Genetically encoded near-infrared fluorescent protein probes based on biliverdin enable optical imaging of injury-associated phosphatidylserine in biological systems.

Optical imaging of injury-associated membrane events remains challenging because conventional fluorescent probes are often limited by tissue autofluorescence, shallow light penetration, and heterogeneous dye-to-protein labeling. Here, we report two genetically encoded, biliverdin-based near-infrared fluorescent protein probes for visualizing phosphatidylserine exposure, a conserved membrane signal associated with injury-induced membrane perturbation. The probes were constructed by fusing a version of red fluorescence protein (miRFP670nano3) with a small phosphatidylserine-targeting protein (lactadherin C2 domain or Annexin V), yielding LMB and AMB, respectively. This single-polypeptide design provides a defined molecular architecture, near-infrared fluorescence, and a fixed relationship between the phosphatidylserine-recognition and optical reporter. Spectroscopic analysis showed absorption and emission maxima at 645 and 675 nm, respectively, and lipid-film assays demonstrated preferential binding to phosphatidylserine. In chemically injured cells, both probes reported phosphatidylserine exposure in a dose-dependent manner, with AMB producing stronger responses in adherent-cell imaging and LMB labeling a larger fraction of suspension cells by flow cytometry. In an ex vivo skin explant model, LMB and AMB generated 2.3- and 2.7-fold higher signals than the non-targeted control, respectively. In mouse cutaneous wounds, topical application produced localized signals with 18.6- and 22.2-fold enhancement. After systemic administration in a deep neck muscle injury model, LMB and AMB yielded 2.6- and 4.5-fold higher signals than the control probe, and targeted signals decreased during tissue repair. These results establish near-infrared fluorescent protein probes as photochemically defined optical tools for relative and longitudinal imaging of injury-associated phosphatidylserine exposure in complex biological systems.