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

## TL;DR

This study explores how monomeric and dimeric bacterial phytochromes respond to two-photon light, showing that the monomeric form is more efficient for imaging and optogenetics.

## Contribution

The study reveals higher two-photon photoconversion efficiency in engineered monomeric DrBphP-PCM compared to its dimeric form.

## Key findings

- Monomeric DrBphP-PCM achieves 73% photoconversion efficiency, outperforming the dimeric form at 57%.
- Monomeric DrBphP-PCM fluorescence brightness increases by 182% under long-wavelength excitation at 37°C.
- Molecular mechanical calculations confirm the higher 2P absorption cross-section for the monomeric form.

## 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/cm2) for the PCM of DrBphP bacterial phytochrome. Our findings reveal substantially higher photoconversion efficiency for the engineered monomeric DrBphP‐PCM (73%) compared to the natural dimeric DrBphP‐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/cm2. For SWE, we observe linear degradation of fluorescence for both DrBphP‐PCMs, decreasing by 32% as the temperature rises from 19 to 38°C. Conversely, under LWE, the monomeric DrBphP‐PCM's brightness increases up to 182% (at 37°C), surpassing the dimeric form's fluorescence rise by 39%. These findings establish the monomeric DrBphP‐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.