A set of monomeric near-infrared fluorescent proteins for multicolor imaging across scales

Abstract

Bright monomeric near-infrared (NIR) fluorescent proteins (FPs) are in high demand as protein tags for multicolor microscopy and in vivo imaging. Here we apply rational design to engineer a complete set of monomeric NIR FPs, which are the brightest genetically encoded NIR probes. We demonstrate that the enhanced miRFP series of NIR FPs, which combine high effective brightness in mammalian cells and monomeric state, perform well in both nanometer-scale imaging with diffraction unlimited stimulated emission depletion (STED) microscopy and centimeter-scale imaging in mice. In STED we achieve~40 nm resolution in live cells. In living mice we detect~10 5 fluorescent cells in deep tissues. Using spectrally distinct monomeric NIR FP variants, we perform two-color live-cell STED microscopy and two-color imaging in vivo. Having emission peaks from 670 nm to 720 nm, the next generation of miRFPs should become versatile NIR probes for multiplexed imaging across spatial scales in different modalities.

Near-infrared fluorescent protein and bioluminescence-based probes for high-resolution in vivo optical imaging

“…Also, because of their monomeric nature, the localization efficiency of these NIR FPs is better than that of dimer NIR FPs. The first set of bright monomeric NIR FPs engineered from bacterial phytochromes are mIFP 22 and miRFPs (miRFP670, miRFP670-2, emiRFP670, miRFP680, miRFP682, miRFP703, emiRFP703, miRFP709, miRFP713 and miRFP720) 19,21,42 (Fig. 3).…”

“…Other bright and photostable NIR-FPs such as emiRFP670 and emiRFP703 with enhanced photophysical characteristics have been successfully utilized for single and multiplex non-invasive whole body imaging of mice. 42 However, miRFP713 and miRFP720 are more preferable for deep-tissue in vivo imaging because of their NIR-shifted emission maximum (Em max : 713 nm and Em max : 720 nm, respectively). These proteins can be used for dual-colour multiplex imaging with other spectrally far NIR-FP bright variants of miRFPs like miRFP 680, miRFP670 and emiRFP670 for dual-colour in vivo imaging.…”

“…9 The enhanced brightness and photostability of the newly developed emiRFP670 and emiRFP703 worked perfectly well in single and dual-colour STED imaging with an 11-36 fold decrease in the STED illumination dose in mammalian cells compared to their previously existing monomer NIR-FPs. 42…”

“…8). 42 Development of bright photoswitchable NIR FPs will provide opportunity to users to reduce the laser power to extend the imaging time and resolution. 4.3.3.…”

“…Among the NIR FPs designed from the bacterial phytochrome photoreceptor, miRFP680 is the brightest NIR-FP in mammalian cells. 42 The brightness of miRFP613 and miRFP720 is approximately B30% lower than miRFP680, however their B33 nm and B40 nm red-shifted emission spectra would be advantageous for in vivo imaging and multiplex imaging with other spectrally distinct NIR-FPs. Another recently developed NIR-FP, miRFP670nano, has nearly similar brightness to miRFP720 in mammalian cells but its monomeric nature and smallest size are the advantage for use as a fusion tag for live in vivo imaging.…”