Analytical Cryo-Scanning Electron Microscopy of Hydrated Polymers and Microgels

Despite the fact that scanning electron microscopes (SEM) coupled with energy-dispersive X-ray microanalysis (EDS) has been commercially available for more than a half-century, SEM/EDS continues to develop and open new opportunities to study the morphology of advanced materials. This is particularly true in applications to hydrated soft matter. Developments in field-emission electron sources that enable low-voltage imaging of uncoated polymers, silicon-drift detectors that enable high-efficiency collection of X-rays characteristic of light elements, and cryogenic methods to effectively cryo-fix hydrated samples have opened new opportunities to apply techniques relatively well established in hard-materials applications to challenging new problems involving synthetic polymers. We have applied cryo-SEM imaging and spatially resolved EDS to collect new information characterizing polyelectrolyte microgels. These are charged gel particles with dimensions in the range of 0.1-100 μm. Perhaps most notable is the fact that the high hydration levels-the samples are mostly water-allow robust calibration curves to be generated using frozen-hydrated buffers with known salt and/or hydrocarbon compositions. Such calibration curves enable quantitative composition measurements in the low-concentration extremes associated with high-swelling hydrogels. We use an experimentally derived carbon calibration curve to determine the microgel swell ratio, Q. The swell ratio, arguably, is the single most important gel characteristic because it is directly related to the mesh size of the networked polymer, which in turn determines many of the gel's mechanical and transport properties. While Q can be experimentally measured in macroscopic gels based on weight measurements in the dry and hydrated states, it is very difficult to measure in a microgel, and the fact that EDS in a cryo-SEM can determine Q from a single X-ray spectrum is significant. Furthermore, because of the electrostatic charge distributed along the polymer chains, the presence and concentration of counter-ions play a critical role in polyelectrolyte systems. While conceptually understood for decades, experimental measurements of counter-ion concentrations have been largely limited to a relatively small set of materials that involve macroscopic samples. By developing calibration curves from frozen-hydrated buffer of known ionic strength, we measure the concentration of Na counter-ions in microgels of poly(acrylic acid) (PAA) with a limit of detection of ∼0.014 M. Such measurements may help resolve some long-standing questions in polyelectrolyte science concerning counter-ion condensation. Even in the absence of a calibration curve, we show that spatially resolved X-ray spectroscopy can map the spatial distribution of a cationic oligopeptide complexed within a hydrated PAA microgel because of the nitrogen fingerprint that, albeit at very low concentration, is unique to the peptide. We look specifically at the case of a microgel with a so-called core-shell structure, where, again, the underlying polyelectrolyte science responsible for core-shell formation remains incompletely understood. These examples highlight how a modern cryo-SEM can be exploited to quantitatively characterize hydrated soft matter. The approach is almost certain to continue its development and impact as the base of experienced practitioners, the accessibility to well-configured microscopes, and the abundance of challenging problems involving hydrated soft matter all continue to grow.