Cryo-fracturing and cryo-planing for in-lens cryo-SEM, using a newly designed diamond knife.

High-pressure frozen rat pancreas tissue samples were cryo-fractured and cryo-sectioned with a diamond knife in the microtome of a freeze-etching device. The bulk fracture faces and block faces were investigated in the frozen-hydrated state by use of a cryo-stage in an in-lens SEM. With this combination, relevant biological structures can be investigated with a few nanometers resolution in a near lifelike state, preventing the artifacts of conventional fixation techniques. Compared to TEM replica techniques, the presented method is more versatile since no replica cleaning is necessary. Additional structures can be made visible by controlled sublimation of ice, leading to a better understanding of the three-dimensional organization of organelles, such as the endoplasmic reticulum.