In situ TEM to Study Iron Crystallization Mechanism in Ferritin

Ferritin is an iron storage protein that regulates iron, stores excess iron, and prevents free radical formation in the human body. The iron in stored as iron oxides inside the protein through the process of biomineralization. Despite several years of research, the iron biomineralization mechanism in ferritin is sparsely known. Further, with evidences of brain iron accumulation in neurological conditions, it is important to understand the iron regulation mechanism in ferritin. Transmission electron microscopy has revolutionized our current understanding of nanostructures and chemistry of ferritin. While it is possible to obtain high spatial resolution with traditional electron microscopy, the application was limited to material sciences for a long time. The need to dry and condition the sample before imaging, restricted its application towards certain specific biological applications. While the evolution of Cryo-electron microscopy tapped the potential to study the structure of hydrated proteins such as ferritin, the applications were still limited to the structural aspects. With recent advancements in microfluidics and chip design, it has become possible to study the dynamics of biomolecules. Although this technology provided several opportunities to study ferritins in liquid phase, it also raises question about the resolution that the microfluidic technology can offer. On the other hand, advancements in graphene liquid cell-transmission electron microscopy (GLC-TEM) enabled atomic resolution imaging of ferritin without compromising the liquid environment of the protein. xxvii The focus of this dissertation will be, firstly to introduce ferritins and its structure, morphology, chemistry and biology as discussed in Chapter 1. Further, the focus will be on the TEM sample preparation techniques utilized to study the ferritin’s structure as discussed in Chapter 2. Different techniques, its advantages and benefits are discussed in this chapter. Further, this chapter also emphasizes the utilization of GLC-TEM to study ferritins in hydrated state. Chapter 2 is further extended as the characterization of GLC-TEM and its significance in imaging biological materials such as ferritin is emphasized in Chapter 3. By utilizing GLC-TEM, the morphology, structural, and chemical characteristics of iron oxides in human ferritins were compared as described in Chapter 4. The significance of this work is to emphasize the role of naturally distinct ratios of amino acid subunits in different organ ferritins in producing iron oxides of different structural and chemical composition. Further, GLC-TEM technique was utilized to study in situ biomineralization in ferritin in realtime as described in Chapter 5. The results obtained through the novel in situ GLC-TEM technique can result in new frontiers in science to study biological structures and better understand human health which is discussed in Chapter 6.