Near-infrared Fluorescent Probes and Optogenetic Tools

ABSTRACT High optical transparency, low light-scattering and low autofluorescence of mammalian tissues in the near- infrared spectral range open up the possibility for non-invasive imaging, sensing and light-regulation of cellular biochemistry, tissue metabolism and physiology in mammals. Due to near-infrared absorption and the use of heme-derived biliverdin produced in all mammalian tissues as a chromophore, bacterial phytochromes are currently the preferred molecular templates for the development of genetically encoded optical reagents for in vivo applications. Here we propose a variety of near-infrared imaging probes and optogenetic tools. We will focus on the development of fluorescent proteins with higher brightness, greater bathochromic shift and exhibiting photoconversion, as well as on the search for new natural near-infrared absorbing proteins as initial templates. These probes should lead to deeper and spatiotemporally precise in vivo imaging. We will also focus on the visualization and manipulation of endogenous molecules in cells and whole organisms using antigen-stabilized fusions of near-infrared fluorescent proteins with genetically encoded nanobodies. This will allow background-free detection of various intracellular antigens and antigen-dependent regulation of protein and cellular functions. In addition, we will develop near-infrared optogenetic modules with improved performance in mammalian cells and use them to engineer light-controlled enzymes, receptors and cells. This should open up possibilities for user-defined regulation of cellular and organismal functions in vivo. All proposed near-infrared probes will spectrally complement existing reagents in the visible range. Planned studies will also expand our basic knowledge of photochemistry and light-induced signaling of phytochrome photoreceptors. Novel genetically encoded near-infrared fluorescent probes and optogenetic tools will drive the development of more sensitive in vivo imaging and light-control technologies, optimization of gene delivery strategies, development of targeted non-invasive illumination, and refinement of optical readouts in tissues and animals.