Nonlinear optical properties of photosensory core modules of monomeric and dimeric bacterial phytochromes

Abstract Near‐infrared (NIR) fluorescent proteins and optogenetic tools derived from bacterial phytochromes' photosensory core modules (PCMs) operate within the first (NIR‐I) tissue transparency window under single‐photon activation. Leveraging two‐photon (2P) light in the second transparency window (NIR‐II) for photoswitching bacterial phytochromes between Pr and Pfr absorption states offers significant advantages, including enhanced tissue penetration, spatial resolution, and signal‐to‐noise ratio. However, 2P photoconversion of bacterial phytochromes remains understudied. Here, we study the non‐linear Pr to Pfr photoconversion's dependence on irradiation wavelength (1180–1360 nm) and energy fluence (41–339 mJ/cm 2 ) for the PCM of Dr BphP bacterial phytochrome. Our findings reveal substantially higher photoconversion efficiency for the engineered monomeric Dr BphP‐PCM (73%) compared to the natural dimeric Dr BphP‐PCM (57%). Molecular mechanical calculations, based on experimentally determined 2P absorption cross‐section coefficients for the monomer (167 GM) and dimer (170 GM), further verify these results. We demonstrate both short‐ (SWE) and long‐wavelength excitation (LWE) fluorescence of the Soret band using 405 and 810–890 nm laser sources, respectively. Under LWE, fluorescence emission (724 nm) exhibits saturation at a peak power density of 1.5 GW/cm 2 . For SWE, we observe linear degradation of fluorescence for both Dr BphP‐PCMs, decreasing by 32% as the temperature rises from 19 to 38°C. Conversely, under LWE, the monomeric Dr BphP‐PCM's brightness increases up to 182% (at 37°C), surpassing the dimeric form's fluorescence rise by 39%. These findings establish the monomeric Dr BphP‐PCM as a promising template for developing NIR imaging and optogenetic probes operating under the determined optimal parameters for its 2P photoconversion and LWE fluorescence.