Dry Ice Self-Pressurised Rapid Freezing (DryIce SPRF): all you need to cool for cryofixation of nematodes is dry ice

Abstract

Summary Cryofixation immediately arrests all biochemical, physiological and dynamic processes underway in the sample in their present state, resulting in both excellent preservation of the specimen’s ultrastructure and its antigenicity. Cryofixation involves extremely rapid cooling of specimens, creating an amorphous, or ‘non-crystalline’, state of water containing no detectable ice crystals, a process dependent on pressure, medium composition and temperature. Self-Pressurised Rapid Freezing (SPRF) employs plunge freezing of specimens in a sealed copper tube into a cryogen such as nitrogen slush (−210°C), liquid nitrogen (−196°C), ethane (−183°C) or propane (−120°C). In this study we have explored the use of SPRF with cooled acetone on dry ice (−80°C) as the cryogen, a method named DryIce SPRF. Although with this relatively high temperature amorphous water cannot be formed, we have demonstrated that the ultrastructural and antigenicity results after DryIce SPRF on Caenorhabditis elegans are perfectly comparable with those achieved using High Pressure Freezing and SPRF. Thus, with sufficient pressure optimal results, with ice crystals below the resolution of transmission electron microscopy, can be achieved even at −78°C. Furthermore, a huge advantage of DryIce SPRF over other techniques is its use of affordable, easily available and safe products.