Characterizing biological processes with microscopy techniques that allow one to directly visualize the complexity of life is an important component of understanding both physiological function and structure. The wide spectrum of biological structures from individual proteins to whole ecosystems necessitates that multiple techniques are used to characterize all levels of organization. While existing techniques cover portions of this spectrum, continued improvement of established methods and development of new techniques is needed. This dissertation outlines my journey in enabling new approaches for imaging biosystems at various scales. Chapters 1 and 2 provide motivation for bioimaging and background for the use of electron microscopy and microfabrication techniques for imaging applications. Chapter 3 highlights my use of established electron microscopy techniques for structural biology. Cell-free expression is described as a biomass production method for generating proteins of sufficient yield and purity for structural analysis. Here I demonstrate a 2D projection map of the protein CcmK at 14 Å resolution using 2D electron crystallography as well as the expression of the membrane protein DGAT into liposomes for use in single particle electron microscopy studies. I also present nanoscale cryogenic imaging of whole cells in their native state using electron tomography. Chapter 4 introduces the concept of imaging liquids with electron microscopy and its potential for capturing dynamic processes in biology and chemistry in real-time. Details for fabrication of the devices used for imaging are included. Radiation viii chemistry and numerical simulation is used to predict pH changes of the solution depending on the solutes present in the aqueous solution. Investigation of the role of electron irradiation effects on liquid samples is continued in Chapter 5 where new multiwindow devices are fabricated and used to characterize the effects of repeated irradiation events on a sample. While the multiwindow devices described in Chapter 5 expand on the scientific utility of the commercially available platforms they are used in, a new platform for directed-flow imaging of liquid and samples in solution with liquid-cell electron microscopy is presented in Chapter 6. This platform allows for increased environmental control, greater experimental reproducibility, and allows for unique mixing and flow experiments not possible prior to the development of this platform. Finally, efforts to optimize imaging conditions for thick, low contrast, and electron beam sensitive samples is presented in Chapter 7 where the image signal-to-noise ratio is quantified across different electron imaging modalities to determine which strategy should be used for optimal liquid imaging. Furthermore, I compare the effects of electron irradiation damage on liquid samples versus cryogenically preserved samples.
Overall, the compilation of research from Chapters 3 through 7 describe findings that provide a basis for future work in advancing the liquid-cell electron microscopy field, from expanding experimental reproducibility, optimizing imaging conditions for future work, and laying the groundwork for establishing irradiation limits for biological structures to enable dynamic imaging at high-resolution and for providing correlative holistic imaging across modalities.
|Advisor:||Shokuhfar, Tolou, Friedrich, Craig|
|Commitee:||Evans, James, Gretz, Michael, Tiwari, Ashutosh|
|School:||Michigan Technological University|
|Department:||Mechanical Engineering-Engineering Mechanics|
|School Location:||United States -- Michigan|
|Source:||DAI-B 80/06(E), Dissertation Abstracts International|
|Keywords:||Structural biology, Transmission electron microscopy|
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