## 4 Discussion
Conversely, ZnQ\({}_{2}\) did not penetrate inside cells, and so we concluded that it is not suitable for in vivo incorporation. This could be in principle due to a strong Zn complex-complex aggregation in solution, leading to a decrease in its bioavailability. Cell viability was also investigated indirectly by exploring the biological property of _Ph. tricornutum_ diatoms to adhere on substrates. As shown in Figure 4, bare cells increased their density on glass substrates in 4 days, clustering at 7 days of culture. Conversely, iridium and aluminum-doped cells formed smaller clusters due to cell-cell interactions. Ruthenium-doped cells appeared as adherent cells, but they lost the capability of forming cell clusters, likely due to a decrease in cell-cell interaction in the suspension as a possible defense mechanism. As with all diatom species, _Pluodactylum tricornutum_ cells exhibit a silica cell wall with polysaccharides impregnating the silica matrix. This cell wall protects the inner protoplasm. To confirm the staining of biosilica after the in vivo incorporation of complexes and isolate luminescent frustules, the inner protoplasm, made of chloroplasts and organic matter of cytoplasm, was removed. Two methods of purification were tested to remove the organic matter and retain the luminescence of complexes into biosilica shells, thus recovering luminescent valves with retained morphology. A soft purification protocol reported by [PERSON] et al. [32] based on washing with sodium dodecyl sulfate (SDS 5% \(V/V\)) and ethylene diamino tetraacetic acid (EDTA 100 mM) for 15 min at 60 \({}^{\circ}\)C did not successfully remove the chloroplasts and the organic protoplasm from biosilica. Contrary to our previous results reported for _Thalassia wewissflogii_ frustules, a harsh cleaning protocol based on sulfuric acid (98%, 50 \(\mu\)L), chloridic acid (34%, 50 \(\mu\)L), and hydrogen peroxide H\({}_{2}\)O\({}_{2}\) (50 \(\mu\)L for 3 times) at 55 \({}^{\circ}\)C for 30 min turned out to be very efficient to remove the protoplasm and chloroplasts and purify the Ir, Ru, and Al-doped biosilica samples, after in vivo incorporation experiments. In living diatoms, red chloroplasts only contributed to the red emission of bare cells (Figure 5 ai), while the iridium complex stained silica and its emission was also localized in chloroplasts (Figure 5 bi). In the case of Ru and Al-doped cells, some empty cells (insets in Figure 5 ci,di) and chloroplast-active cells were observed (Figure 5 ci,di). After three cycles of acid-oxidative treatment, empty stained valves incorporating the complexes were isolated and spotted over glass slides (Figure 5 bi,ci,dii). Control valves exhibited a weak blue autofluorescence mainly due to silica and residual organic matter (Figure 5 aii). Confocal microscopy was also used to record photoluminescence spectra for each sample of doped biosilica after purification, with the aim to compare them with those of neat films of the organometallic complexes. All samples were obtained after 7 days of feeding. Emission spectra were recorded in the 450-750 nm spectral range, exciting (\(\lambda_{\text{exc}}\) = 405 nm) entire valves. In all cases, the photoluminescence of complexes inside doped valves was slightly blue-shifted versus that of the neat film of the complex (Figure 6a-c). This was likely due to an effect of entrapment of complexes in the silica bulk, or of interaction between the complex photoluminescence and the silica nanostructure, or reduced aggregation compared to the complex as a neat film. As shown in the insets of Figure 7, all purified luminescent valves retained their morphological integrity. Evidence of the efficacy of the cleaning procedure of biosilica valves was also given by Fourier Transformed Infrared-Attenuated Total Reflectance (FTIR-ATR) spectroscopy (Figure 7).