Recent developments in high-resolution scanning electron microscopy (HR-SEM) have made it an ideal tool for the study of nanoparticles and collids in viscous systems or in systems containing large objects, hundreds of nanometers and larger, in which small (nanometric) features are to be imaged, e.g., hydrogels or body cells.  Such system cannot be studied by cryo- TEM.  Liquid nanostructured systems can now be studied by cryogenic-temperature scanning electron microscopy (cryo-SEM), using much-improved cryogenic specimen holders and transfer systems, even without conductive coating.  In recent years we have developed a novel specimen preparation methodology for cryo-SEM specimens that preserve the original nanostructure of labile complex liquids at specified composition and temperature, quite similarly to  what has been done in cryo-TEM.

In my talk I will describe briefly the principles and the state-of-the-technology of cryo-SEM, and, through examples of our recent work, will demonstrate various variants of the methodology that allow us to study a wide range of soft matter systems, taking advantage also of the combination of cryo-TEM, cryo-SEM, and non-imaging, e.g., scattering techniques.

Synthetic and biological soft materials present an array of challenging morphological problems over length scales - nano and micro - that are well suited for various methods of scanning electron microscopy.  However, soft-matter SEM brings challenges associated with intrinsically weak electron-optical image contrast, structural and chemical sensitivity to the incident ionizing radiation, and, in many cases, levels of hydration that are essential to the material's functional performance.  This presentation addresses these various points using examples relevant to the design and development of surfaces to control bacterial colonization.  We have, for example, used FIB tomography coupled with traditional biological fixation, staining, embedding, and microtomy methods to show that shape of individual staphylococcal bacteria changes when they adhere to a synthetic surface and, furthermore, that the magnitude of the shape change depends on the chemical properties of the underlying surface.  Extending such a method to unstained frozen-hydrated specimens introduces a number of important challenges.  one centers on the lack of contrast.  Mild sublimation generates compelling topographic contrast but does not lend itself well to the automated serial sectioning required for tomographic reconstruction.  Another centers on specimen preparation.  Again using bacterial biofilms, we show that simply plunging a hydrated specimen into liquid nitrogen introduces significant artifactual structure due to water crystallization.  The alternate method of high-pressure freezing (HPF) can avoid water crystallization and perserve the intrinsic biofilm morphology, though questions remain about how the molecular-scale network structure of the biofilm extracellular matrix responds during low-voltage SEM image after gentle sublimation.  Our most recent work centers on cryo-SEM of microgels, prepared by high-pressure freezing of colloidal microgel solutions, to make precise measurements of microgel swell ratio and characterize their water-binding properties.

Focused ion beam (FIB) technology, already a powerful tool in materials sciences, is now carving a niche in nanoscale-resolution 3D imaging of biological specimens.  Combined with a scanning electron microscope (SEM) and a host of powerful hardware and software advances, a FIB-SEM instrument can now be used to investigate the 3D ultrastructure of stained, resin-embedded biological samples too thick to be easily imaged in the transmission electron microscope (TEM).  The latest solutions allow long and stable runs using mostly automated protocols, resulting in large image volumes rich in ultrastructural detail, with few artifacts and at near-TEM resolutions.  The FIB can also be used to mill away targeted areas of biological specimens to make previously inaccessible volumes available to chemical imaging techniques.  Finally, as FIB milling can be used to thin rapidly frozen cellular samples; these can then be imaged by an SEM or TEM in near-native states.  Together, these advances make FIB technology an increasingly significant addition to the imaging toolkit in biology.