In situ graphene liquid cell-transmission electron microscopy study of insulin secretion in pancreatic islet cells.

BACKGROUND: Islet cell transplantation is one of the key treatments for type 1 diabetes. Understanding the mechanisms of insulin fusion and exocytosis are of utmost importance for the improvement of the current islet cell transplantation and treatment of diabetes. These phenomena have not been fully evaluated due either to the lack of proper dynamic imaging, or the lack of proper cell preservation during imaging at nanoscales. METHODS: By maintaining the native environment of pancreatic ß-cells between two graphene monolayer sheets, we were able to monitor the subcellular events using in situ graphene liquid cell (GLC)-transmission electron microscopy (TEM) with both high temporal and high spatial resolution. RESULTS: For the first time, the nucleation and growth of insulin particles until the later stages of fusion were imaged at nanometer scales. The release of insulin from plasma membrane involves the degradation of plasma membrane and drastic reductions in the shorter axis of the insulin particles. Sequential exocytosis results indicated the nucleation, growth and attachment of the new insulin particles to the already anchored ones, which is thermodynamically favorable due to the reduction in total surface, further reducing the Gibbs free energy. The retraction of the already anchored insulin toward the cell is also monitored for the first time live at nanoscale resolution. CONCLUSION: Investigation of insulin granule dynamics in ß-cells can be investigated via GLC-TEM. Our findings with this technology open new realms for the development of novel drugs on pathological pancreatic ß-cells, because this approach facilitates observing the effects of the stimuli on the live cells and insulin granules.

Investigation of the magnetosome biomineralization in magnetotactic bacteria using graphene liquid cell - transmission electron microscopy.

Understanding the biomineralization pathways in living biological species is a grand challenge owing to the difficulties in monitoring the mineralization process at sub-nanometer scales. Here, we monitored the nucleation and growth of magnetosome nanoparticles in bacteria and in real time using a transmission electron microscope (TEM). To enable biomineralization within the bacteria, we subcultured magnetotactic bacteria grown in iron-depleted medium and then mixed them with iron-rich medium within graphene liquid cells (GLCs) right before imaging the bacteria under the microscope. Using in situ electron energy loss spectroscopy (EELS), the oxidation state of iron in the biomineralized magnetosome was analysed to be magnetite with trace amount of hematite. The increase of mass density of biomineralized magnetosomes as a function of incubation time indicated that the bacteria maintained their functionality during the in situ TEM imaging. Our results underpin that GLCs enables a new platform to observe biomineralization events in living biological species at unprecedented spatial resolution. Understanding the biomineralization processes in living organisms facilitates the design of biomimetic materials, and will enable a paradigm shift in understanding the evolution of biological species.