A copper block method for freezing non-cryoprotected tissue to produce ice-crystal-free regions for electron microscopy. I. Evaluation using freeze-substitution.

A wide variety of plant and animal tissues were prepared for electron microscopy by freeze-substitution, after rapid freezing on a liquid nitrogen cooled copper block by the van Harreveld method. Measurements were made of ice crystal size versus depth in tissues that had not been treated with any cryoprotectants. Ice crystal size increased exponentially with depth. It was confirmed that a narrow surface band increased exponentially with depth. It was confirmed that a narrow surface band (approximately 12 mum) was frozen sufficiently rapidly to prevent the formation of electron microscopically-visible ice crystals.