BPS2026 – The two-photon polarization ratio explains unusual shape of the two-photon absorption spectra of fluorescent dyes and detects weak excitonic coupling in fluorescent protein dimers

Two-photon absorption (2PA) is a nonlinear optical process widely used in biological microscopy because of its high spatial selectivity and deep tissue penetration. Understanding physical mechanisms of the 2PA process can guide more efficient excitation of fluorescent probes in two-photon microscopy. A critical parameter characterizing 2PA is the two-photon polarization ratio (Ω), which describes molecular response to circularly vs. linearly polarized light and provides insight into the symmetry of vibronic and excitonic transitions. In this work, we present a theoretical model based on sum-over-states approach to 2PA and few-state approximations to derive analytical expressions for Ω as a function of different molecular dipole moment matrix elements and the angles between them. The model describes vibronic contributions via Herzberg-Teller coupling of the permanent dipole moments to bond-length alternation (BLA) vibrations. Using this framework, we analyze both monomeric fluorophores—such as rhodamine 6G, bodipy, and the fluorescent proteins (FPs) chromophore in FP venus—and dimeric systems, such as tandem dimer of FP Venus, where two chromophores demonstrate a weak excitonic coupling. Our model explains unusual shape of the 2PA spectra of commonly used fluorescent dyes and FPs and detects weak excitonic coupling in FP dimers.