Theoretical regulation of firefly bioluminescence spectra: Exploring new frontiers in NIR-II imaging

Abstract

Firefly bioluminescence (BL) imaging holds significant promise for non-invasive, real-time monitoring of biological processes. To improve its efficiency, considerable efforts have been made to achieve red-shifting of the emission wavelength. In this work, we systematically investigated the fluorescence properties of the light emitter oxyluciferin (oLu) dependent on the electrostatic effects using the TD DFT method. oLu is a typical donor–acceptor (D-A) charge transfer system, with the benzothiazole group acting as the electron donor and the thiazole group as the electron acceptor. Therefore, we focused on the significant impact of external charges on the fluorescence wavelength (λ_F), where the negative charge around the benzothiazole (donor) and/or the positive charge around the thiazole (acceptor) side promotes a red-shift in λ_F. Basing on these insights, we theoretically explored modifications to the oLu structure and mutations in luciferase to enhance D and A properties of oLu for facilitating the λ_F red-shift. Besides, we also inserted conjugation structures between D and A of oLu to format D-π-A systems for further red-shifting the λ_F. By integrating oLu modifications with luciferase mutations, we successfully designed a new nova-I351D using MD and QM/MM methods, a bioluminescent system that emits brightly in the near-infrared II (NIR-II) region. This system demonstrates significant potential for deep tissue imaging, providing a promising advancement in bioluminescence-based imaging technologies.