Immersion Freezing of Biological Specimens: Rationale, Principles, and Instrumentation

INTRODUCTION

Cryo-transmission electron microscopy (cryo-TEM) allows the visualization of biological specimens within their native, hydrated environment at nanometer resolution. To prevent the formation of destructive ice crystals, an extremely high cooling rate has to be achieved in the freezing process. For samples that are inherently thin enough, ranging from macromolecules to peripheral parts of spread cells, this can be accomplished by plunging the sample into a cryogen. This approach, known as “immersion freezing,” is an essential preparation technique in structural, molecular, and cell biology. In this article, we discuss the advantages of cryo-EM, the scope of specimens that can be visualized using this technique, the method of immersion freezing, and the instrumentation available. In particular, we focus on the new semiautomatic immersion freezer by Leica Microsystems (“EM GP”).