## 3 Results and Discussion
### Two-Photon-Induced Luminescence Imaging and Calcein-AM Assay on Human Glioblastoma Cells  
To evidence the efficacy of the 2P-PDT effect, a cell experiment was conducted. In particular, the calcein-AM assay was performed on human glioblastoma U87-MG cells to determine the viability after incubation with **1** followed by two-photon excitation exposure. The acetoxymethyl derivate of calcein (calcein-AM) is a non-fluorescent hydrophobic compound that easily penetrates into viable cells. It was then converted by intracellular esterases to calcein, a strongly fluorescent compound that was retained in the cytoplasm of cells with intact membranes, producing an intense green signal (500-550 nm), whereas it was rapidly extruded from damaged or dead cells [44]. U87-MG cells were incubated with calcein-AM or with compound **1** and calcein-AM. An area of the cell medium was exposed to two-photon irradiation at 800 nm and the remaining region kept in the dark. Then, confocal laser scanning microscopy images were carried out detecting the luminescence signals arising from both calcein (green channel) and Ir(III) complex (red channel), by exciting the probe at 488 nm and **1** at 800 nm (Figure 4). As clearly shown in Figure 4a-c, upon excitation at 488 nm a strong and intense green signal in small vesicles and in the cytoplasm was observed in U87-MG cells incubated only with calcein-AM and exposed to irradiation, indicating their viability and no toxic effects due to the NIR exposure. In the further control experiment (Figure 4d-f)-cells were incubated with both calcein-AM/**1** and not exposed to radiation--the luminescence of the organometallic complex, localized mainly in the cytoplasm, was clearly visible by exciting at 800 nm (Figure 4d). Then, upon excitation at 488 nm, a huge luminescence arising from the calcein probe was also detected (Figure 4e), highlighting the cell viability and consequently no cytotoxicity of **1** without NIR-mediated activation. \begin{table}
\begin{tabular}{c c c c} \hline \hline
**Sample** & **ABDA mol** & **ABDA mol** & **1O2 mol** \\  & **(0)** & **(15 min)** & **6.2\(\cdot\)10\({}^{-9}\)** \\ \hline
1 & 7.62\(\cdot\)10\({}^{-8}\) & 7.00\(\cdot\)10\({}^{-8}\) & 6.2\(\cdot\)10\({}^{-9}\) \\ \hline \hline \end{tabular}
\end{table} 
Table 1: Moles of 1O2 generated after light exposure of **1** (\(\lambda_{\text{ex}}\) 800 nm-100 mW-15 min) and calculated from the amount of reacted ABDA molecules. Figure 3: Singlet oxygen generation detected by ABDA method. (**a**) Photobleaching of ABDA by singlet oxygen at different irradiation times (0–15 min) in water, and (**b**) in presence of **1** (8\(\cdot\)10\({}^{-5}\) M). The experiments were carried out by using an ABDA concentration of 2.4\(\cdot\)10\({}^{-4}\) M and a laser beam at 800 nm (100 mW) as excitation source; (**c**) plotting of ABDA absorption at 378 nm as a function of the illumination time. On the contrary, in the area containing cells incubated with both calcein-AM/**1** followed by NIR exposure (Figure 4g-i), the green luminescence intensity of the viability marker results changed, becoming very weak and diffuse (Figure 4h), pointing out a loss of cell viability. Since no cell death was recorded in the control groups, the results confirmed that the reduced cell viability in the experimental group was clearly due to a photocytotoxic effect of the organometallic compound after two-photon irradiation. Moreover, a change in the luminescence intensity signal arising from **1** was also observed (Figure 4g). In particular--comparing the confocal view of Figure 4g,d--a strong increase in the emission intensity of the cell viability was observed (Figure 4g). The increase in the fluorescence intensity of the cell viability was observed (Figure 4g). The increase in fluorescence intensity of the cell viability was observed (Figure 4g). sion signal of 1 was easily observed in the area exposed to laser irradiation (Figure 4g) with respect to the unexposed one (Figure 4d).