Compact red-shifted near-infrared fluorescent proteins enable deep-tissue SWIR imaging with in vivo optical clearing.

Manoilov, K. Y.; Xu, Y.; Luo, J.; Oliinyk, O. S.; Carey, E.; Tokarchuk, K. O.; Zhang, J.; Hong, G.; Nimmerjahn, A.; Yao, J.; Verkhusha, V. V.

doi:10.64898/2026.07.25.740731

bioRxiv · 10.64898/2026.07.25.740731

Compact red-shifted near-infrared fluorescent proteins enable deep-tissue SWIR imaging with in vivo optical clearing.

Manoilov, K. Y.·Xu, Y.·Luo, J.·Oliinyk, O. S.·Carey, E.·Tokarchuk, K. O.·Zhang, J.·Hong, G.·Nimmerjahn, A.·Yao, J.·Verkhusha, V. V.

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Abstract

Compact 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 Leptolyngbya sp. into three monomeric, biliverdin-binding 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 1,000 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-{kappa}B and AP-1 transcriptional activities. It enabled single-laser, dual-color three-photon imaging with EGFP to depths of [~]950 m in cortex and [~]300 m 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.

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Manoilov, K. Y., Xu, Y., Luo, J., Oliinyk, O. S., Carey, E., Tokarchuk, K. O., Zhang, J., Hong, G., Nimmerjahn, A., Yao, J., Verkhusha, V. V.. 2026-07-27. Compact red-shifted near-infrared fluorescent proteins enable deep-tissue SWIR imaging with in vivo optical clearing.. https://doi.org/10.64898/2026.07.25.740731

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