Insulin is a peptide hormone produced in pancreatic beta cells which is responsible for glucose metabolism regulation in mammals.
Insulin molecules are stored in 200-300 nm single membrane granules inside cytoplasm of beta cell. As we and others previously demonstrated [1,2], insulin molecules in secretory granules are organized into crystalline particles.
We have used correlative light-electron microscopy workflow under cryogenic conditions to characterize insulin granules in the cellular context and under near native conditions.
Rat beta cells INS 1E were seeded onto golden electron microscopy grid with carbon foil and incubated overnight to allow them to adhere on the grid surface. Adhered cells were fluorescently stained with live cell imaging dyes (nucleus, insulin granules and cell membrane) and plunge frozen into the liquid ethane. Vitrified cells were transferred into the cryo-plasma focused ion beam scanning electron microscope (PFIB-SEM) with integrated light microscope. Optical data of the cell of interest were obtained and correlated with SEM data.
Cellular lamella in the region of insulin fluorescent signal was prepared by xenon plasma FIB to the final thickness 200 nm. Optical data in the polished lamella were collected to verify the presence of insulin granules. Grids with lamellae were subsequently tranferred into transmission electron cryo-microscope operating for cryo-electron tomography. Collected electron tomography data confirm crystalline of the secretory granules.