Dual-wavelength 3D photoacoustic imaging of mammalian cells using a photoswitchable phytochrome reporter protein

Abstract Photoacoustic imaging has been shown to provide high-resolution images of genetically labelled cells at depths that are inaccessible to optical microscopy. While the detection of genetic reporters, such as fluorescent proteins and pigments, has been demonstrated using multiwavelength imaging and spectral unmixing, these approaches remain challenging due to their large computational scale. In this study we report a method based on a reversibly photoswitchable phytochrome-based reporter protein (AGP1) and dual-wavelength interleaved image acquisition for obtaining difference images with unambiguous reporter-specific contrast. Detailed, full 3D images of tumours formed of cells lentivirally transduced to express AGP1 were acquired in vivo in deep tissue in a longitudinal study. This method represents a powerful new approach to studying cellular and genetic processes which, due to its experimental simplicity, can be implemented in a wide range of existing photoacoustic imaging platforms.