# Mapping Nanoparticles in Hydrogels: A Comparison of Preparation Methods for Electron Microscopy
## 1 Introduction  
The ability of a hydrogel to absorb large amounts of water without being dissolved has shown to be useful in a significant number of applications, such as drug delivery systems, wound healing, protein purification, crystallization of minerals, distillation, pollutant capturing, and sensor technologies [1; 2; 3; 4; 5; 6; 7; 8; 9; 10]. The hydrogel properties can be designed by controlling the crosslinking density, the polymer hydrophilicity, the type of recognition entities, polymer ionicity, or polymer elasticity. The common functions of the polymer network consist of responding volumetrically to external stimuli such as pH [11; 12; 13], temperature [14; 15; 16; 17], ionic strength [18; 19; 20], or receptor-analyte recombinations [21; 22; 23; 24]. Some of these stimuli-responsive features have shown to be very useful in controlling drug release [1] or to be useful for label-free and specific sensing of biomolecules [22; 25]. By filling the hydrogel with composite materials such as polymeric fibers, carbon-based or metal nanostructures, and amorphous or crystalline inorganic materials, the stability of stimuli-responsivehydrogels as well as its homogeneity can be enhanced, and new functionalities can be added in terms of sensor selectivity, sensor specificity, and new sensor techniques [26; 27; 28; 29]. These nanocomposite hydrogels with noble metal nanoparticles (NMNPs) are fascinating materials, often utilized for wound healing [30; 31], but are also impressive materials as nanoplasmonic systems [32; 33]. In membrane technology, NMNPs have also shown to be a potential candidate for development in thermoplasmonics that may improve nanofiltration, pervaporation processes, or oil-in-water nanoemulsions [34; 35; 36; 37]. Hydrogels with NMNPs as composite materials exhibit intense optical scattering at specific light frequencies. These phenomena arise from light interacting with confined collective oscillations of electron clouds in the NMNPs at a resonance frequency, also known as localized surface plasmon resonance (LSPR) [38; 39; 40]. The LSPR of NMNPs may occur in the visible or infrared light-range and are frequently used in label-free biosensor applications due to fast response times, high sensitivity, high selectivity, and the possibility for multianalyte sensing in complex mixtures [38; 41; 42; 43; 44]. The LSPR of NMNP-nanocomposite hydrogels are dependent on the particle shape, the size, the refractive index (RI) of the surrounding medium, and the interparticle distances [44; 45]. Coupling may occur between the resonant modes of the localized surface plasmons of NMNPs in close proximity to each other [44; 46; 47; 48; 49]. The scattering of NMNPs in a hydrogel may have an LSPR frequency for a random distribution of particles that is different from the LSPR frequency for an inhomogeneous particle distribution. We have in earlier work developed proof-of-concepts of fiber optic (FO) sensors based on using NMNPs immobilized in polyacrylamide hydrogels and combining LSPR and interferometric sensing modalities [50; 51; 52; 53; 25]. The interferometric sensing modality detects the change in volume of a stimuli-responsive hydrogel, whereas the LSPR sensing modality detects receptor-analyte recombinations on the NMNP surface. The dipole-dipole coupling and RI sensitivity of the LSPR in NMNPs may as well be utilized for detecting the change in volume of stimuli-responsive hydrogels for significant swelling and deswelling. The LSPR response of spherical or spheroidal gold nanoparticles (GNP) in hydrogels has shown to deviate from the LSPR response of randomly distributed spherical or spheroidal SNPs [51; 52; 53]. The plasmon coupling between spherical GNPs occurred for an interparticle distance that was larger than the estimated plasmon coupling distance [51]. This was found to be due to the inhomogeneous distribution of the particles in the hydrogel. The LSPR response for decreasing interparticle distances, where dominated by the side-by-side plasmon coupling between spheroidal GNPs [53].