## 4 Discussion  
Cosmetic Regulation (EC) No. 1223/2009 defines nanomaterials in cosmetic products as intentionally manufactured, insoluble, biopersistent substances, such as metals, metal oxides, or carbon materials, with sizes between 1 and 100 nm. In the sunscreen we applied to skin, the nanomaterials were a rutile of titanium dioxide (TiO\({}_{2}\)) nanoparticles with an apparent size of 42 \(\pm\) 10 nm by 9 \(\pm\) 2 nm. To observe NPs using electron microscopy and SIMS, it is essential to incorporate steps for fixing and dehydrating the biological tissues, embedding them in resin, cutting the sections, and depositing them on a suitable support. The objectives of these preparations are manifold: (1) to preserve ultrastructures, (2) to prevent the displacement of diffusible elements, (3) to make sample sections compatible with the physical constraints of the microscopes, (4) to render biological structures visible in TEM, and (5) to ensure that preparation methods do not introduce bias. Here, sections are deposited on electron microscopy grids for which a SIMS sample holder has been specially designed. The thickness of the sections must be as thin as possible (60 to 90 nm) so that electrons from the electron beam can pass through without blurring the image. Conversely, since NanoSIMS is a destructive analysis, the section has to be thick enough (the standard thickness is about 200 nm). In multi-instrument imaging studies that rely on different physical principles and, consequently, have different requirements, it is common practice to use serial sections, with each section's thickness adjusted to suit the specific instrument [49]. However, in this case, the analyzed areas were not exactly the same. Since nanoparticles are smaller than the section's thickness, nanoparticle analysis requires working on the same section [50], ensuring that the same NPs are analyzed using different instruments [51]. It was, therefore, essential in our study to use sample sections compatible with the two instruments. We, therefore, observed the same 90-nm-thick section with both instruments. In the TEM images, the NPs are clearly visible and distinguished from skin structures with a few rare exceptions. The NPs are noticeable by their size, their aggregate shape, and especially the contrast inherent in their chemical composition. The SIMS images of skin surfaces on which sunscreen had been applied show large aggregates of TiO\({}_{2}\) NPs. As we tested one, two, and three successive applications of sunscreen on the skin over 24 h, we were able to observe that the number of aggregates and their density on the skin surface increased with the amount of cumulated applied cream (not shown). This finding is encouraging, as it means that (1) the cream remained on the skin's surface despite the sample preparation for microscopy, (2) if there is a loss of NPs, it does not disrupt the NPs' proportionality with the number of applications, and (3) the fixation/dehydration/inclusion protocol did not induce a consequent NP loss. We could, therefore, extrapolate that NPs on the skin surface or in the skin were not relocated during the sample preparation process. Thus, we chose to present the results from samples exposed to three applications during 24 h because they were overloaded with NPs to increase the chance of NPs' penetration into the skin (if, indeed, NPs did penetrate). There are numerous studies on the localization of TiO\({}_{2}\) NPs applied to the skin, often employing two (and sometimes more) imaging techniques. However, their differing resolutions can occasionally limit the precision of NP localization. With the PIXE methods, it is difficult to detect Ti, as detection limits are reached [40] with a resolution of the order of 1-2 \(\upmu\)m. In our study, we aimed to detect and localize small NPs within biological ultrastructures. Combining the high-sensitivity imaging of NanoSIMS50 with TEM imaging appeared to be a relevant approach.