Improving Biospecimen Imaging in Liquid Phase Electron Microscopy
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This thesis highlights the progress made in LPEM and the study of biosamples. The introduction provides a comprehensive overview of the challenges encountered in investigating molecular processes and traces the historical evolution of microscopy, leading up to the development of transmission electron microscopy (TEM), cryogenic electron microscopy (cryo EM), and LPEM. Two major hurdles in LPEM are outlined and addressed in this thesis: LC transmissibility, and electron-induced biosample damage. Low electron transmission in thick silicon nitride (SiNx) LCs reduces the achievable resolution during imaging. To address this, I developed a technique to fabricate thin SiNx windows. These windows exhibit reduced bulging, higher transmission, and higher resolution imaging in the liquid phase than currently available SiNx cells. Implementation of thin SiNx LCs was demonstrated through imaging of apoferritin, adeno-associated virus (AAV), pAAV, and vesicles at high resolution. Though future work remains to produce thin LCs of reproducible liquid thickness, this work demonstrates a breakthrough in the capabilities of LPEM imaging. Another principal hurdle in LPEM lies in sample damage inflicted by the electron beam. The concept of radiolysis and its impact on biosamples is elucidated and demonstrated, showing the need to mitigate sample damage during imaging. The thesis addresses this challenge through the evaluation of inpainting techniques, which can be used to perform reduced-dose imaging. In silico analysis showed that algorithmic inpainting can be used to inpaint biosample images up to 80% with high accuracy. The analysis also showed that spiral inpainting does not significantly compromise image quality, indicating its potential v for performing low-dose imaging when spiral distortion is corrected. Overall, this thesis provides invaluable technical insights into LPEM and the cutting edge advancements made in this work. It sets the stage for future research endeavours and groundbreaking discoveries in the realm of in situ bioimaging.
Cite this version of the work
Nicolette Shaw (2023). Improving Biospecimen Imaging in Liquid Phase Electron Microscopy. UWSpace. http://hdl.handle.net/10012/19749