Nearinfrared fluorescent proteins for multicolor in vivo imaging

Near-infrared fluorescent proteins are in high demand for in vivo imaging. We developed four spectrally distinct fluorescent proteins, iRFP670, iRFP682, iRFP702, and iRFP720, from bacterial phytochromes. iRFPs exhibit high brightness in mammalian cells and tissues and are suitable for long-term studies. iRFP670 and iRFP720 enable two-color imaging in living cells and mice using standard approaches. Five iRFPs including previously engineered iRFP713 allow multicolor imaging in living mice with spectral unmixing. Deep optical imaging in mammalian tissues and whole mammals requires near-infrared (NIR) fluorescent probes. In a so called NIR “optical window ” (from 650 nm to 900 nm) the mammalian tissue is more transparent to light because combined absorption of hemoglobin, melanin and water is minimal1. In addition, reduced autofluorescence and low light scattering makes this spectral region preferable for deep-tissue imaging. Efforts to engineer GFP-like fluorescent proteins (FPs) into far-red probes have resulted in several fluorescent FPs shown to be useful in mammals, such as E2-Crimson2, mNeptune3, TagRFP6574, eqFP6505 and eqFP6705. Among them TagRFP657 exhibits the most red-shifted excitation maximum at 611 nm but suffers from low brightness. The photoconverted form of the photoswitchable PSmOrange6 has an excitation peak at 634 nm, but to appear this form requires photoconversion of the FP with blue light, which poorly penetrates mammalian tissues. Bacterial phytochrome photoreceptors (BphPs) are promising templates for engineering of NIR FPs7. Phytochromes, found in plants, fungi, bacteria, and cyanobacteria, are a superfamily of photoreceptors, which use different types of tetrapyrroles as chromophores. Users may view, print, copy, and download text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use:http://www.nature.com/authors/editorial_policies/license.html#terms Correspondence should be addressed to V.V.V