In vivo photoacoustic imaging of a nonfluorescent E2 crimson genetic reporter in mammalian tissues

Significance: Green-fluorescent protein (GFP)-like fluorescent proteins are used extensively as genetic reporters in fluorescence imaging due to their distinctive ability to form chromophores independent of external enzymes or cofactors. However, their use for photoacoustic (PA) imaging has not been demonstrated in mammalian tissues because they possess low PA signal generation efficiency in their native state. By engineering them to become nonfluorescent (NF), their PA generation efficiency was increased. This enabled the generation of in vivo contrast in mice, making it possible for GFP-like proteins to be used as PA genetic reporters in mammalian tissues. Aim: The aim was to develop a darkened GFP-like protein reporter by modifying E2 crimson fluorescent protein (FP) in order to generate NF mutant proteins with high PA signal generation efficiency for in vivo imaging. Approach: The absorbance, fluorescence, and PA amplitude spectra of purified protein solutions of the FP and engineered NF mutants were measured in order to identify the mutant with the highest PA signal generation efficiency. This mutant, referred to as NFA, and the native FP were then stably expressed in LS174T human colorectal tumor cells using a retroviral vector and tested for photostability under continuous pulsed illumination. To demonstrate the improvement in PA signal generation in vivo, cells expressing the FP and NFA mutant were injected subcutaneously in mice and imaged using a Fabry–Perot based PA scanner. Results: The NF mutants of E2 crimson exhibited fluorescence that was 2 orders of magnitude lower than the FP and a higher PA signal generation efficiency; the NFA-generated PA signal was approximately three times higher than the FP. Tumor cells expressing the NFA mutant provided sufficient image contrast to be visualized in vivo against a background of strong vascular contrast, whereas the FP-expressing cells did not generate visible contrast. Conclusion: A GFP-like protein has been demonstrated as a genetic reporter for PA imaging in mammalian tissue for the first time. This was achieved by a mutation, which darkened the FP and increased the PA signal generation efficiency. The approach taken suggests that GFP-like proteins could be a promising addition to the current cohort of genetic reporters available for in vivo PA imaging.