Dual-wavelength photoacoustic imaging of a photoswitchable reporter protein

Photoacoustic (PA) imaging has been shown to provide detailed 3-D images of genetically expressed reporters, such as fluorescent proteins and tyrosinase-induced melanin. Their unambiguous detection in vivo is a vital prerequisite for molecular imaging of biological processes at a cellular and molecular level. This typically requires multiwavelength imaging and spectral unmixing techniques, which can be computationally expensive. In addition, fluorescent proteins often exhibit fluence-dependent ground state depopulation and photobleaching which can adversely affect the specificity of unmixing methods. To overcome these problems, a phytochrome-based reporter protein and a dual-wavelength excitation method have been developed to obtain reporter-specific PA contrast. Phytochromes are non-fluorescent proteins that exhibit two isomeric states with different absorption spectra. Using dual-wavelength excitation pulses in the red and near-infrared wavelength region, these states can be switched, resulting in a modulation of the total absorption coefficient, and hence the PA signal amplitude. Since this is not observed in endogenous chromophores, signals acquired using simultaneous pulses can be subtracted from the sum of signals obtained from separate pulses to provide a reporterspecific contrast mechanism and elimination of the tissue background. PA signals measured in protein solutions using separate and simultaneous excitation pulses at 670 nm and 755 nm ( -2 ) showed a difference in amplitude of a factor of five. Photobleaching was not observed. To demonstrate suitability for in vivo applications, mammalian cells were transduced virally to express phytochrome, and imaged in tissue phantoms and in mice in an initial preclinical study. The results show that this method has the potential to enable deep-tissue PA reporter gene imaging with high specificity.