Quantification of Bound Microbubbles in Ultrasound Molecular Imaging

Molecular markers associated with diseases can be visualized and quantified noninvasively with targeted ultrasound contrast agent (t-UCA) consisting of microbubbles (MBs) that can bind to specific molecular targets. Techniques used for quantifying t-UCA assume that all unbound MBs are taken out of the blood pool few minutes after injection and only MBs bound to the molecular markers remain. However, differences in physiology, diseases, and experimental conditions can increase the longevity of unbound MBs. In such conditions, unbound MBs will falsely be quantified as bound MBs. We have developed a novel technique to distinguish and classify bound from unbound MBs. In the post-processing steps, first, tissue motion was compensated using block-matching (BM) techniques. To preserve only stationary contrast signals, a minimum intensity projection (MinIP) or 20th-percentile intensity projection (PerIP) was applied. The after-flash MinIP or PerIP was subtracted from the before-flash MinIP or PerIP. In this way, tissue artifacts in contrast images were suppressed. In the next step, bound MB candidates were detected. Finally, detected objects were tracked to classify the candidates as unbound or bound MBs based on their displacement. This technique was validated in vitro, followed by two in vivo experiments in mice. Tumors (n = 2) and salivary glands of hypercholesterolemic mice (n = 8) were imaged using a commercially available scanner. Boluses of 100 mu L of a commercially available t-UCA targeted to angiogenesis markers and untargeted control UCA were injected separately. Our results show considerable reduction in misclassification of unbound MBs as bound ones. Using our method, the ratio of bound MBs in salivary gland for images with targeted UCA versus control UCA was improved by up to two times compared with unprocessed images

Quantification of bound microbubbles in ultrasound molecular imaging

Molecular markers associated with diseases can be visualized and quantified noninvasively with targeted ultrasound contrast agent (t-UCA) consisting of microbubbles (MBs) that can bind to specific molecular targets. Techniques used for quantifying t-UCA assume that all unbound MBs are taken out of the blood pool few minutes after injection and only MBs bound to the molecular markers remain. However, differences in physiology, diseases, and experimental conditions can increase the longevity of unbound MBs. In such conditions, unbound MBs will falsely be quantified as bound MBs. We have developed a novel technique to distinguish and classify bound from unbound MBs. In the post-processing steps, first, tissue motion was compensated using block-matching (BM) techniques. To preserve only stationary contrast signals, a minimum intensity projection (MinIP) or 20th-percentile intensity projection (PerIP) was applied. The after-flash MinIP or PerIP was subtracted from the before-flash MinIP or PerIP. In this way, tissue artifacts in contrast images were suppressed. In the next step, bound MB candidates were detected. Finally, detected objects were tracked to classify the candidates as unbound or bound MBs based on their displacement. This technique was validated in vitro, followed by two in vivo experiments in mice. Tumors (n = 2) and salivary glands of hypercholesterolemic mice (n = 8) were imaged using a commercially available scanner. Boluses of 100 μL of a commercially available t-UCA targeted to angiogenesis markers and untargeted control UCA were injected separately. Our results show considerable reduction in misclassification of unbound MBs as bound ones. Using our method, the ratio of bound MBs in salivary gland for images with targeted UCA versus control UCA was improved by up to two times compared with unprocessed images

Quantification of Endothelial αvβ3 Expression with High-Frequency Ultrasound and Targeted Microbubbles: In Vitro and In Vivo Studies

Angiogenesis is a critical feature of plaque development in atherosclerosis and might play a key role in both the initiation and later rupture of plaques. The precursory molecular or cellular pro-angiogenic events that initiate plaque growth and that ultimately contribute to plaque instability, however, cannot be detected directly with any current diagnostic modality. This study was designed to investigate the feasibility of ultrasound molecular imaging of endothelial αvβ3 expression in vitro and in vivo using αvβ3-targeted ultrasound contrast agents (UCAs). In the in vitro study, αvβ3 expression was confirmed by immunofluorescence in a murine endothelial cell line and detected using the targeted UCA and ultrasound imaging at 18-MHz transmit frequency. In the in vivo study, expression of endothelial αvβ3 integrin in murine carotid artery vessels and microvessels of the salivary gland was quantified using targeted UCA and high-frequency ultrasound in seven animals. Our results indicated that endothelial αvβ3 expression was significantly higher in the carotid arterial wall containing atherosclerotic lesions than in arterial segments without any lesions. We also found that the salivary gland can be used as an internal positive control for successful binding of targeted UCA to αvβ3 integrin. In conclusion, αvβ3-targeted UCA allows non-invasive assessment of the expression levels of αvβ3 on the vascular endothelium and may provide potential insights into early atherosclerotic plaque detection and treatment monitorin