data indicate that PCCA-specific imaging is more sensitive to small numbers of bound agents than conventional contrast imaging. However, data also revealed that contrast from targeted microbubbles was greater than that provided by PCCAs. Both control and targeted PCCAs were observed to be retained in tissue post-vaporization, which was expected for targeted agents but not expected for control agents. The exact mechanism underlying this observation remains unknown.}, number={1}, journal={ULTRASOUND IN MEDICINE AND BIOLOGY}, author={Rojas, Juan D. and Dayton, Paul A.}, year={2019}, month={Jan}, pages={177–191} }
 @article{rojas_papadopoulou_czernuszewicz_rajamahendiran_chytil_chiang_chong_bautch_rathmell_aylward_et al._2019, title={Ultrasound Measurement of Vascular Density to Evaluate Response to Anti-Angiogenic Therapy in Renal Cell Carcinoma}, volume={66}, ISSN={["1558-2531"]}, DOI={10.1109/TBME.2018.2860932}, abstractNote={<italic>Background:</italic> Functional and molecular changes often precede gross anatomical changes, so early assessment of a tumor's functional and molecular response to therapy can help reduce a patient's exposure to the side effects of ineffective chemotherapeutics or other treatment strategies. <italic>Objective:</italic> Our intent was to test the hypothesis that an ultrasound microvascular imaging approach might provide indications of response to therapy prior to assessment of tumor size. <italic>Methods:</italic> Mice bearing clear-cell renal cell carcinoma xenograft tumors were treated with antiangiogenic and Notch inhibition therapies. An ultrasound measurement of microvascular density was used to serially track the tumor response to therapy. <italic>Results:</italic> Data indicated that ultrasound-derived microvascular density can indicate response to therapy a week prior to changes in tumor volume and is strongly correlated with physiological characteristics of the tumors as measured by histology (<inline-formula><tex-math notation="LaTeX">$\rho = {\text{0.75}}$</tex-math></inline-formula>). Furthermore, data demonstrated that ultrasound measurements of vascular density can determine response to therapy and classify between-treatment groups with high sensitivity and specificity. <italic>Conclusion/Significance:</italic> Results suggests that future applications utilizing ultrasound imaging to monitor tumor response to therapy may be able to provide earlier insight into tumor behavior from metrics of microvascular density rather than anatomical tumor size measurements.}, number={3}, journal={IEEE TRANSACTIONS ON BIOMEDICAL ENGINEERING}, author={Rojas, Juan D. and Papadopoulou, Virginie and Czernuszewicz, Tomasz J. and Rajamahendiran, Rajalekha M. and Chytil, Anna and Chiang, Yun-Chen and Chong, Diana C. and Bautch, Victoria L. and Rathmell, W. Kimryn and Aylward, Stephen and et al.}, year={2019}, month={Mar}, pages={873–880} }
 @article{rojas_dayton_2019, title={VAPORIZATION DETECTIONIMAGING: A TECHNIQUE FOR IMAGING LOW-BOILING-POINTPHASE-CHANGE ONTRAST AGENTS WITH A HIGH DEPTH OF PENETRATION AND CONTRAST-TO-TISSUERATIO}, volume={45}, ISSN={["1879-291X"]}, DOI={10.1016/j.ultrasmedbio.2018.08.017}, abstractNote={Phase-change contrast agents (PCCAs) possess advantages over microbubble contrast agents, such as the ability to extravasate and circulate longer in the vasculature that could enhance the diagnostic capabilities of contrast-enhanced ultrasound. PCCAs typically have a liquid perfluorocarbon (PFC) core that can be vaporized into echogenic microbubbles. Vaporization of submicron agents filled with liquid PFCs at body temperature usually requires therapeutic pressures higher than typically used for diagnostic imaging, but low-boiling-point PCCAs using decafluorobutane or octafluoropropane can be vaporized using pressures in the diagnostic imaging regime. Low-boiling-point PCCAs produce a unique acoustic signature that can be separated from tissue and bubble signals to make images with high contrast-to-tissue ratios. In this work, we explore the effect of pulse length and concentration on the vaporization signal of PCCAs and a new technique to capture and use the signals to make high contrast-to-tissue ratio images in vivo. The results indicate that using a short pulse may be ideal for imaging because it does not interact with created bubbles but still produces strong signals for making images. Furthermore, it was found that capturing PCCA vaporization signals produced higher contrast-to-tissue ratio values and better depth of penetration than imaging the bubbles generated by droplet activation using conventional contrast imaging techniques. The resolution of the vaporization signal images is poor because of the low frequency of the signals, but their high sensitivity may be used for applications such as molecular imaging, where the detection of small numbers of contrast agents is important.}, number={1}, journal={ULTRASOUND IN MEDICINE AND BIOLOGY}, author={Rojas, Juan D. and Dayton, Paul A.}, year={2019}, month={Jan}, pages={192–207} }
