title: In-lens cryo-high resolution scanning electron microscopy: methodologies for molecular imaging of self-assembled organic hydrogels.
abstract: 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 degrees C) that hold 5-10 microl 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.
authors.affiliation: Integrated Microscopy and Microanalytical Facility, Emory University, Atlanta, GA 30322, USA. rapkari@emory.edu
authors.forename: Robert P
authors.initials: RP
authors.lastname: Apkarian
authors.forename: Elizabeth R
authors.initials: ER
authors.lastname: Wright
authors.forename: Victor A
authors.initials: VA
authors.lastname: Seredyuk
authors.forename: Susan
authors.initials: S
authors.lastname: Eustis
authors.forename: L Andrew
authors.initials: LA
authors.lastname: Lyon
authors.forename: Vincent P
authors.initials: VP
authors.lastname: Conticello
authors.forename: Fredric M
authors.initials: FM
authors.lastname: Menger