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

Abstract

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.