The micro- and nanoarchitectures of water-swollen hydrogels were routinely analyzed in three dimensions at very high resolution by two cryopreparation methods that provide stable low-temperature specimens for in-lens high magnification recordings. Gemini surfactants (gS), poly-N-isopropylacrylamides (p-NIP Am), and elastin-mimetic di- (db-E) and triblock (tb-E) copolymer proteins that form hydrogels have been routinely analyzed to the sub-10-nm level in a single day. After they were quench or high pressure frozen, samples in bulk planchets were subsequently chromium coated and observed at low temperature in an in-lens field emission SEM. Pre-equilibrated planchets (4–40°C) that hold 5–10 μl of hydrogel facilitate dynamic morphological studies above and below their transition temperatures. Rapidly frozen samples were fractured under liquid nitrogen, low-temperature metal coated, and observed in-lens to assess the dispersion characteristics of micelles and fragile colloidal assemblies within bulk frozen water. Utilizing the same planchet freezing system, the cryoetch-HRSEM technique removed bulk frozen water from the hydrogel matrix by low-temperature, high-vacuum sublimation. The remaining frozen solid-state sample faithfully represented the hydrogel matrix. Cryo- and cryoetch-HRSEM provided vast vistas of hydrogels at low and intermediate magnifications whereas high magnification recordings and anaglyphs (stereo images) provided a three-dimensional prospective and measurements on a molecular level.

Elastin-mimetic block copolymers were produced by genetic engineering. Genetically driven synthesis permitted control of the final physiochemical characteristics of the block copolymers. We designed BB and BAB block copolymers in which the A-block was hydrophilic and the B-block was hydrophobic. By designing the copolymers in this manner, it was proposed that they would self-assemble into micellar aggregates that, at high concentration, would form thermoreversible hydrogels. To analyze the three-dimensional fine surface morphology of the copolymers, to the resolution level of a few nanometers, we employed cryo-HRSEM. This method provided vast expanses of the specimen in its frozen hydrated state for survey. In our initial cryo-HRSEM studies, we observed the protein filaments and micelles surrounded by lakes of vitreous ice. Upon examination at low and intermediate magnifications, there was an extensive honeycomb-like filamentous network. To delineate the fine morphology of the hydrogel network at high magnification and to greater depths, we cryoetched away unbound water from the sample surface, in high vacuum, prior to chromium deposition. By using this technique, we were able to visualize for characterization purposes the fine fibril networks formed from the micellar aggregates over the surface of the hydrogel.