Rational control of two-photon absorption through π-bridge engineering: theoretical study on the π-conjugation effects on the optical properties of fluorescent probes

Although two-photon-excited fluorescent (TPEF) probes offer significant advantages for deep tissue imaging, their practical application remains constrained by limited two-photon absorption (TPA) cross sections (σ) and low fluorescence quantum yields (Φ). This study systematically investigates the effects of introducing different π-conjugated structures (π-extension and π-shortening) between the fluorophore and reaction site on the optical properties of fluorescent probes. Theoretical calculations reveal that π-extension induces spectral red-shift and enhances σ by approximately 5–10-fold, but simultaneously accelerates excited-state geometric relaxation, causing Φ to decrease by several-fold to tens of fold; π-shortening induces spectral blue shifts, weakening σ by approximately 5–10 fold, yet effectively enhances molecular rigidity and suppresses nonradiative decay, thereby increasing Φ by several-fold to tens of fold and prolonging fluorescence lifetime. The study further reveals that regulating the frontier molecular orbital energy gap, transition dipole moment, and reorganization energy is the key mechanism influencing TPA efficiency and luminescence performance. This work provides explicit guidance for the rational design of TPEF probes: π-extension is suitable for deep tissue imaging requiring high absorption cross sections, while π-shortening is more appropriate for cellular-level detection demanding high luminescence efficiency and stability.