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

Mikhail E. Matlashov, Daria M. Shcherbakova, Jonatan Alvelid, Mikhail Baloban, Francesca Pennacchietti, Anton A. Shemetov, Ilaria Testa, Vladislav V. Verkhusha

Research output: Contribution to journalArticlepeer-review

101 Scopus citations


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 ~105 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.

Original languageEnglish (US)
Article number239
JournalNature communications
Issue number1
StatePublished - Dec 1 2020

ASJC Scopus subject areas

  • General Chemistry
  • General Biochemistry, Genetics and Molecular Biology
  • General Physics and Astronomy


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