 @article{lin_tsuruta_rojas_dayton_2017, title={OPTIMIZING SENSITIVITY OF ULTRASOUND CONTRAST-ENHANCED SUPER-RESOLUTION IMAGING BY TAILORING SIZE DISTRIBUTION OF MICROBUBBLE CONTRAST AGENT}, volume={43}, ISSN={["1879-291X"]}, DOI={10.1016/j.ultrasmedbio.2017.05.014}, abstractNote={Ultrasound contrast-enhanced super-resolution imaging has recently attracted attention because of its extraordinary ability to image vascular features much smaller than the ultrasound diffraction limit. This method requires sensitive detection of separable microbubble events despite a noisy tissue background to indicate the microvasculature, and any approach that could improve the sensitivity of the ultrasound system to individual microbubbles would be highly beneficial. In this study, we evaluated the effect of varying microbubble size on super-resolution imaging sensitivity. Microbubble preparations were size sorted into different mean diameters and then were imaged at equal concentrations. Commercially manufactured Definity and Optison were also imaged for comparison. Both in vitro experiments in phantom vessels and in vivo experiments imaging rat tumors revealed that the sensitivity of contrast-enhanced super-resolution imaging can be improved by using microbubbles with a larger diameter.}, number={10}, journal={ULTRASOUND IN MEDICINE AND BIOLOGY}, author={Lin, Fanglue and Tsuruta, James K. and Rojas, Juan D. and Dayton, Paul A.}, year={2017}, month={Oct}, pages={2488–2493} }
 @article{tsuruta_schaub_rojas_streeter_klauber-demore_dayton_2017, title={Optimizing ultrasound molecular imaging of secreted frizzled related protein 2 expression in angiosarcoma}, volume={12}, ISSN={["1932-6203"]}, DOI={10.1371/journal.pone.0174281}, abstractNote={Secreted frizzled related protein 2 (SFRP2) is a tumor endothelial marker expressed in angiosarcoma. Previously, we showed ultrasound molecular imaging with SFRP2-targeted contrast increased average video pixel intensity (VI) of angiosarcoma vessels by 2.2 ± 0.6 VI versus streptavidin contrast. We hypothesized that redesigning our contrast agents would increase imaging performance. Improved molecular imaging reagents were created by combining NeutrAvidin™-functionalized microbubbles with biotinylated SFRP2 or IgY control antibodies. When angiosarcoma tumors in nude mice reached 8 mm, time-intensity, antibody loading, and microbubble dose experiments optimized molecular imaging. 10 minutes after injection, the control-subtracted time-intensity curve (TIC) for SFRP2-targeted contrast reached a maximum, after subtracting the contribution of free-flowing contrast. SFRP2 antibody-targeted VI was greater when contrast was formulated with 10-fold molar excess of maleimide-activated NeutrAvidin™ versus 3-fold (4.5 ± 0.18 vs. 0.32 ± 0.15, VI ± SEM, 5 x 106 dose, p < 0.001). Tumor vasculature returned greater average video pixel intensity using 5 x 107 versus 5 x 106 microbubbles (21.2 ± 2.5 vs. 4.5 ± 0.18, p = 0.0011). Specificity for tumor vasculature was confirmed by low VI for SFRP2-targeted, and control contrast in peri-tumoral vasculature (3.2 ± 0.52 vs. 1.6 ± 0.71, p = 0.92). After optimization, average video pixel intensity of tumor vasculature was 14.2 ± 3.0 VI units higher with SFRP2-targeted contrast versus IgY-targeted control (22.1 ± 2.5 vs. 7.9 ± 1.6, p < 0.001). After log decompression, 14.2 ΔVI was equal to ~70% higher signal, in arbitray acoustic units (AU), for SFRP2 versus IgY. This provided ~18- fold higher acoustic signal enhancement than provided previously by 2.2 ΔVI. Basing our targeted contrast on NeutrAvidin™-functionalized microbubbles, using IgY antibodies for our control contrast, and optimizing our imaging protocol significantly increased the SFRP2-specific signal returned from angiosarcoma vasculature, and may provide new opportunities for targeted molecular imaging.}, number={3}, journal={PLOS ONE}, author={Tsuruta, James K. and Schaub, Nicholas P. and Rojas, Juan D. and Streeter, Jason and Klauber-DeMore, Nancy and Dayton, Paul}, year={2017}, month={Mar} }
