INVESTIGATION OF CRYOGENIC SURFACE CHARGING DURING OFF-AXIS ELECTRON HOLOGRAPHY
Cryogenic electron microscopy (cryo-EM) is an increasingly popular technique for determining the structure of solutions, especially of those with protein solutes. The ability to immobilize a fluid and attain atomic resolution of solutes within has propelled the technique to the forefront of electron microscopy. Regular advancements in resolution, specimen fabrication, data collection, and analysis mean cryo-EM will continue to expand and find new and far-reaching applications. In this dissertation, a model system is proposed to address interface charging effects that are typically ignored when analyzing cryo-EM data. Cryogenic specimen fabrication rapidly solidifies the solution through cooling the native fluidic state, preventing short- and long-range ordering from occurring, compared to slower cooling processes. The resulting vitreous state is effectively a static representation of a liquid trapped in a solid state. In this work, cryo-EM samples are fabricated using water, and then more complex solutions, to create a baseline understanding of the extent of surface charging from secondary electron emission. Subsequent solutions are investigated to expand the understanding of surface charging to include dispersed ions within the specimen. The interferometry technique of off-axis electron holography is used to measure the relative electron-wave phase shift produced by the specimen, which directly probes the electromagnetic fields in and around the specimen. The holography data are compared to finite-element simulations revealing that a steady-state surface charge of 1.5E-4 ± 1.6E-5 C/m2 is generated on each surface of the vitreous solution during cryo-EM imaging. The work is repeated with a solution of functionalized Au nanoparticles roughly 30nm in diameter suspended in DI water as a model system for more complex fluids. The mean inner potential of Au nanoparticles is measured in two experiments with values of 22.3V and 22.9V and the uncertainty in both values is 2.5V from phase sensitivity. Compared with literature, these values agree with prior measurements and demonstrate cryo-EM combined with off-axis electron holography as a powerful technique to measure electric potentials in cryogenic specimens.
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