## 3 Results and Discussion  
### Two-Photon-Induced Luminescence  
We previously reported and extensively discussed the absorption spectrum of **1** in different solvents [33] and for a better visualization, the absorption profile in aqueous solution is displayed in Figure 2a. The compound intensely absorbed in the UV region (250-300 nm), with weaker bands in the visible (390-450 nm), and shoulders between 460 and 480 nm due to a direct, spin-forbidden population of the triplet-excited states(\({}^{3}\)MLCT and \({}^{3}\)LC mixed transitions). Under one-photon excitation at 400 nm, **1** exhibited a strong luminescence in the range 470-620 nm (Figure 2a); the emission quantum yield in air-equilibrated and deaerated aqueous solution was 0.1 and 0.96, respectively, with lifetimes on the time scale of hundreds of nanoseconds (395 ns in air-equilibrated water) to microseconds (2.37 \(\upmu\)s in deaerated water) [32; 36]. The long lifetime values confirmed the triplet nature of the emissive state. The significant quenching effect on the luminescence quantum yield and lifetime in the air-equilibrated condition confirmed that molecular oxygen in its ground state was able to interact with the triplet excited state of **1**, highlighting, indirectly, the huge photosensitizing abilities of the organometallic compound. The two-photon-induced luminescence properties of **1** were investigated in air-equilibrated aqueous solution by using a femtosecond Ti/sapphire laser as excitation source. As shown  
Figure 2: (**a**) Absorption and emission spectra of **1** in water solution (2\(\cdot\)10\({}^{-5}\) M). The emission spectrum was acquired under one-photon excitation at 400 nm. (**b**) Emission spectrum and (**c**) time-resolved fluorescence decay of **1** in water solution (1.54\(\cdot\)10\({}^{-2}\) M) under two-photon excitation at 800 nm. The used fitting equation was I(t) = I0 + \(\alpha_{1}\) exp (\(-\)t/\(\tau_{1}\)) + \(\alpha_{2}\) exp (t/\(\tau_{2}\)), with I0 = 621.51, \(\alpha_{1}\) (%) = 32, \(\alpha_{2}\) (%) = 68, \(\tau_{1}\) = 0.21 ns, \(\tau_{2}\) = 388.4 ns and a \(\chi^{2}\) = 0.973.  
in Figure 2b, the obtained emission profile, upon two-photon excitation at 800 nm, was similar in shape and energy to that of the linearly-excited emission (Figure 2a), indicating that the same excited state was reached by either one or two-photon excitation. Then, the fluorescence lifetime of **1** was obtained using a time-correlated, single-photon counting technique. The resulting decay curve shown in Figure 2c, exhibits a bi-exponential behavior consisting of a slow component (388 ns)--which dominates the decay profile--and a very fast component (0.21 ns) with a relative intensity of 32%. The lifetime value of the long component was identical (within experimental error) to that reported by exciting the sample under one-photon excitation at 400 nm (395 ns), and was ascribed to the phosphorescence arising from the excited 3 MLCT state. Regarding instead the fast component, in agreement with what was observed for the parent complex Ir(pppy)\({}_{3}\) under two-photon excitation [38], it could be attributed to a fluorescence from the 1 MLCT state populated by internal conversion from higher energy states, reached by two-photon absorption processes. To investigate the 2 PA properties of **1** in water, the 2 PA cross section at 800 nm was determined by the two-photon excited fluorescence (TPEF) technique [14], using Rhodamine B as standard. The measured \(\alpha_{2}\) value of **1** was 3.5 GM (1 GM = 10\({}^{-50}\) cm\({}^{4}\) s photon\({}^{-1}\)). As suggested by [PERSON] et al., \(\alpha_{2}\) values should be higher than 0.1 GM for optical imaging applications in live specimens [39]. The \(\alpha_{2}\) value of **1** not only met the requirement mentioned above but it was higher than that of the clinical photosensitizer H\({}_{2}\)TPP (\(\alpha_{2}\) = 2.2 GM at 800 nm in chloroform solution) [40].