## 3 Results
### 3D Distribution of NPs  
If one aims to study the localization of a nanoparticle within a biological object, it is valuable to represent the 3D distribution of this NP within the biological material using SIMS data. For this reason, we extracted the signal plane by plane to reconstruct the sputtered volume in the three X, Y, and Z-axes. Figure 6A,B are the \"sum\" images of the 30 planes acquired at masses \({}^{12}\)C\({}^{14}\)N and \({}^{48}\)Ti\({}^{16}\)O, respectively. Note that, as these images are here dissociated from the TEM image, the orientation is that of the original SIMS images, which (Figure 6A) shows the corneocyte outgrowth (the yellow arrow) and the furrow (the white arrow). Figure 5: STEM–HAADF image of TiO\({}_{2}\) NPs on corneocytes. This region of interest corresponds to the outgrowth and the furrow mentioned in Figure 4C. O: outgrowth, F: furrow. The arrows indicate NPs. Scale bar: 100 nm. In Figure 6C, the top side of the parallelepiped represents the projection onto the XY plane (the sum of the \({}^{12}\)C\({}^{14}\)N planes), as shown in Figure 6A. The other two sides represent the projections onto the YZ plane (the right side of the parallelepiped) and the XZ plane (the front side of the parallelepiped) of the 3D \({}^{12}\)C\({}^{14}\)N data. It is important to note that, for improved readability, particularly along the Z-axis, the Z scale has been adjusted (see \"Materials and Methods\"). Figure 6D-F shows overlays of the \({}^{48}\)Ti\({}^{6}\)O \"sum\" image on the \({}^{12}\)C\({}^{14}\)N \"sum\" image on the XY (Figure 6D), ZY (Figure 6E), and XZ (Figure 6F) projections. A large number of false positive pixels were scattered across the surface and at depth, as already discussed in Figure 4E. Figure 6G-I shows the corresponding SCP-filtered images. The \({}^{48}\)Ti\({}^{6}\)O signal on the XY projection clearly shows three pixel clusters (Figure 6G): the largest (the red circle) of 47 pixels on the surface of the corneocyte outgrowth, a smaller one of 7 pixels (the pink circle) in the corneocyte furrow, and the third cluster of 2 pixels (the green circle) on the opposite and lower edge of the corneocyte. In the ZY and XZ projections (Figure 6H,I), the largest cluster was distributed from plane 3 to plane 30. The corresponding NP signal extended over approximately 300 nm on the X-axis and 400 nm on the Y-axis, and it was at least 5.1 nm in depth (on the Z-axis). Given the apparent size of the NPs (rutile form) at \(42\pm 10\) by \(9\pm 2\) nm, we estimated that there were several tens of NPs in this aggregate. We assumed that not all of the NPs were packed in a continuous aggregate, as groups of two or  
Figure 6: Three-dimensional distribution of NPs in the vicinity of a corneocyte. This ROI corresponds to the outgrowth, the furrow of the corneocyte mentioned in Figure 4. (**A**) XY projection of the \({}^{12}\)C\({}^{14}\)N image. Yellow dotted arrow: outgrowth; white dashed line: furrow. (**B**) XY projection of the \({}^{48}\)Ti\({}^{6}\)O image of the same area as (**A**). (**C**) Representation of the analyzed volume. The XY projection of the \({}^{12}\)C\({}^{14}\)N image is shown on the top side, the YZ projection of the \({}^{12}\)C\({}^{14}\)N image is shown on the right side, and the XZ projection of the \({}^{12}\)C\({}^{14}\)N image is shown on the front side. (**D–I**) Overlay of the \({}^{48}\)Ti\({}^{6}\)O images on the \({}^{12}\)C\({}^{14}\)N images’ XY projections. (**D**,**G**): XY projection. (**E**,**H**) YZ projection. (**F**,**I**) XZ projection. (**D–F**) are images without an SCP filter, and (**G–I**) are images with an SCP filter. The colored circles indicate clusters of positive pixels. The SIMS analyzed volume was \(38\times 50\times 30\) pixels (\(1.5\) μm \(\times\)\(2\) μm \(\times\)\(5.7\) nm) from the SIMS analysis shown in Figure 4. Dwell time: \(1\) ms/pixel; raster: \(10\) μm \(\times\)\(10\) μm, \(256\times 256\) pixels, \(30\) plans. three pixels stood out from the main cluster over several tens of nanometers on the Y-axis (Figure 6H).