Applicable technical method for lower temperature freeze-substitution of cryo-fixed cells

Cryo-fixation and freeze substitution followed by microscopy are commonly used sample preparation methods for visualizing the morphology of intracellular organelles. Freeze substitution is an especially important preparative step because it enables the preservation of intracellular structures in cryo-fixed cells close to the living states for visualization. In this study, we describe a novel freeze substitution method and device that uses both dry ice and liquid nitrogen as coolants rather than dry ice alone. We found that our approach achieved lower temperatures for longer periods of time than conventional devices and therefore allows major improvements in preservation quality, ultimately facilitating high contrast imaging of intracellular organelles. The technical approaches that are realized by this valuable approach are simple, rapid, and applicable for visualizing cellular events of interest.