Cryo-Electron Microscopy for the Study of Hybrid Materials and Biological Specimens

Cryo-electron microscopy has grown overwhelmingly in popularity in recent years. Instrumental developments enable higher- and higher-resolution studies of biological molecules to near-atomic resolution using single particle analysis, while cryo-TEM tomography gives three-dimensional information about cellular structure. This thesis applies cryo-EM in less-conventional studies: in applications to fields outside of biology, and studies of cryo-scanning TEM (STEM) techniques with new detector technology. Vitrification methods for cryo-TEM perfectly preserve native structure in solution. It is used here to study the early formation processes of mesoporous silica nanoparticles, which are templated by self-assembling surfactant molecules in solution. Direct imaging of this hybrid inorganic/organic material with organics intact would be difficult without vitrifying the reaction solution. Through cryo-TEM imaging of different stages of the formation process we identify synthesis parameters affecting the properties of the fully-formed nanoparticles: relative reactant concentrations, stirring rate, and types of silica precursors. We study formation of hierarchical structures of single-pore particles, from their initial formation to alignment in a 1D cylinder, higher-order nanosheet, and helical structures. Structural preservation by cryo-EM is also utilized to quantitatively describe a shape change in hexagonal silica particles using cryo-STEM imaging, including three dimensional structure determined using cryo-STEM tomography. STEM has been the technique of choice for high-resolution imaging of materials, however conventional detectors discard much of the incident electron dose making the technique's use difficult for radiation-sensitive cryo-EM specimens. A new direct detector for STEM, the Electron Microscope Pixel Array Detector (EMPAD) records the full diffraction pattern at each scan position, allowing nearly all electrons incident on a specimen to be used for imaging. In particular, we describe a new technique for bright field imaging, where pixelated detection over the central diffraction disk allows collection of many images at different relative tilts to the optical axis. Because the images are detected separately, we can measure the tilt-induced image shift at each pixel and correct for the shifts, creating a coherent image with improved signal-to-noise ratio and resolution over both BF-STEM and conventional TEM imaging. The improvement is greater for thick specimens, for which effects due to chromatic aberrations are greatly reduced in STEM compared to TEM. At very low doses, tcBF-STEM outperforms TEM, which will lead to improvements in tomography where the total dose must be fractioned over all frames in the tilt series