Resonance Enhancement of Two-Photon Absorption in Fluorescent Proteins

We measure two-photon absorption (2PA) spectra of wild-type green fluorescent protein, cyan fluorescent protein, and monomeric red fluorescent protein in absolute cross section values in a wide spectral range (λ2PA = 550 − 1300 nm), and find, for the first time to our knowledge, a new S0 → S n 2PA transition in all three proteins in the short-wavelength region. This transition is strongly resonantly enhanced, showing 2PA cross section values of ∼20−160 GM, which are at least 2−4 times higher than those measured in the lowest energy (S0 → S1) transition of corresponding proteins. We also show that the change of permanent dipole moment upon S0 → S1 excitation (|Δμ10|) can be deduced from 2PA cross section, providing a new tool for fast evaluation of |Δμ10| in physiological conditions.