3.3 Electrostatic effect of polar solvents. Next, we seek to understand how the fluorescence spectrum of HFOH may be affected by electrostatic effect. First, we investigated the effect of solvent polarity, which can be used to detect the polarity of the environment surrounding the fluorophore. TD-DFT calculations were performed using the polarizable continuum model (PCM) to account for the change in electronic properties due to solvent polarity. This approach has been commonly used to study the effect of the luciferase cavity on bioluminescence. To exclude conformational effect, the electrostatic effect calculations were studied with System III based on a fixed geometry, namely the planar QM/MMoptimized geometry shown in Figure . Figure shows the shift of the fluorescence spectrum under the influence of different polar solvents. Generally, the emission maximum λ F is red-shifted to longer wavelengths as the solvent polarity increases. To be more specific, the dielectric effect can be divided into regimes of low (ε < 25) and high (ε > 25) polarity. In the low-polarity regime, the spectral shifting due to polarity change is significant. Conversely, in the highpolarity regime, the change in emission wavelength is much smaller or even negligible. Intriguingly, the variation trend is consistent with the one observed for the fluorophore in the firefly system, indicating the generality of this spectral regulation rule by solvent polarity. Figure shows the variation of spectral intensity upon the change of solvent polarity. The intensity increases as the solvent polarity increases with a stronger dependence in the low-polarity regime compared to the high-polarity regime (Figure ). The computed energies of the S 0 and S 1 states (E 0 and E 1 , respectively) under different solvents (Figure ) show that: (1) polar solvents stabilize both S 0 and S 1 states; (2) the stabilization effect is stronger for solvents with larger dielectric constants (ε); (3) the stabilization effect is slightly stronger for the S 1 state than the S 0 state. Therefore, it is the different stabilization of the S 0 and S 1 states that leads to the observed red-shift in the emission spectrum with increasing solvent polarity. It is well-known that the electrostatic interaction directly correlates with the magnitude of the dipole moment, a larger dipole moment corresponding to a stronger stabilization. To understand the variation trend of E 0 and E 1 , the dipole moments of the S 0 and S 1 states (μ 0 and μ 1 , respectively) under different solvents were calculated. As shown in Figure , both μ 1 and μ 0 increase with increasing ε, and μ 1 is always larger than μ 0 . The increase of μ 1 and μ 0 results in the decrease of E 1 and E 0 , respectively. Moreover, the larger value of μ 1 compared to μ 0 readily explains the stronger stabilization of the S 1 state compared to the S 0 state. Thus, the variation trend of E 1 and E 0 shown in Figure can be well explained by the change of the dipole moments shown in Figure , which accounts for the observed variation in the fluorescence spectrum with solvent polarity shown in Figure .