Freeze-fracture-etching Electron Microscopy for Facile Analysis of Yeast Ultrastructure

We describe a streamlined method that enables the quick observation of yeast ultrastructure by electron microscopy (EM). Yeast cells are high-pressure frozen, freeze-fractured to cut across the cytoplasm, and freeze-etched to sublimate ice in the cytosol and the organelle lumen. The cellular structures delineated by these procedures are coated by a thin layer of platinum and carbon deposited by vacuum evaporation, and this platinum-carbon layer, or replica, is observed by transmission EM. The method differs from the deep-etching of pre-extracted samples in that intact live cells are processed without any chemical treatment. Lipid droplets made of unetchable lipid esters are observed most prominently, but other organelles-the nucleus, endoplasmic reticulum, Golgi, vacuoles, mitochondria-and their mutual relationships can be analyzed readily. It is of note that the entire procedure, from quick-freezing to EM observation, can be performed within a day.