Non-Degenerate Two-Photon Absorption of Fluorescent Protein Chromophores.

Two-photon absorption (2PA), where a pair of photons are absorbed simultaneously, is recognized as a potent bioimaging technique, which depends on the quantified 2PA probability, defined as cross-section (σ). The absorbed photons either have equivalent (ω = ω) or different frequencies (ω ≠ ω), where the former is degenerate 2PA (D-2PA) and the latter is nondegenerate 2PA (ND-2PA). ND-2PA is of particular interest since it is a promising imaging technology with flexibility of photon frequencies and enhanced cross sections, however, it remains a relatively unexplored area compared to D-2PA. This work utilizes time-dependent density functional theory (TD-DFT) and second-order approximate coupled-cluster with the resolution-of-identity approximation (RI-CC2), for the excitation from S to S, to investigate σ and σ of FP chromophore models. Interestingly, comparing CAM-B3LYP with the RI-CC2 computations shows qualitative and, in fact, near quantitative agreement in the computed improvements of σ for comparable (relative) frequency detunings, despite the known underestimations of 2PA cross sections, for TD-DFT results relative to RI-CC2 values. As expected from the 2-state model, the computed values of σ are quantitatively larger than σ, where chromophores with the largest values of σ show greater potential for σ improvement. Anionic chromophores demonstrated improvements up to 14%, while substantial enhancements were observed in neutral chromophores with some achieving a 30% increase. This work investigates the ND-2PA photophysical characteristics of FP chromophores and identifies qualitative patterns in the computed properties of ND-2PA relative to D-2PA.