High-pressure freezing of mechanically stretched cells.

Abstract

High-pressure freezing (HPF) is an electron microscopy (EM) preparation technique with superb ultrastructural preservation. Combined with EM tomography it provides virtual EM serial sections with extraordinary spatial resolution. For HPF, cells are usually cultured on a rigid sapphire disc that provides a tight fit in the holding bracket of the HPF apparatus. Since we are using extensible elastic silicone membranes as a growth support to perform cell stretch experiments, we developed a method to clamp the stretched silicone membrane and place it instead of the sapphire disc into the HPF holding bracket. Compared to chemical fixation the HPF immobilised cells showed improved structural preservation, partly even on a molecular level. However, the outstanding quality of HPF immobilised cells on sapphire discs was not achieved. Moreover, regions with obvious freezing artefacts seemed to be more abundant in the HPF silicone membranes, probably caused by lower heat transfer rates of the silicone membrane during the HPF process. Taken together, we have shown that HPF immobilisation can be performed on growth supports different than sapphire discs. Since even stretched membranes can be used with the new method, also other unconventional growth supports should not pose a problem.