Illuminating the origins of two‐photon absorption properties in fluorescent protein chromophores

We provide here a structural impact on two‐photon absorption cross‐section ( σ TPA ) for 22 distinct fluorescent protein (FP) chromophores. By employing time‐dependent density functional theory, we gain insight into two‐photon absorption (TPA) process by investigating relationship between σ TPA and one‐photon electronic transition dipole moment and permanent dipole moment change (Δ μ ) upon transition. Our results reveal that for the S 1 excited state, σ TPA is proportional to (Δ μ ) 2 in agreement with two‐state model of TPA process. On the contrary, the TPA spectroscopy of higher excited states ( S n , n  > 1) is much more complex. We do not find a main driving force of large σ TPA that would be common for investigated chromophores. Instead, it seems that channel interference between one‐photon transition dipole moment vectors is responsible for enhancement or diminishment of σ TPA . Our in vacuo results may serve as a benchmark to investigate a role of chromophore‐protein interaction in shaping TPA spectra of FPs.