Bright Monomeric Near-Infrared Fluorescent Proteins for Multiscale Imaging

Monomeric near-infrared (NIR) fluorescent proteins (FPs) and biosensors are in high demand as additional colors for microscopy and, particularly, for deep-tissue imaging. We engineered three bright and spectrally distinct monomeric NIR FPs, called miRFPs, miRFPs are 2-5-fold brighter in mammalian cells than the only available monomeric NIR FP and perform well in protein fusions, allowing multicolor structured illumination microscopy. Starting from miRFPs, we engineered the first monomeric multicolor fluorescence complementation reporters for protein-protein interactions and RNA labeling. We then designed NIR IκBα signaling reporter for canonical NF-κB signaling in both cells and animals. We further developed NIR cell cycle biosensor for detection of proliferation status of individual cells and cell populations. For the first time, miRFPs enable non-invasive visualization of biological processes at different scales, from super-resolution microscopy to in vivo imaging, using the same probes.