Extending Cryo-EM to Nonaqueous Liquid Systems

[Image: see text] Cryogenic-temperature transmission electron microscopy (cryo-TEM) of aqueous systems has become a widely used methodology, especially in the study of biological systems and synthetic aqueous systems, such as amphiphile and polymer solutions. Cryogenic-temperature scanning electron microscopy (cryo-SEM), while not as widely used as cryo-TEM, is also found in many laboratories of basic and applied research. The application of these methodologies, referred to collectively as cryogenic-temperature electron microscopy (cryo-EM) for direct nanostructural studies of nonaqueous liquid systems is much more limited, although such systems are important in basic research and are found in a very large spectrum of commercial applications. The study of nonaqueous liquid systems by cryo-EM poses many technical challenges. Specimen preparation under controlled conditions of air saturation around the specimen cannot be performed by the currently available commercial system, and the most effective cryogen, freezing ethane, cannot be used for most such liquid systems. Imaging is often complicated by low micrograph contrast and high sensitivity of the specimens to the electron beam. At the beginning of this Account, we describe the basic principles of cryo-EM, emphasizing factors that are essential for successful direct imaging by cryo-TEM and cryo-SEM. We discuss the peculiarities of nonaqueous liquid nanostructured systems when studied with these methodologies and how the technical difficulties in imaging nonaqueous systems, from oil-based to strong acid-based liquids, have been overcome, and the applicability of cryo-TEM and cryo-SEM has been expanded in recent years. Modern cryo-EM has been advanced by a number of instrumental developments, which we describe. In the TEM, these include improved electron field emission guns (FEGs) and microscope optics, the Volta phase plate to enhance image contrast by converting phase differences to amplitude differences without the loss of resolution by an objective lens strong underfocus, and highly sensitive image cameras that allow the recording of TEM images with minimal electron exposure. In the SEM, we take advantage of improved FEGs that allow imaging at a low (around 1 kV) electron acceleration voltage that is essential for high-resolution imaging and for avoiding specimen charging of uncoated nonconductive specimens, better optics, and a variety of sensitive detectors that have considerably improved resolution and, under the proper conditions, give excellent contrast even between elements quite close on the periodic table of the elements, such as the most important oxygen and carbon atoms. Finally we present and analyze several examples from our recent studies, which illustrate the issues presented above, including the remarkable progress made in recent years in this field and the strength and applicability of cryo-EM methodologies.