Chemistry

Materials science

Fluorescence

Optical imaging

Preclinical imaging

Biochemistry

Physics

Cell

Fluorescence microscope

Biotechnology

Biology

Optics

Near-infrared spectroscopy

Fluorescence-lifetime imaging microscopy

STED microscopy

Microscopy

Live cell imaging

Brightness

Molecular imaging

In vivo

Stimulated emission

Laser

Nature Communications

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.

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