Near-infrared probes · Near-infrared fluorescent probes based on bacterial phytochromes for in vivo imaging

Non-invasive monitoring of deep-tissue developmental, metabolic and pathogenic processes will advance modern biology. Imaging of live mammals using fluorescent probes is more feasible within the near-infrared (NIR) transparency window (NIRW: 650-900 nm) where hemoglobin and melanin absorbance significantly decreases, and water absorbance is still low. The most red-shifted fluorescent proteins (FPs) of the GFP-like family exhibit fluorescence outside of the NIRW and suffer from low brightness and modest photostability. Natural bacterial phytochrome photoreceptors (BphPs) utilize low molecular weight biliverdin as a chromophore and provide many advantages over other chromophore binding proteins. First, unlike the chromophores of non-bacterial phytochromes, biliverdin is ubiquitous in mammals. This makes BphP applications in mammalian cells, tissues and mammals as easy as conventional GFP-like FPs, without supplying chromophore through an external solution. Second, BphPs exhibit NIR absorbance and fluorescence, which are red-shifted relative to that of any other phytochromes, and lie within the NIRW. This makes BphPs spectrally complementary to GFP-like FPs and available optogenetic tools. Third, independent domain architecture and conformational changes upon biliverdin photoisomerization make BphPs attractive templates to design various photoactivatable probes. Based on the analysis of the photochemistry and structural changes of BphPs we plan to develop three new types of the BphP-based probes. These include bright and spectrally resolvable permanently fluorescent NIRFPs, NIRFPs photoswitchable either irreversibly or repeatedly with non-phototoxic NIR light, and NIR reporters and biosensors. The resulting NIR probes will extend fluorescence imaging methods to deep-tissue in vivo macroscopy including multicolor cell and tissue labeling, cell photoactivation and tracking, detection of enzymatic activities and protein interactions in mammalian tissues and whole animals.