# Mapping Nanoparticles in Hydrogels: A Comparison of Preparation Methods for Electron Microscopy
## 4 Conclusions  
The morphology of low-weight-percentage polyacrylamide and poly(acrylamide-co-acrylic acid) hydrogels, with and without GNRs, has been characterized using conventional SEM. A comparison was made between the CPD and HMDS drying methods of hydrogels with and without GNRs. Results of CPD and HMDS techniques show comparable pore structures, estimated by computing the mean Feret diameter in ImageJ. For acrylamide gels, the pore sizes were also comparable with and without GNRs. The use of an HMDS solvent for drying may serve as a suitable alternative to the CPD technique. The HMDS technique is a cost-effective substitution of the CPD technique that omits equipment use as well as being less time consuming. The poly(acrylamide-co-acrylic \begin{table}
\begin{tabular}{c c c c c} \hline \hline
**Hydrogels** & **AAM-BIS** & **AAM-BIS-GNR** & **AAM-AAC-BIS** & **AAM-AAC-BIS-GNR** \\ \hline CPD & \(0.1405\pm 0.1006\) & \(0.2236\pm 0.1942\) & \(0.6240\pm 0.4775\) & \(0.1219\pm 0.1050\) \\ HMDS & \(0.1750\pm 0.1320\) & \(0.1421\pm 0.1156\) & \(0.4190\pm 0.3570\) & \(0.0932\pm 0.0908\) \\ \hline \hline \end{tabular}
\end{table} 
Table 1: Mean and standard deviation of Feret diameters of hydrogel pore structures in \(\upmu\)m. CPD: Critical Point Drying. HMDS: Hexamethyldisilazane. acid) hydrogels have slightly smaller pores sizes than polyacrylamide hydrogels with branched polymer structure. This difference can be assumed to be due to the more substantial hydrophilicity of poly(acrylamide-co-acrylic acid) anionic hydrogel. The pore structures of the dehydrated and dried hydrogels are likely smaller than the pore structures of hydrated hydrogels due to the water exchange with ethanol in the dehydration. The dehydration and drying of hydrogels may result in an uncertain degree of collapsing characteristics [57; 58; 62; 63; 87]. By comparing the morphology of dried hydrogels with cryoSEM or environmental SEM images of hydrated hydrogels [54; 55], it is possible to estimate the presence of these collapsing features. Small-sized contrast elements can be observed from the BSE-mode SEM images of GNR-nanocomposite hydrogels and may indicate the presence of GNRs since they are denser than the polymer network. The contrast of these segments was not significant enough to distinguish them from artifacts associated with the sample preparations [80; 81]. These results are consistent with similar studies in [82; 85]. Additionally, the identification of GNRs with threshold filters was not feasible due to the small contrast differences. The contrast of NMNPs in gel-like samples may be improved by omitting the sample coating and by including highly conductive sample holders instead [83], or by using both SE and BSE detectors to obtain density-dependent contrasts that highlights the high densities areas in the image [84]. Further work is needed to increase the contrast of the metal particles in the polymer matrix so that a mapping of the GNP positions in hydrogels can be determined. However, an evaluation of preparation methods may elucidate new aspects concerning hydrogel drying techniques and GNR identification in the hydrogel polymer network. Since the pore sizes were comparable for acrylamide hydrogels with and without GNRs, it may also be that the hydrogel structure was nearly unaffected by adding GNPs to its matrix. Future work will consist of developing methods for enhancing the contrast of high-density materials in a low-density matrix for conventional SEM imaging. Furthermore, volume-imaging methods such as the FIB-SEM or the SBFSEM techniques will be used to map the positions of metal particles in nanocomposite hydrogels. [PERSON], [PERSON], and [PERSON] conceived and designed the experiments; [PERSON] and [PERSON] performed the experiments; [PERSON], [PERSON], and [PERSON] analyzed the data; [PERSON], [PERSON], and [PERSON] wrote the paper. This work was supported by strategic research funding from Norwegian University of Science and Technology (NTNU).