 @article{lindsey_shelton_martin_ozgun_rojas_foster_dayton_2017, title={High Resolution Ultrasound Superharmonic Perfusion Imaging: In Vivo Feasibility and Quantification of Dynamic Contrast-Enhanced Acoustic Angiography}, volume={45}, ISSN={["1573-9686"]}, url={https://europepmc.org/articles/PMC5682933}, DOI={10.1007/s10439-016-1753-9}, abstractNote={Mapping blood perfusion quantitatively allows localization of abnormal physiology and can improve understanding of disease progression. Dynamic contrast-enhanced ultrasound is a low-cost, real-time technique for imaging perfusion dynamics with microbubble contrast agents. Previously, we have demonstrated another contrast agent-specific ultrasound imaging technique, acoustic angiography, which forms static anatomical images of the superharmonic signal produced by microbubbles. In this work, we seek to determine whether acoustic angiography can be utilized for high resolution perfusion imaging in vivo by examining the effect of acquisition rate on superharmonic imaging at low flow rates and demonstrating the feasibility of dynamic contrast-enhanced superharmonic perfusion imaging for the first time. Results in the chorioallantoic membrane model indicate that frame rate and frame averaging do not affect the measured diameter of individual vessels observed, but that frame rate does influence the detection of vessels near and below the resolution limit. The highest number of resolvable vessels was observed at an intermediate frame rate of 3 Hz using a mechanically-steered prototype transducer. We also demonstrate the feasibility of quantitatively mapping perfusion rate in 2D in a mouse model with spatial resolution of ~100 μm. This type of imaging could provide non-invasive, high resolution quantification of microvascular function at penetration depths of several centimeters.}, number={4}, journal={ANNALS OF BIOMEDICAL ENGINEERING}, author={Lindsey, Brooks D. and Shelton, Sarah E. and Martin, K. Heath and Ozgun, Kathryn A. and Rojas, Juan D. and Foster, F. Stuart and Dayton, Paul A.}, year={2017}, month={Apr}, pages={939–948} }
 @article{rojas_dayton_2017, title={Optimizing Acoustic Activation of Phase Change Contrast Agents With the Activation Pressure Matching Method: A Review}, volume={64}, ISSN={["1525-8955"]}, DOI={10.1109/tuffc.2016.2616304}, abstractNote={Submicrometer phase-change contrast agents (PCCAs) consist of a liquid perfluorocarbon (PFC) core that can be vaporized by ultrasound (acoustic droplet vaporization) to generate contrast with excellent spatial and temporal control. When these agents, commonly referred to as nanodroplets, are formulated with cores of low boiling-point PFCs such as decafluorobutane and octafluoropropane, they can be activated with low-mechanical-index (MI) imaging pulses for diagnostic applications. Since the utilization of minimum MI is often desirable to avoid unnecessary biological effects, enabling consistent activation of these agents in an acoustic field is a challenge because the energy that must be delivered to achieve the vaporization threshold increases with depth due to attenuation. A novel vaporization approach called activation pressure matching (APM) has been developed to deliver the same pressure throughout a field of view in order to produce uniform nanodroplet vaporization and to limit the amount of energy that is delivered. In this paper, we discuss the application of this method with a Verasonics V1 Research Ultrasound System to modulate the output pressure from an ATL L11-5 transducer. Vaporization-pulse spacing optimization can be used in addition to matching the activation pressure through depth, and we demonstrate the feasibility of this approach both in vivo and in vitro. The use of optimized vaporization parameters increases the amount of time a single bolus of nanodroplets can generate useful contrast and provides consistent image enhancement in vivo. Therefore, APM is a useful technique for maximizing the efficacy of PCCA while minimizing delivered acoustic energy.}, number={1}, journal={IEEE TRANSACTIONS ON ULTRASONICS FERROELECTRICS AND FREQUENCY CONTROL}, author={Rojas, Juan D. and Dayton, Paul A.}, year={2017}, month={Jan}, pages={264–272} }
 @article{lindsey_rojas_dayton_2015, title={ON THE RELATIONSHIP BETWEEN MICROBUBBLE FRAGMENTATION, DEFLATION AND BROADBAND SUPERHARMONIC SIGNAL PRODUCTION}, volume={41}, ISSN={["1879-291X"]}, DOI={10.1016/j.ultrasmedbio.2014.12.668}, abstractNote={Acoustic angiography imaging of microbubble contrast agents uses the superharmonic energy produced from excited microbubbles and enables high-contrast, high-resolution imaging. However, the exact mechanism by which broadband harmonic energy is produced is not fully understood. To elucidate the role of microbubble shell fragmentation in superharmonic signal production, simultaneous optical and acoustic measurements were performed on individual microbubbles at transmit frequencies from 1.75 to 3.75 MHz and pressures near the shell fragmentation threshold for