Flash Melting Amorphous Ice: Electron Diffraction of Water in No Man's Land and Methods for Time-Resolved cryo-EM

Abstract

The origins of the anomalous behavior of water are believed to lie in the deeply supercooled regime, where rapid crystallization has prevented further investigation, giving it the name no man's land.' This thesis uses a modified electron microscope to study this supercooled regime by flash melting amorphous ice. Water vapor is deposited onto a cryogenic sample in situ, flash melted by a heating laser and then probed using short electron pulses. This method developed to study 'no man's land' is then applied to the study of microsecond time-resolved cryo-electron microscopy to obtain the critical heating rate of amorphous ice. Finally, this method is used to show how it can overcome the problem of orientation bias proteins typically exhibit in cryo-electron microscopy. Chapter 1 provides the foundational information, including the questions that drive this research, followed by chapter 2, which gives the experimental details of the flash melting process. Chapter 3 reveals the structure of liquid water for the entire temperature regime of no man's land.' It provides the first experimental application of using the flash melting process to study supercooled water. A shaped laser pulse is used to heat an amorphous ice sample to well defined temperatures and is then probed using electron diffraction with microsecond electron pulses. This temperature range has never been measured before. The results shed light on modern theories about the origin of the anomalous behaviors of water. Chapter 4 describes the experiments used to measure the critical heating rate of water - the rate at which an amorphous ice sample must be warmed at to prevent crystallization. By shaping the heating laser pulses with differing leading-edge intensities, different heating rates can be obtained. The crystallization is then determined by using microsecond electron pulses to probe the sample while it is being heated. This experiment provides the microsecond time-resolved electron microscopy community with the rate at which a biological sample must be warmed at to prevent any detrimental effects due to crystallization. Chapter 5 provides multiple flash melting techniques that can be used to overcome the problem of orientation bias of proteins in cryo-electron microscopy. Many proteins exhibit an orientation bias, limiting the number of viewing angles. It is shown that by in situ depositing thin layers of amorphous ice and/or using shaped heating laser pulses, the proteins can be redistributed, allowing for a more uniform angular distribution, overcoming preferred orientation. Chapter 6 looks to the future possibilities of the flash melting process and how it can be used to further study supercooled water and microsecond time-resolved cryo-electron microscopy.