Recent progress in freeze-fracturing of high-pressure frozen samples.

Pancreatic tissue, bacteria and lipid vesicles were high-pressure frozen and freeze-fractured. In addition to the normal holder, a new type of high-pressure freezing holder was used that is particularly suitable for suspensions. This holder can take up an EM grid that has been dipped in the suspension and clamped in between two low-mass copper platelets, as used for propane-jet freezing. Both the standard and the new suspension holder allowed us to make cryo-fractures without visible ice crystal damage. High-pressure frozen rat pancreas tissue samples were cryo-fractured and cryo-sectioned with a new type diamond knife in the microtome of a freeze-etching device. The bulk fracture faces and blockfaces were investigated in the frozen-hydrated state by use of a cryo-stage in an in-lens SEM. Additional structures can be made visible by controlled sublimation of ice ('etching'), leading to a better understanding of the three-dimensional organization of organelles, such as the endoplasmic reticulum. With this approach, relevant biological structures can be investigated with a few nanometre resolution in a near life-like state, preventing the artefacts associated with conventional fixation techniques.