# Mapping Nanoparticles in Hydrogels: A Comparison of Preparation Methods for Electron Microscopy
## 1 Introduction
This was likely due to an ordered tendency of the spheroidal particles in the hydrogel. To further understand and engineer the LSPR properties of GNP-nanocomposite hydrogels for biosensing purposes, it is desirable to identify how the particles are ordered and distributed from morphological characterizations. Morphological characterizations of hydrogels are often performed by using scanning electron microscopy (SEM). By using specialized microscopy techniques, such as cryoSEM, environmental-SEM, or time-of-flight secondary ion mass spectrometry (TOF-SIMS), it is possible to characterize the morphology of hydrogels in their swelled state [54; 55; 56]. However, specialized microscopy techniques may have limited accessibility due to being a state-of-the-art technology that increases the user costs. Conventional SEM techniques require the samples to be in a vacuum, which can pose challenges for conserving the native hydrated state of the hydrogel since its structure is contained by the water absorbed in the polymer network. To prevent a complete collapse of the hydrogel in the SEM vacuum chamber, one can use drying methods such as high or low temperature drying [17; 57; 58; 59] or critical point drying (CPD) [55; 58; 60; 61] to conserve its pore structure. Different collapsing characteristics occur depending on the drying methods used and the composition of the hydrogel [58; 62; 63]. By using transmission electron microscopy (TEM), the high contrast of metal nanoparticles embedded in hydrogels can be observed; however, only a two-dimensional particle positions can be quantized [64; 65]. To map the three-dimensional positions of particles in hydrogels with volume imaging methods, such as the focused ion beam-SEM technique (FIB-SEM) [66; 67; 68], conventional drying methods must be used. The CPD technique is a common method for drying hydrogels or biological materials to conserve the morphology in SEM imaging [55; 58; 60; 61]. An alternative to CPD is to dry the samples with hexamethyldisilazane (HMDS) solvent in air. The surface tension of the sample is reduced by using HMDS, and it may also cross-link proteins for biological materials [69]. This is adding strength to the sample during air-drying and prevents collapse or fracturing. A comparison of samples dried with CPD or HMDS for SEM have in previous work often been performed for biological specimens [70; 71; 72; 73]. The air-drying with HMDS as an alternative to the CPD technique has its advantages: (1) less effort and fewer variables are involved; (2) it is cost-effective; and (3) the method requires no equipment use such as for controlling carbon dioxide phase changes. The HMDS may reduce surface tension for all hydrophilic materials and, therefore, serve as a useful drying method for hydrogels. The HMDS drying of fibrin or elastin hydrogels have been described in earlier work [74; 75; 76; 77]. Additionally, the sputter coating of the sample may have to be optimized concerning coating thickness and coating material. Samples with significant differences in the topography may require a thicker coating than for samples with small differences in topography; i.e., the SEM imaging of the GNPs and the hydrogel structure may have conflicting requirements. The optimization of the sputter coating is beyond the scope of this article. Furthermore, since swelling and molecular transport properties of acrylamide hydrogels have been discussed in earlier work [25; 50], the characterization of mechanical and rheological properties have not been included. Here, the morphology of 10 wt % polyacrylamide (AAM-BIS) and poly(acrylamide-co-acrylic acid) (AAM-AAC-BIS) hydrogels, with and without gold nanorods (GNRs) as composite materials, was studied using conventional SEM. Low GNR densities were used (10/um3) as for the earlier FO sensor studies in [51; 52; 53]. The hydrogels were prepared for SEM using CPD or the drying with HMDS and characterized concerning pore structures and GNR positions in the polymer network.