Abstract
<jats:p> Compact near-infrared (NIR) fluorescent proteins (FPs) with red-shifted emission are needed for deep-tissue short-wavelength infrared (SWIR) imaging. We engineered a GAF domain from the JSC1 cyanobacteriochrome of thermophilic <jats:italic>Leptolyngbya sp.</jats:italic> into three monomeric, biliverdin-binding NIR FPs of 19.1 kDa: miRFP729nano, miRFP732nano and miRFP735nano, with excitation/emission maxima of 714/729, 716/732 and 719/735 nm, respectively. Their off-peak fluorescence beyond 1000 nm was several-fold higher than that of miRFP718nano previously used for SWIR imaging. miRFP732nano functioned as a fusion tag, a component of target-stabilized nanobodies, and a reporter of NF-kappaB and AP-1 transcriptional activities. It enabled single-laser, dual-color three-photon imaging with EGFP to depths of ~950 micrometers in cortex and ~300 micrometers in spinal cord. In mice, miRFP732nano supported SWIR imaging of skeletal muscle, inflammatory signaling and intracellular targets. Combining SWIR detection with biocompatible 4-aminoantipyrine-based in vivo tissue clearing enhanced signal and image sharpness. These red-shifted NIR FPs expand the genetically encoded toolkit for deep-tissue imaging. </jats:p>