On day 7 after the intraocular inoculation of tumors, in vivo high-frequency, contrast-enhanced ultrasound (HF-CE-US) imaging was performed with a dedicated small-animal, high-resolution imaging system (VisualSonics, Inc., Toronto, ON, Canada) with a 40-MHz transducer. Mice in group 1 (n = 8) were imaged with Vevo 770 2D contrast mode, and mice in group 2 (n = 6) were imaged with Vevo 2100 3D contrast mode. Before the imaging session, mice were anesthetized as described previously and were placed on a flat heating pad. The heart rate was monitored and remained at approximately 400 beats per minute. Sterile ultrasound gel (Aquasonic 100; Parker Laboratories, Fairfield, NJ) was placed on the right eye bearing the uveal melanoma xenograft. The probe (RMV 707B; VisualSonics, Inc.) was positioned so that the cornea was to the superior of B-scan imaging, and the probe was then adjusted until the tumor was visible centrally on the screen of the ultrasound unit within the focal zone. Thereafter, the probe was fixed mechanically to improve the accuracy of intensity comparison and subtraction in a given slice. As suggested by the manufacturer, the parameters of the ultrasound system were initially optimized as described here for standard values for imaging small animal tissues: 50% transmitted power, 52-dB dynamic range, and 6.00 × 6.00-mm field of view. Baseline images without contrast agent were recorded. For contrast-enhanced imaging, a vial of contrast agent (MicroMarker; VisualSonics) was reconstituted with sterile saline and gently agitated. A 50-μL bolus containing a final count of 1.0 × 108 microbubbles, which had been optimized for tumor imaging, was injected through the mice tail vein with a fixed needle (Butterfly 625; Abbott, Dublin, Ireland), and a real-time cine loop of contrast-mode imaging was immediately recorded. The 3D volumetric data were obtained according to the image acquisition protocol with 3D image reconstruction and visualization software. When the 3D images were being processed, the ultrasound scan head linearly translated across the mouse's eye on the 3D motor system, and 2D images were acquired at regular spatial intervals so that they were parallel and uniformly spaced at 30-μm intervals over the entire image. Thereafter, 3D images were constructed and displayed through the parallel 2D image planes. At the end of all studies, the tumor-bearing right eyes were oriented, marked, enucleated, and submitted for routine histology processing.