The TPA and 3PA spectra are calculated, respectively, by reading the excitation energies, oscillator strengths, and two-and three-photon transition strengths from the Dalton outputs, summing up to the third excited state (i = 3 in Eqns. 2-3), and applying Lorentzian broadening with γ i = 0.1 with the help of Dalton Project. The resulting spectra are shown in Fig. , and numbers are summarized in Table 2. The OPA spectra for the QM-sampled moiety closely aligns with the experimental results, showing deviations of 0.17 and -0.3 eV for the first and second excited states, respectively. In contrast, the MM-sampled moiety exhibits larger deviations of 1.02 and 0.74 eV. The experiment shows an intense peak at 4.5 eV, which is not reproduced theoretically, potentially due to contributions from tryptophan residues in the ChR2, which contains up to 10 tryptophans in total absorbing in this region. The TPA spectra for the QM-and MMsampled moieties are shown in Fig. . The differences in excitation energies between both moieties are summarized in Table , showing deviations of 0.38 and 0.34 eV for the first and second excited states, respectively. To the best of our knowledge, no experimental TPA spectra have been reported for this system for direct comparison. Fig. compares the OPA and TPA spectra with and without considering the environment. While the line shape and intensities differ significantly, the position of the peaks show only minor deviations. For the OPA spectra, the first and second excited states exhibit differences of 0.37 and 0.62 eV, respectively. In the case of the TPA spectra, the deviations are smaller, with differences of 0.21 and 0.06 eV for the first and second excited states, respectively.