Fig. 1. Mechanism of two-photon excited photodynamic therapy (2P-PDT). (a) Jablonski diagram of singlet oxygen generation by photosensitizers with one-photon, two-photon, and three-photon absorption, where S₀ represents ground state, S₁ represents singlet excited state, IC represents internal conversion, ISC represents intersystem crossing, T₁ represents the first triplet excited state, ¹O₂ represents singlet oxygen, and ³O₂ represents molecular oxygen; (b) spatial distribution of one-photon and two-photon excited fluorescence signals^[18]; (c) absorption spectra of oxyhaemoglobin and deoxyhaemoglobin in visible and near-infrared regions^[25]; (d) illustration of penetrability and activation distribution of UV-vis light excited one-photon photodynamic and near-infrared light excited two-photon photodynamic in tissue

Fig. 2. Examples of chemical structures of typical photosensitizers

Fig. 3. Improvement of ROS generation efficiency. (a) Improving ROS generation efficiency through HOMO-LUMO separation^[49]; (b) adjustment of ΔE_ST and spin-orbit coupling (SOC) for enhanced ROS generation efficiency^[50]

Fig. 4. Enhancement of two-photon absorption. (a) FRET techniques for two-photon absorption improvement^[52-53]; (b) enhancement of two-photon absorption through extended conjugation in donor-acceptor systems^[57-59,61]

Fig. 5. In vitro real time detection of ROS generation under two-photon excitation^[65]

Fig. 6. Aggregation-induced emission characteristics^[71]. Fluorescence images of DDPD and TPE molecules in the mixtures of tetrahydrofuran and water with different ratios under UV light: (a) DDPD; (b) TPE. Fluorescence brightness and ROS generation efficiency changes of Ce6 and TPEDC in the nanoparticle formulations with varied loading percentages: (c) fluorescence brightness; (d) ROS generation efficiency

Fig. 7. 2P-PDT induced blood vessel closure. (a) Bright field and (b) fluorescence images of blood vessels in a skinfold window of a living mouse injected with conjugated porphyrin dimers after femtosecond laser excitation at 920 nm^[84]: (a) bright field image; (b) fluorescence image. In vivo two-photon fluorescence images of mouse brain blood vessels labeled with TPEDC nanoparticles before and after femtosecond laser irradiation at 800 nm^[86]: (c) before irradiation; (d) after irradiation

Fig. 8. Polymerization-enhanced two-photon photosensitization^[65]. (a) Chemical structures of small molecule and conjugated polymer photosensitizers; (b) in vitro¹O₂ detection of photosensitizer dots in aqueous media under two-photon excitation; (c) intracellular ¹O₂ detection of photosensitizer dots-stained HeLa cells under two-photon excitation; (d) in vitro 2P-PDT on PTPEDC2 dots-stained HeLa cells under different laser scanning numbers, green represents live cells and red represents dead cells; (e) in vivo 2P-PDT on PTPEDC2 dots targeted zebrafish liver tumor

Fig. 9. NIR-II excited 2P-PDT^[92]. (a) Scattering coefficient of brain tissue as a function of wavelength in 700－1300 nm; (b) normalized photo fraction of light after passing through a brain tissue with thickness of 1 mm; (c) photosensitizer TQ-BTPE for lysosome targeted and NIR-II excited 2P-PDT; (d) comparison of NIR-I and NIR-II excited ROS generation of TQ-BTPE in aqueous media; (e) intracellular ROS generation of TQ-BTPE targeted HeLa cells under NIR-II excitation; (f) comparison of NIR-I and NIR-II excited intracellular ROS generation; (g) comparison of NIR-I and NIR-II excited 2P-PDT induced ablation of TQ-BTPE labeled cells

Fig. 10. 2P-PDT for solid tumors. (a) Three-dimensional tumor spheroids model^[99]; (b) activable photosensitizer Ace-DSB for 2P-PDT^[102]; (c) stereotactic 2P-PDT^[103]