Cryogenic electron microscopy (cryo-EM) is a powerful technique used to visualize the inside of cells and to study specific protein complexes. Within this thesis, I describe the use of various cryo... Show moreCryogenic electron microscopy (cryo-EM) is a powerful technique used to visualize the inside of cells and to study specific protein complexes. Within this thesis, I describe the use of various cryo-EM techniques to gain insight into the structural changes of the human pathogen, Vibrio cholerae, as it transitions between different environments. A combination of established and novel techniques is used to prepare the individual cells for cryogenic electron tomography (cryo-ET). For example, I designed a manual plunge freezing apparatus to prepare cryo-EM samples off site and subsequently image them with cryo-ET. Furthermore, I used light microscopy and serial block face scanning EM imaging to visualize changes to the cells’ morphology and structure when transitioning from the environment, into the natural host, the zebrafish (Danio rerio), and back into the environment. In addition, this thesis demonstrates how ultraviolet-C radiation of cryo-EM samples of V. cholerae and the ICP1 bacteriophage can be used to inactivate the pathogen while retaining their ultrastructural details. Lastly, this thesis outlines current and novel methods for processing of larger, more complex samples for cryo-ET. These techniques, together with new models of host-pathogen interactions, offer new tools for exploring microbial interactions with their environments. Show less
Biological molecules, especially the proteins, have a special and important function. We study their structure to understand their functions, and further make application, like the medical... Show moreBiological molecules, especially the proteins, have a special and important function. We study their structure to understand their functions, and further make application, like the medical research. The routine method is diffraction, but not work for molecules which cannot grow into crystal and molecules which their crystal are too small. Cryo-EM technique provides another way to solve their structures through their images, it does not need crystals. Meanwhile, electron diffraction can work small crystals (micro- and nano-crystals) after the sample preparation was improved. Hence, we try to build a method that can restore the structure from the crystal’s image. We collect images of protein nano-crystal, and these images were processed to enhance their contrast. The key step is to find the orientations of these images in the procedure of reconstruction, therefore, we create a method that calculates these orientations. Nano-crystals, which cannot be used in diffraction method, then can be used in this method. Show less
