## 3 Results
### Toward Isolated NPs' Detection  
In Figure 4E, numerous pixels with a low-intensity \({}^{48}\)Ti\({}^{16}\)O signal are relatively evenly and randomly distributed. They were derived from background noise. In the context of an exploration of isolated NPs, these non-specific signal pixels were considered false positives. If isolated NPs were present in the analyzed zone (true positive pixels), they produced a low signal that was difficult to distinguish from the false positive pixels. To distinguish true positive pixels with a very low signal (one or two counts on the \"sum\" image in Figure 4B) from false positives, the pixels were filtered using a probabilistic approach. We measured, on control images, the frequency of false positive pixels (non-zero pixels) at 0.00047. Considering the 30 analyzed planes (30 images of 256 \(\times\) 256 pixels), the sputtered volume was roughly equivalent to 2.10\({}^{6}\) pixels. Thus, we could expect to observe an average of 900 false positive pixels (across the entire volume). In the \"sum\" image (Figure 4B), all  
Figure 4: Localization of TiO\({}_{2}\) NPs close to corneocytes. (**A**,**B**) SIMS images of the skin/NP sample section in the “Cameca” color scale. (**A**) “Sum” image at mass \({}^{12}\)C\({}^{14}\)N. (**B**) “Sum” image at mass \({}^{48}\)Ti\({}^{16}\)O. The purple square represents the region of interest used in figures (**C**–**F**). (**C**) Scheme of the corresponding ROI. O: Outgrowth; F: Furrow; C1, C2, and C3: Corneocytes 1, 2, and 3; arrow: inter-cornecyc contact. (**D**) MET image acquired via Jeol JEM 2010. The arrows indicate NP aggregates. (**E**,**F**) Overlay of the \({}^{12}\)C\({}^{14}\)N (magenta) and \({}^{48}\)Ti\({}^{16}\)O (“Cameca” color scale) on the TEM image without an SCP filter (**E**) and with an SCP filter (**F**). (**D**–**F**) The arrows indicate NPs, and the circles indicate clusters of false positive pixels. SIMS images: dwell time, 1 ms/pixel; raster, 10 \(\upmu\)m \(\times\) 10 \(\upmu\)m, 256 \(\times\) 256 pixels, sum of 30 plans. Scale bar: 1 \(\upmu\)m (**A**,**B**); 500 nm (**C**–**F**). false positive pixels are stacked with a density of 1 per 80 pixels. Considering the sputtered volume, the probability of two contiguous false positive pixels in three-dimensional space was, therefore, around 25.10\({}^{-8}\). Given that this probability was low, we applied a filter named \"Selection of Contiguous Pixels\" (SCP) to the \({}^{48}\)Ti\({}^{16}\)O images. The principle was to consider as true positive pixels only those positive pixels (a non-zero \({}^{48}\)Ti\({}^{16}\)O signal) that had at least one non-zero neighboring pixel (a contiguous pixel in three-dimensional space). Figure 4F shows the overlay produced from this filtered \({}^{48}\)Ti\({}^{16}\)O \"sum\" image. The resulting image shows fewer scattered pixels. The three large clusters (the red and pink arrows) initially identified (Figure 4D) are clearly visible. Some pixel clusters disappeared after the SCP filter was applied because they were not contiguous pixels on the X, Y, and Z-axes despite appearances (the circles in Figure 4E). On the other hand, the application of the SCP filter revealed a few true positive pixels (the green arrows in Figure 4F), which were not distinguished from the false positives in Figure 4E. These NPs were not detected via TEM. Figure 5 is a STEM-HAADF image of the same sample (but with a smaller ROI) performed before the NanoSIMS analysis. The truncated corneocyte outgrowth and furrow (shown in Figure 4C) were clearly recognizable. The aggregate at the edge of the corneocyte (the red arrow), as previously reported via TEM and SIMS (Figure 4D-F), could now be observed with an improved resolution and measured 60 nm \(\times\) 140 nm. The 93 nm \(\times\) 33 nm aggregate at the base of the outgrowth (the yellow arrow), where the NPs appear aligned, is the aggregate already observed in TEM (the yellow arrow in Figure 4D).