Final Report Summary - NEAR-INFRARED PROBES (Near-infrared fluorescent probes based on bacterial phytochromes for in vivo imaging)
Near-infrared fluorescent proteins are in high demand as protein tags and components of biosensors for multicolor microscopy. They are also highly required for non-invasive imaging of deep-tissue developmental, metabolic and pathogenic processes. Imaging using fluorescent probes is more feasible within the near-infrared spectral region where hemoglobin and melanin absorbance significantly decreases, cellular autofluorescence also decreases, and water absorbance is still low. Based on the analysis of photochemistry and structural changes of bacterial phytochromes we proposed to develop several new types of genetically encoded fluorescent proteins, reporters and biosensors. As the result of the project, we enabled to engineer the collection of advanced multicolor fluorescent probes, which are as versatile as GFP-like fluorescent proteins and substantially expand imaging capabilities into near-infrared spectral region in live cells, tissues and in vivo. We applied these proteins to study tumor growth and metastases in live mice by using fluorescence and bioluminescence imaging, as well as photoacoustic tomography. We also studied the structural, biochemical and photophysical properties of these and other near-infrared genetically encoded probes developed from bacterial phytochromes in order to increase their brightness and make them multicolor. Importantly, the developed near-infrared fluorescent proteins and biosensors allow imaging and sensing of developmental, metabolic and pathogenic processes across scales: from subcellular level using live-cell high-resolution microscopy to tissue and organ levels using whole-body imaging of living animals. Furthermore, the designed probes extend the common fluorescence microscopy methods to deep-tissue in vivo macroscopy. Moreover, because both fluorescence excitation and emission of these probes stay in the near-infrared spectral region, they can be cross-talk free used in multiplexing experiments with various GFP-like probes and opsin-like optogenetic tools, which operate in the visible spectral range, thus, enabling all-optical noninvasive types of assays. The developed in the project near-infrared proteins and biosensors will be useful in various applications that deal with imaging of different cell types, tissues and animal models, as well as should help to resolve long-standing and important problems in basic biology and biomedicine.