In vivo photoacoustic difference-spectra imaging of bacteria using photoswitchable chromoproteins

Photoacoustic (PA) imaging offers great promise for deep molecular imaging of optical reporters but has difficulties in imaging multiple molecular probes simultaneously in a strong blood background. Photoswitchable chromoproteins like BphP1 have recently allowed for sensitive PA detection by reducing high-blood background signals but lack multiplexing capabilities. We propose a method known as difference-spectra demixing for multiplexing multiple photoswitchable chromoproteins and introduce a second photoswitchable chromoprotein, sGPC2. sGPC2 has a far-red and orange state with peaks at 700 and 630 nm, respectively. It is roughly one-tenth the size of BphP1 and photoswitches four times as fast (2.4% per mJ  /  cm2). We simultaneously image Escherichia coli expressing sGPC2 and BphP1 injected in mice in vivo. Difference-spectra demixing obtained successful multiplexed images of photoswitchable molecular probes, resulting in a 21.6-fold increase in contrast-to-noise ratio in vivo over traditional PA imaging and an 8% to 40% reduction in erroneously demixed signals in comparison with traditional spectral demixing. PA imaging and characterization were conducted using a custom-built photoswitching PA imaging system.