microbubbles of varying diameter. High-amplitude, broadband superharmonic signals were produced with shell fragmentation, whereas weaker signals (approximately 25% of peak amplitude) were observed in the presence of shrinking bubbles. Furthermore, when populations of stationary microbubbles were imaged with a dual-frequency ultrasound imaging system, a sharper decline in image intensity with respect to frame number was observed for 1-μm bubbles than for 4-μm bubbles. Finally, in a study of two rodents, increasing frame rate from 4 to 7 Hz resulted in decreases in mean steady-state image intensity of 27% at 1000 kPa and 29% at 1300 kPa. Although the existence of superharmonic signals when bubbles shrink has the potential to prolong the imaging efficacy of microbubbles, parameters such as frame rate and peak pressure must be balanced with expected re-perfusion rate to maintain adequate contrast during in vivo imaging.}, number={6}, journal={ULTRASOUND IN MEDICINE AND BIOLOGY}, author={Lindsey, Brooks D. and Rojas, Juan D. and Dayton, Paul A.}, year={2015}, month={Jun}, pages={1711–1725} }
 @article{sheeran_rojas_puett_hjelmquist_arena_dayton_2014, title={In Vivo Quantification of Image Enhancement and Circulation Kinetics for Phase Change Perfluorocarbon Agents Using Custom Pulse Sequences}, ISSN={["1948-5719"]}, DOI={10.1109/ultsym.2014.0103}, abstractNote={Few investigations have been published demonstrating the in vivo contrast characteristics of phase-change perfluorocarbon droplets. In this study, we examine the properties of low boiling point nanoscale droplets compared to microbubbles with respect to image enhancement and circulation time. To accomplish this, we develop a custom pulse sequence to vaporize and image droplets using the Verasonics research platform. Results show that droplets can produce similar contrast compared to microbubbles, and can circulate for significantly longer than microbubbles, depending on formulation. Finally, this study demonstrates a novel concept in contrast-enhanced ultrasound: capture of droplet-generated contrast wash-out in the target organ.}, journal={2014 IEEE INTERNATIONAL ULTRASONICS SYMPOSIUM (IUS)}, author={Sheeran, Paul S. and Rojas, Juan D. and Puett, Connor and Hjelmquist, Jordan and Arena, Christopher B. and Dayton, Paul A.}, year={2014}, pages={417–420} }
 @article{lindsey_rojas_martin_shelton_dayton_2014, title={Optimization of Contrast-to-tissue Ratio and Role of Bubble Destruction in Dual-Frequency Contrast-Specific "Acoustic Angiography" Imaging}, ISSN={["1948-5719"]}, DOI={10.1109/ultsym.2014.0440}, abstractNote={Recently, dual-frequency transducers have enabled high-spatial resolution, high-contrast imaging of microvasculature by transmitting at a low frequency and receiving broadband superharmonic echoes from microbubble contrast agents at a higher frequency. In this work, we examine the imaging parameters for optimizing contrast-to-tissue ratio (CTR) for dual-frequency imaging and the relationship between bubble destruction and broadband harmonic signal production. CTR was assessed in vitro by acquiring scattered echoes by bubbles and beef muscle for transmit pressures up to 2 MPa, transmit frequencies from 1.5-8 MHz, and receive frequencies from 7.5 to 25 MHz. Optimum CTR (25.5 dB) was found to occur at the lowest transmit frequencies, though a broad peak exists within the 1.5-3.5 MHz range. At these frequencies, CTR is optimized when receiving at a center frequency of 10 - 15 MHz. A 4 μm-diameter microbubble population yielded ~5 dB higher CTR than a 1 μm population. Single bubble behavior was assessed with simultaneous acoustic and optical recordings. For n=250 single bubbles subjected to five consecutive single-cycle pulses (100-500 kPa), three primary categories of bubble behavior were observed optically: 1) no change in bubble diameter, 2) bubble shrinking (deflation), and 3) immediate bubble destruction (fragmentation). Matched acoustic data indicate that superharmonic signals having the broadest bandwidth and highest energy are associated with shell fragmentation. In the deflation case, a weaker superharmonic signal is produced with an amplitude approximately 25% of the signal in the shell fragmentation case. Similar regimes were observed in vivo, suggesting that bubble diameter, transmit frequency, peak negative pressure, and frame rate must be selected in light of the intended application, accounting for attenuation and local perfusion rate in the region of interest.}, journal={2014 IEEE INTERNATIONAL ULTRASONICS SYMPOSIUM (IUS)}, author={Lindsey, Brooks D. and Rojas, Juan D. and Martin, K. Heath and Shelton, Sarah E. and Dayton, Paul A.}, year={2014}, pages={1774–1777} }