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

Ryan K. W. Chee, Yan Li, Wei Zhang, Robert E. Campbell
Journal of Biomedical Optics2018
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.
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