# Nanoparticles--Plant Interaction: What We Know, Where We Are?
## 4 Transmission Electron Microscope (TEM) Analysis of the NP in the Symplast and Apoplast  
Without an appropriate characterization of the chemical composition of NP, the TEM images of the NP in plant cells have to be treated with extreme caution [19,83]. For example, \"rolled\" PD, which are caused by the long and complicated procedure of sample preparation that can cause such an artefact, can be seen in the TEM (Figure 1A and inset). Without a specific determination concerning the diffraction pattern, chemical composition (using energy-dispersive X-ray spectroscopy, EDS), the high-resolution images that can reveal the characteristic morphology/structure of the tested NP, the presence of NP in the plant material cannot be confirmed. Thus, without the above-mentioned assays, it is not possible to predict whether the PD is filled with NP, which is clearly visible in Figure 1A. In the PD electron dense spots were visible very often (Figure 1B-D,G), however, an analysis of their diffraction pattern indicated that these were not NP (Figure 1A and inset, Figure 1H). Similar caution must be taken when analyzing the cell wall after NP are applied, especially when the studies concern the primary cell wall. Namely, in such walls, the pectins are abundantly present and their appearance in a TEM is electron dense (Figure 1B,E). The middle lamella region was electron dense and the diffraction pattern confirmed that these areas in the cell walls were not NP (Figure 1E,F). Confirmation of the importance of knowledge about the chemical composition is shown in Figure 1I. The electron-dense spots look like NP (Figure 1I), but an analysis revealed that they were amorphous materials (Figure 1I). The diffraction pattern that indicates the presence of Au-NP is visible in Figure 1L, which confirms that the electron-dense spots that are visible in Figure 1K are Au-NP. The importance of such a validation was proven by [PERSON] et al. [32], who showed that the electron-dense NP that were seen in the plants were in fact yttrium phosphate (YbPO4) precipitates and not nano-Yb2O3. Staining agents and buffers can produce similar artefacts, especially osmium tetroxide, cacodylate, lead citrate or uranyl acetate [83]. The \"fingerprints\" of the agents that were used for the sample procedure for the TEM analysis can cause misunderstandings (Figure 2). The characteristics of lead citrate (Figure 2A), uranyl acetate (Figure 2B) and osmium tetroxide (Figure 2C) are specific for these reagents and should be taken into account during studies on presence of NP in plant cells. Therefore, it is recommended that at least high resolution TEM be used to identify the crystalline structure or the distinct shape of metal-based NP. Figure 1: High-resolution transmission electron microscope (HRTEM) images of sections from barley roots that had been treated with the Au-NP. (**A**) The PD in the wall. The appearance of the PD is an artefact that was caused by mistakes in the sample preparation procedure; inset—diffraction pattern that that indicates that the black color (indicated by red arrow) is not NP. (**B**) The PD with electron-dense material inside (indicated by black arrow), but as was proven by the diffraction pattern (see inset on (**A**)), the electron-dense material was not NP. (**C**) Cell wall traversed by the PD with electron-dense spots (indicated by black open arrow), which are not NP proved by diffraction pattern. Many electron-dense dots were present in the cytoplasm but were also not NP (black arrow). Note that the dark colouration of the cell walls resulted from the presence of pectins. (**D**) Similar to Figure (**C**), none of electron-dense particles were NP (black arrow; diffraction patterns were the same as for (**A**)). (**E**) Cross-section through the root cell walls. Note the electron-dense bands in the wall (black double arrow), which were not NP, but just a higher amount of pectins compared to the other parts of the walls (cw). Such an image is especially characteristic for the middle lamella.