In-Lens Cryo-High Resolution Scanning Electron Microscopy: Methodologies for Molecular Imaging of Self-Assembled Organic Hydrogels

• Published in: Microscopy and microanalysis 2003-08, Vol.9 (4), p.286-295
• Main Authors: Apkarian, Robert P., Wright, Elizabeth R., Seredyuk, Victor A., Eustis, Susan, Lyon, L. Andrew, Conticello, Vincent P., Menger, Fredric M.
• Format: Article
• Language: English
• Subjects: High Resolution Cryo-SEM
• ISSN: 1431-9276; 1435-8115
• DOI: 10.1017/S1431927603030551

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.