*2.2. Microbubble Production*

Biotinylated lipid-coated microbubbles with a C4F<sup>10</sup> gas core were made as described previously [27], by probe sonication at 20 kHz with a Sonicator ultrasonic processor XL2020 at a power setting 10 (HeatSystems, Farmingdale, NY, USA) for 10 s. Three types of microbubbles were made by altering the production method or adding cholesterol to the microbubble coating. For microbubbles without cholesterol, the coating components (84.8 mol% DSPC; 8.2 mol% PEG40-stearate; 5.9 mol% DSPE-PEG2000; 1.1 mol% DSPE-PEG2000-biotin) were prepared with either an indirect or a direct method as described previously [16]. In short, for the indirect method, the components were dissolved in chloroform/methanol (9:1 vol/vol), the solvent was evaporated using argon gas, and the obtained lipid film was dried overnight under vacuum. The lipid film was then dispersed in saline solution (0.9% NaCl, saturated with C4F10) with a final concentration of 2.5 mg/mL DSPC, 0.625 mg/mL PEG40-stearate, 0.625 mg/mL DSPE-PEG2000 and 0.125 mg/mL DSPE-PEG2000-biotin. The fluorescent dye rhodamine-DHPE (0.01 mol%) was added to image the lipid phase separation in the microbubble coating. The solution was placed in a sonicator bath for 10 min, and the probe sonicator was used at power setting 3 for 5 min. For the direct method, the coating components (84.8 mol% DSPC; 8.2 mol% PEG40-stearate; 5.9 mol% DSPE-PEG2000; 1.1 mol% DSPE-PEG2000 biotin) were dispersed directly in C4F10-saturated saline solution with a final concentration of 2.5 mg/mL DSPC, 0.625 mg/mL PEG40-stearate, 0.625 mg/mL DSPE-PEG2000 and 0.125 mg/mL DSPE-PEG2000-biotin. Fluorescent dye rhodamine-DHPE (0.01 mol%) was added before sonication.

Microbubbles with cholesterol, referred to as DSPC-cholesterol microbubbles, were produced with the indirect method only since cholesterol is insoluble in an aqueous medium, and the organic solvent was required to mix all microbubble coating components [28]. Cholesterol was added (7, 10, 12, 14, or 32 mol%) to the ternary mixture of coating components: DSPC, PEG40-stearate, DSPE-PEG2000, and DSPE-PEG2000-biotin (molar ratio 84.8/8.2/5.9/1.1) in chloroform/methanol (9:1 vol/vol). The lipids were then dried to form a lipid film and dispersed in saline solution, as described above, with 0.02 mol% rhodamine-DHPE added for fluorescent labeling of the microbubbles. All types of microbubbles were produced by sonicating under a constant flow of C4F10.
