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Microfluidic Sample Preparation for High-Resolution EM and Single Cell Analysis

Rima, Luca. Microfluidic Sample Preparation for High-Resolution EM and Single Cell Analysis. 2021, Doctoral Thesis, University of Basel, Faculty of Science.

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Abstract

To understand and explain biological processes, the functions of the involved biological macromolecules must be known. These functions are directly linked to their three-dimensional structures and conformations. Therefore, knowledge of these structures and their dynamics is essential for understanding and can be used, for example, for the development of pharmaceutical products. In the past, X-ray crystallography (XRC) or nuclear magnetic resonance (NMR) were commonly used for structure elucidation. However, both techniques make high demands on the sample, which greatly limits specimen selection. In recent years, cryogenic electron microscopy (cryo-EM) has gained more and more importance. Formerly a niche technology, it has become the method of choice for many scientists due to continued developments in instrumentation, electron detectors, and data processing. Among its advantages are that smaller sample sizes are required and that crystallization is unnecessary. This allows the investigation of new, previously unavailable samples. However, not all samples can be analyzed, and cryo-EM is also limited if problems occur with expression, purification, or sample preparation. Yet these areas have hardly changed compared to the otherwise enormous progress that has been made in the field and therefore represent the biggest bottleneck in the cryo-EM pipeline today. Therefore, new methods are required to overcome the existing limitations. One approach is the use of microfluidic systems for sample preparation. This is possible because the number of particles required for successful structure elucidation is ~$10^{4}$ to ~$10^{6}$, and the required volumes are in the nanolitre range. Such a microfluidic approach is now the focus of the following thesis. Primarily, the system for sample preparation is presented. Alongside samples for cryo-EM, samples for negative staining EM can also be prepared. The system is also capable of lysing single cells and processing their lysate. Afterward, a new module of this system will be discussed, which improves the EM grid preparation and offers new possibilities to influence the sample preparation process. Furthermore, a method is presented that allows the isolation of proteins from minute amounts of starting material, which can then be directly prepared for structure elucidation by cryo-EM. The last part is dedicated to the combination of the microfluidic system with other techniques that can provide complementary information to EM analysis.
We demonstrate that the use of this microfluidic system for sample purification and sample preparation has decisive advantages over classical methods. Therefore, it will help the future elucidation of structures that are currently still unattainable. Additionally, we show that the system is very versatile and thus opens up completely new experimental possibilities that go beyond structural elucidation.
Advisors:Braun, Thomas and Stahlberg, Henning and Maier, Timm and Kukulski, Wanda
Faculties and Departments:05 Faculty of Science > Departement Biozentrum > Structural Biology & Biophysics > Structural Biology (Braun)
UniBasel Contributors:Braun, Thomas and Stahlberg, Henning and Maier, Timm
Item Type:Thesis
Thesis Subtype:Doctoral Thesis
Thesis no:14566
Thesis status:Complete
Number of Pages:vi, 142
Language:English
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edoc DOI:
Last Modified:13 Jan 2022 05:30
Deposited On:12 Jan 2022 09:51

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