Nanofluidic Liquid-Phase Electron Microscopy of Microtubule Dynamics
Development of a next-generation LP-EM system
N. Andrea (TU Delft - Applied Sciences)
A.M. Dogterom – Promotor (TU Delft - Applied Sciences)
Gregory Schneider – Promotor (Universiteit Leiden)
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Abstract
In this dissertation we describe the systematic steps taken towards the observation of microtubule dynamics inside the electron microscope.
• In Chapter 2 we explore two different established methods to do liquid-phase EM. We find that neither method offers a clear path towards allowing the visualisation of microtubule dynamics. We also present two microfabrication-based methods that aim to remedy some of the shortcoming of the methods tried.
• In Chapter 3 we discuss our efforts to integrate the graphene growth process into the microfluidic chip device fabrication. We find that the thermal crystallisation of candidate membrane materials is the limiting factor with molybdenum-based graphene growth.
• In Chapter 4 we describe a next-generation micro- and nanofluidic device for use in liquid-phase electron microscopy. We show how this new device is designed around fluorescence observation and with graphene compatibility. We then describe the remaining challenges in the fabrication approach.
• In Chapter 5 we explore methods to incorporate a high number of microtubules inside nanochannels. To this end, we develop a simplified method to produce nanochannels. We also try to address challenges related to nonspecific adhesion of proteins and channel clogging in microfabricated chips, in both those of Insight Chips and in chips of our own making.
• In Chapter 6 We use our custom made chips to observe liquid water in the electron microscope. We attempt to image microtubules in a commercial system, but are unsuccessful in identifying any filaments. We discuss the limitations of these preliminary TEM studies and our plans to overcome those limitations.
We conclude this work by reflecting on the obtained results in the context of the original aims, and within the context of the range of biological questions that could be addressed with LP-EM. We address competing techniques and future improvements that could be made to both the design of the chips and to the imaging approach — providing a perspective on the future of the LP-EM field.
4TU ResearchData Collection - The data associated with this dissertation and source files of the document of will be available at the 4TU.ResearchData archive: https://doi.org/10.4121/f53fbc37-fb3f-4228-8a3a-7d8ed2cca8a7
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