# Mapping Nanoparticles in Hydrogels: A Comparison of Preparation Methods for Electron Microscopy
## 3 Results and Discussion
### Morphology of AAM-AAC-BIS Hydrogel With and Without GNRs  
SEM images of CPD and HMDS-dried hydrogels, with and without GNRs, in SE and BSE mode are shown in Figure 3.  
Figure 3A,B show CPD hydrogels in SE mode, while Figure 3C,D show HMDS-dried hydrogels in BSE mode. The mean and standard deviation of the Feret diameter of the pore sizes for the CPD hydrogels are 0.6240 \(\pm\) 0.4775 \(\upmu\)m and 0.1219 \(\pm\) 0.1050 \(\upmu\)m for the hydrogel without and with GNRs, respectively. The corresponding mean and standard deviation of the Feret diameter of the pore sizes for the HMDS-dried hydrogels are 0.4190 \(\pm\) 0.3570 \(\upmu\)m and 0.0932 \(\pm\) 0.0908 \(\upmu\)m for the  
Figure 3: **(A)** SEM image of CPD AAM-AAC-BIS hydrogel without GNRs in SE mode; **(B)** SEM image of CPD AAM-AAC-BIS hydrogel with GNRs in SE mode; **(C)** SEM image of HMDS-dried AAM-AAC-BIS hydrogel without GNP in BSE mode; **(D)** SEM image of HMDS-dried AAM-AAC-BIS hydrogel with GNP in BSE mode. Note that the scalebar in the images is 5 \(\upmu\)m. The white arrows show the small-sized contrast features observed that could indicate GNR positions. Figure 2: **(A)** SEM image of CPD AAM-BIS hydrogel with GNRs in SE mode; **(B)** air-dried GNR solution on carbon tape in SE mode for comparison with SEM images of GNP-hydrogels; **(C)** SEM image of HMDS-dried AAM-BIS hydrogel with GNP in BSE mode. The white arrows show the small-sized contrast features observed that could indicate GNR positions. hydrogel without and with GNRs, respectively. The significant increase in Feret diameter for hydrogel without GNRs may indicate a hydrogel collapse where many smaller pores have merged into larger pores. The collapse could be a result of insufficient dehydration considering that the AAM-AAC-BIS hydrogels have 2/1 (AAC/AAM) molar ratio of hydrophilic anionic AAC co-monomers. For CPD, the hydrogels may have been rehydrated due to the humidity at room temperature while transferring it to the CPD. Ethanol will only exchange efficiently with liquid carbon dioxide, so a partly rehydrated hydrogel will be more susceptible to a collapse. For HMDS drying, the hydrogel may have been rehydrated, while substituting ethanol with HMDS. HMDS may also only exchange efficiently with ethanol. This suggests that the HMDS-dried hydrogel is also susceptible to collapse due to rehydration. For hydrogels with GNRs, the Feret diameter is significantly smaller. This may indicate that the GNR-hydrogel has been less susceptible to a collapse. The Feret diameters for the AAM-AAC-BIS GNR-hydrogels are also smaller than the AAM-BIS hydrogels in Section 3.1. This could be due to the hydrophilicity of the AAM-AAC-BIS hydrogels. The anionic AAC co-monomer may contribute to the absorption of a more substantial fraction of water into small pores of the polymer network in its original state. The preparation methods may also influence the pore structures as discussed in Section 3.3[57; 58]. As for the results obtained in Section 3.1, there are high contrast features along the edges of the polymer networks in Figure 3D in BSE mode (indicated by white arrows), which could represent dense materials. However, the contrast features are similar for hydrogel, with and without GNRs, and make the quantizing of the positions of GNRs in the polymer network uncertain. The sizes and shapes of the contrast features could be a result of the artifacts introduced from the sample preparations[80; 81]. The studies of LSPR of GNPs in AAM-AAC-BIS hydrogels have been discussed in [51; 53]. The LSPR signal did not decay for different hydrogel swelling equilibriums and indicated that the GNPs were contained in the hydrogel. Since the Feret diameters of AAM-AAC-BIS hydrogels with and without GNRs are in the order of 90 to 600 nm, it may be that the gold rods are bound to the polymer network of the hydrogel.