Photoacoustic and Ultrasound Dual-Mode Imaging via Functionalization of Recombinant Protein-Stabilized Microbubbles with Methylene Blue

Contrast-enhanced photoacoustics and ultrasonics are complementary methods of bioimaging. In this study, a flow-focusing junction microfluidic device is used for the generation of uniform microbubbles (<5 mu m) for simultaneous enhancement of photoacoustic and ultrasound imaging. Microbubbles stabilized with a mixture of a recombinant protein and a synthetic amphiphilic block copolymer are functionalized with an FDA-approved photoacoustic dye, methylene blue (MetB). These microbubbles are uniform in size and stable. We show that the ultrasound and photoacoustic signals can be independently controlled by changing the concentration of MetB during microbubble preparation and the concentration of MetB-functionalized microbubbles in the probe suspension. We also perform animal tests to demonstrate the enhancement of ultrasound and acoustic signals upon injection of MetB-functionalized microbubbles in mice. The increase in the sonographic and photoacoustic signals is visibly obvious in the images. Taken together, MetB-functionalized microbubbles represent promising dual-mode ultrasound and photoacoustic imaging contrast agents for theranostic applications.