###### Abstract  
This work reports the study of two-photon induced properties of a highly luminescent cyclometalated Ir(III) complex, [Ir(ppy)\({}_{2}\)(en)] OOCCH\({}_{3}\) (**I**), ppy = 2-phenylpyridine, en = ethylenediamine. Steady-state and time-resolved fluorescence measurements were performed by exciting **1** at the biologically relevant wavelength of 800 nm, whereas, the generation of singlet oxygen (\({}^{1}\)O\({}_{2}\)) was evaluated using 9,10-Anthracenediyl-bis(methylene)dimalonic acid (ABDA) as a detection probe. Preliminary in vitro experiments with U87-MG cells were performed, showing the potential of this compound as a two-photon photodynamic therapy (2P-PDT) agent at NIR wavelengths. cyclometalated Ir(III) complexes; two-photon photodynamic therapy; time-resolved luminescence spectroscopy +
Footnote †: journal: Medical  
excited state of the PS typically populated by the absorption of one photon of around twice the energy. The efficiency of two-photon absorption (2 PA) can be identified as the 2 PA cross-section (\(\sigma_{2}\)) of the PS, which is similar to the molar absorption coefficient in a one-photon absorption process. The first successful in vivo PDT experiment using a PS engineered for efficient two-photon excitation was reported by [PERSON] and co-workers in 2008 [15]. The authors synthesized a new conjugated porphyrin dimer and demonstrated--after injection, followed by two-photon irradiation at 920 nm--the selective closure of blood vessels in mice bearing skinfold window chambers [15]. Over the last few decades, transition metal complexes have attracted a growing interest as PSs and imaging probes for theranostic applications [16] and, more recently, as promising multi-photon absorbing materials [17; 18; 19]. In particular, the presence of a heavy atom, favoring the intersystem crossing process, leads to the population of excited triplet states, usually metal-to-ligand charge transfer (MLCT) excited states. Since these states have a distorted symmetry with respect to that of the ground state, the radiative deactivation is disadvantaged, whereas an energy transfer process towards molecular oxygen is favored, with consequent singlet oxygen generation. Furthermore, the metal center can act as multidimensional scaffold assembling several two-photon active ligands in a variety of arrangements and, depending on the nature of the metal-promoting long-range intramolecular charge transfer processes, which are known to be crucial for improving nonlinear optical properties [20]. Hence, transition metal complexes that exhibit ILCT (intraligand charge transfer) and MLCT transitions, i.e., Ru(II) and Ir(III) compounds, turn out to be promising candidates for two-photon applications [21; 22]. In 2009, [PERSON]'s group reported the first example of in vitro 2P-PDT studies using a water soluble Ru(II) complex [23] as a photosensitizer. A few years later, [PERSON] and colleagues synthesized and evaluated in vitro the first Ir(III)-based molecular dyad as a two-photon singlet oxygen generation agent and fluorescent probe [24]. Since then, a number of luminescent Ru(II) and Ir(III) complexes with two-photon absorption behavior have been developed for application in bioimaging and cancer phototherapy [11; 25; 26; 27; 28; 29; 30; 31; 32]. Previously, we reported the synthesis and characterization of the cyclometalated Ir(III) complex [Ir(pypy)(en)] OOCCH\({}_{3}\), (**1**), where ppy = 2-phenylpyridine and en = ethylenediamine [33] (Figure 1). The presence of a hydrophilic acetate as counterion induces a high water solubility (45 mg/mL), making the organometallic compound very appealing for biological applications. Upon one-photon excitation, **1** displayed remarkably high phosphorescence quantum yield and oxygen photosensitizing ability [33; 34]. Thus, based on the interesting photophysical properties, it was investigated in vitro and in vivo as a PS and lumines  
Figure 1: Chemical structure of [Ir(pppy)\({}_{2}\)(en)] OOCCH\({}_{3}\) compound (**1**).