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It is of great technological interest to control the organization of nanoparticles (NPs) into functional devices that can make use of NP’s properties not found in the bulk form of the solid material. To this end, a major scientific challenge is to further elucidate inter-particle forces that govern spontaneous self-assembly processes in liquid suspensions. Liquid-phase electron microscopy (LPEM) can resolve morphological details of small objects in μm-thick liquid layers with nanometer resolution. The goal of this doctoral thesis has been to develop LPEM towards directly visualizing colloidal self-assembly processes in aqueous suspensions. As a model system, we used a colloidal binary system in which positively charged 30 nm nanoparticles (SiONP) form a shell around 100 nm, negatively charged polystyrene microspheres (PMS). Analytical calculations and Monte-Carlo simulations were performed to optimize experimental parameters and to validate contrast in data obtained with a scanning transmission electron microscope (STEM). The extent of radiolytic damage due to the electron beam (PMS) was directly analyzed from the image data and an acceptable dose range was defined. Within this range, the core-shell structure of the pre-assembled binary system was directly visualized. Finally, a novel liquid cell design was tested which enabled us to initiate colloidal assembly reactions in the confinement of the nanofluidic device.
Electron microscopy of native biological materials is usually hampered by sample preparation procedures such as dehydration and freezing, and by electron beam damage. Liquid phase electron microscopy (LP-EM) allows the observation of biological samples in liquid environments without conventional preparation procedures. However, electron beam damage also occurs in LP-EM, and thresholds for biological samples are not yet fully explored. In this work, the electron dose tolerance of green fluorescent protein (GFP) was analyzed in LP-EM. Protein damage was studied with increasing electron dose, using fluorescence degradation as an indication. Dc < 0.01 e-/Ų and Dc < 0.1 e-/Ų were observed for GFP on silicon nitrite in transmission electron microscopy (TEM) and environmental scanning electron microscopy (ESEM), respectively. In TEM, the dose tolerance was increased by three orders of magnitude when GFP was encapsulated in graphene liquid cells. The dose tolerance of more complex systems was investigated by binding GFP to actin filaments in fixed SKBR3 cells, which showed Dc < 0.1 e-/Ų in TEM and ESEM. In fixed SKBR3 cells, radiation damage was also studied based on the displacement of labeled membrane proteins. At electron doses of D = (7.8 ± 0.4) ∙ 10³ e-/Ų these labels showed a displacement of 0.8%. Procedures for studying biological materials such as proteins and fixed cells in LP-EM are presented in this thesis. Strategies to study and mitigate beam damage are demonstrated.
