Quantitative, in situ visualization of intracellular insulin vesicles in pancreatic beta cells

Characterizing relationships between Zn²⁺, insulin, and insulin vesicles is of vital importance to the study of pancreatic beta cells. However, the precise content of Zn²⁺ and the specific location of insulin inside insulin vesicles are not clear, which hinders a thorough understanding of the insulin secretion process and diseases caused by blood sugar dysregulation. Here, we demonstrated the colocalization of Zn²⁺ and insulin in both single extracellular insulin vesicles and pancreatic beta cells by using an X-ray scanning coherent diffraction imaging (ptychography) technique. We also analyzed the elemental Zn²⁺ and Ca²⁺ contents of insulin vesicles using electron microscopy and energy dispersive spectroscopy (EDS) mapping. We found that the presence of Zn²⁺ is an important characteristic that can be used to distinguish insulin vesicles from other types of vesicles in pancreatic beta cells and that the content of Zn²⁺ is proportional to the size of insulin vesicles. By using dual-energy contrast X-ray microscopy and scanning transmission X-ray microscopy (STXM) image stacks, we observed that insulin accumulates in the off-center position of extracellular insulin vesicles. Furthermore, the spatial distribution of insulin vesicles and their colocalization with other organelles inside pancreatic beta cells were demonstrated using three-dimensional (3D) imaging by combining X-ray ptychography and an equally sloped tomography (EST) algorithm. This study describes a powerful method to univocally describe the location and quantitative analysis of intracellular insulin, which will be of great significance to the study of diabetes and other blood sugar diseases.