## 4 Discussion  
The negative effects of Nd:YAG laser treatment on IOLs surface have been extensively demonstrated with SEM [9; 10; 11; 12]: in fact, in PMMA IOLs, surface star-like craters were observed, while, in silicone IOLs, smooth splash-like craters were present [9]. However, in all these studies, the technique used to dehydrate IOLs prior to SEM examination was not indicated. Only in an experimental work in canine cadaver eyes the examined specimens were treated with CPD: at high energy, in PMMA IOLs, sharp, irregular central pits with radiating fractures were demonstrated [15]. In this study, we confirmed that Nd:YAG laser treatment, even at the low energy level (1.2 mJ) currently used in the clinical practice, induced evident damage on IOL surface, closely related to the number of spots applied. However, no data were available on IOL behavior under physical stress. Therefore, we evaluated the effects of the process of dehydration with CPD, ideal for SEM observation, on both normal and laser-treated IOLs. Most soft biological specimens are damaged when water is allowed to dry by evaporation. The damage is triggered when the air/water meniscus or interface passes through the specimen, creating surface tension forces of 2000 PSI and inducing the collapse of the tissues [16]. To prevent structural damages, prior to CPD water is substituted with ethanol and then with liquid CO\({}_{2}\), which,  
Figure 4: **(A)** Carbon (C) and oxygen (O) counts in control IOLs and in the spots (Spot \(\alpha\) (undamaged surface), Spot \(\beta\) (edge of the surface hit by the laser) and Spot \(\gamma\) (center of the laser injury)) of the laser damaged IOLs. * \(p\) < 0.05 versus C of control and Spot \(\alpha\); § \(p\) < 0.05 versus O of control and Spot \(\alpha\); § \(\mu\) \(p\) < 0.05 versus C of Spot \(\beta\). **(B)** Carbon (C) and oxygen (O) counts ratio in control IOLs and in the spots (Spot \(\alpha\) (undamaged surface), Spot \(\beta\) (edge of the surface hit by the laser) and Spot \(\gamma\) (laser injury)) of the laser damaged IOLs. * \(p\) < 0.05 versus control and Spot \(\alpha\); § \(p\) < 0.05 versus Spot \(\beta\). at a specific temperature and a corresponding pressure (critical point), is converted to gas that is slowly released, eliminating the hazardous air/water interface. For liquid CO\({}_{2}\), the critical point is 31.1 \({}^{\circ}\)C at 1073 PSI [16]. Therefore, CPD technique can reliably preserve the spatial and structural integrity of tissues and soft specimens [14; 17]. After CPD, it was demonstrated that Group 1 IOLs had regular shape, margins and surface. Mild changes were observed in Group 2 IOLs, which showed irregular surface, peripheral blebs and evident cracks starting from the laser-induced holes. On the contrary, in Group 3 IOLs, after an elevated number of laser shots, even the biologically harmless tension force (1073 PSI) created during CPD was followed by collapse and burst of the IOL. Therefore, laser treatment at low energy (1.2 mJ) was able to induce moderate structural changes when few spots were applied, while a higher number of spots induced structural weakness of the IOL, with an evident change of its shape and lower resistance of the materials even to low physical stress [18]. The second target of this study was to evaluate the same type of IOLs after the different laser treatments (two and six spots) with SEM and EDX. This analytical technique provides a chemical characterization of the samples detecting the elements by which they are formed [19]. The spectra indicate the single elements with objective data about their concentration. Recently, IOLs have been examined with SEM and EDX analyses. However, these studies analyzed explanted IOLs after opacification induced by calcification [20]. No data, as far as we know, are currently available on the possible changes in chemical composition of IOL materials after Nd:YAG laser treatment.