Reversibly switchable photoacoustic tomography using a genetically encoded near-infrared phytochrome

Optical imaging of genetically encoded probes has revolutionized biomedical studies by providing valuable information about targeted biological processes. Here, we report a novel imaging technique, termed reversibly switchable photoacoustic tomography (RS-PAT), which exhibits large penetration depth, high detection sensitivity, and super-resolution. RS-PAT combines advanced photoacoustic imaging techniques with, for the first time, a nonfluorescent photoswitchable bacterial phytochrome. This bacterial phytochrome is the most near-infrared shifted genetically encoded probe reported so far. Moreover, this bacterial phytochrome is reversibly photoconvertible between its far-red and near-infrared light absorption states. Taking maximum advantage of the powerful imaging capability of PAT and the unique photochemical properties of the phytochrome, RS-PAT has broken through both the optical diffusion limit for deep-tissue imaging and the optical diffraction limit for super-resolution photoacoustic microscopy. Specifically, with RS-PAT we have achieved an unprecedented detection sensitivity of ∼2 μM, or as few as ∼20 tumor cells, at a centimeter depth. Such high sensitivity is fully demonstrated in our study by monitoring tumor growth and metastasis at whole-body level with ∼100 μm resolution. Moreover, our microscopic implementation of RS-PAT is capable of imaging mammalian cells with a sub-diffraction lateral resolution of ∼140 nm and axial resolution of ∼400 nm, which are respectively ∼2-fold and ∼75-fold finer than those of our conventional photoacoustic microscopy. Overall, RS-PAT is a new and promising imaging technology for studying biological processes at different length scales.