A method for efficient observation of intracellular membranes of monolayer culture cells by quick-freeze and freeze-fracture electron microscopy

We previously developed a method of analyzing the two-dimensional distribution of membrane lipids by combining quick-freezing and freeze-fracture replica labeling (QF-FRL). In principle, this method can be applied to any membrane, but in practice it is not easy to observe cytoplasmic organelles efficiently without ice crystal damage. In this paper, we report a modification of our method that circumvents this problem. In the modified method, cells are cultured on a gold foil scratched with sandpaper and quick-frozen according to a high-pressure freezing method. This technique enables the efficient observation of intracellular structures such as the nucleus, endoplasmic reticulum, Golgi apparatus and mitochondria. Specific labeling of phosphoinositide 4,5-bisphosphate was confirmed in the obtained freeze-fracture replica. The present QF-FRL method is easy to use and should expedite the analysis of intracellular lipid distribution.