# Mapping Nanoparticles in Hydrogels: A Comparison of Preparation Methods for Electron Microscopy
## 3 Results and Discussion  
### Morphology of AAM-BIS Hydrogel With and Without GNRs  
SEM images from CPD and HMDS-dried hydrogels with and without GNRs in SE and BSE mode are shown in Figure 1. Figure 1A,B show hydrogels prepared with CPD, while Figure 1C,D show HMDS-dried hydrogels. The mean and standard deviation of the Feret diameter of the pore sizes for the CPD hydrogels are 0.1405 \(\pm\) 0.1006 \(\upmu\)m and 0.2236 \(\pm\) 0.1942 \(\upmu\)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 0.1750 \(\pm\) 0.1320 \(\upmu\)m and 0.1421 \(\pm\) 0.1156 \(\upmu\)m for the hydrogel without and with GNRs, respectively. Thus, the pore sizes of the HMDS-dried and the CPD hydrogels are comparable, illustrating that using HMDS for drying hydrogels for SEM imaging may serve as a suitable alternative to the CPD technique. Furthermore, the mean pore size changes of \(-0.03/+0.08\)\(\upmu\)m when embedding GNRs in the hydrogel indicates that GNRs have a minor influence on the polymer network structure. We expected to observe high-density materials, such as NMNPs, as high contrast segments in BSE mode at high electron beam voltages, since high-density materials have more substantial electron-backscattering intensities than low-density materials. However, the observed contrast features are similar for hydrogel with and without GNRs, which makes the quantizing of the position of GNRs in the polymer network uncertain. The small-sized contrast features shown with white arrows in Figure 1D could be characterized as GNR positions but may also be difficult to distinguish from artifacts associated with the sample preparation [80; 81]. Without any immobilization, one might suspect that the GNRs will leak out of the hydrogel. However, results from our earlier study of LSPR of gold nanospheres embedded in AAM-BIS hydrogels were consistent with a uniform and stable particle distribution in the hydrogel volume [52]. These results show that the LSPR signal was stable over timeand for different swelling equilibriums. That the Feret diameters of the pore sizes of the AAM-BIS hydrogel are on the order of 200 nm suggests that the GNRs are bound to the polymer network of the hydrogel. Figure 2 shows SEM images of CPD and HMDS-dried hydrogels with GNRs at higher magnification than in Figure 1. The SEM image of GNR solution dried on carbon tape in Figure 2B is included to enable direct comparison of how GNRs can appear in an SEM image. The small-sized contrast features in Figure 2B have lengths and widths similar to the GNR specifications received from the producer. The BSE mode SEM image shows few small-sized contrast features with dimensions comparable to the contrast features in Figure 2B, indicating the presence of GNRs. However, we did not succeed in pinpointing the contrast segments indicating particles using threshold filters on the images. The contrast differences in the SEM images indicating GNPs cannot be proved without additional measurements. This is in accordance with the observation in [82], where cryoSEM was used for characterizing GNPs in polyacrylamide hydrogels. The contrast of NMNPs in gel-like samples may be improved by omitting the metal sample coating and by including highly conductive sample holders instead [83]. The detection of SE and BSE can also be combined to obtain density-dependent contrasts that highlights the high densities areas in the image [84]. Figure 1: **(A) Scanning electron microscope (SEM) image of critical point dried (CPD) AAM-BIS hydrogel without gold nanorods (GNRs) in SE mode; (B) SEM image of CPD AAM-BIS hydrogel with GNRs in SE mode; (C) SEM image of hexamethyldisilazane-dried (HMDS-dried) AAM-BIS hydrogel without GNP in backscattered electron (BSE) mode; (D) SEM image of HMDS-dried AAM-BIS hydrogel with GNP in BSE mode. Note that the scalebar in (B) is different from the other images.