In vivo photoacoustic imaging (PAI)

All mouse imaging experiments were performed using a real-time multispectral optoacoustic tomographic imaging system (Vevo 2100 LAZR system, Visual Sonic Inc. New York, NY) equipped with a 40 MHz, 256-element linear array transducer on tumors. For in vivo PA imaging study, subcutaneous 4T1 tumor bearing Balb/c mice were anesthetized by 1% isoflurane delivered via a nose cone, and then the MBBN (100 μL, 2 mg mL^-1) were injected via the tail vein. In vivo PA images were acquired before injection and at different time points post injection (1 h, 2 h, 4 h, 8 h, 12 h and 24 h) using the multispectral optoacoustic tomography system at a wavelength of 780 nm. A region of interest (ROI) volume consisting of transverse slices with a step size of 0.4 mm, spanning through the tumor region, was selected by manual inspection of live MSOT images. The average PA signal of the tumor area was extracted using the multispectral optoacoustic tomography software.