High-density morphologies of ice in high-pressure frozen biological specimens

Mouse liver tissue was rapidly cooled by high-pressure freezing at a nominal pressure of 2100 bar. Ultrathin cryosections were examined at 110 K with a cryoelectron microscope and the state of water was studied on micrographs and electron diffraction patterns. The results are compared with those from liver specimens cryofixed at ambient pressure by plunge freezing. The high-pressure frozen specimens contained crystalline frozen regions as well as areas which were vitreous. The spot diffraction patterns from crystalline regions are found to differ from those known for hexagonal ice. The comparison with powder diffraction data reveals the presence of the high-density morphologies Ice II and Ice III. Vitreous areas of biological samples frozen at ambient pressure are optically denser than areas where the water crystallised as cubic or hexagonal ice. However, on micrographs from high-pressure frozen samples, no significant contrast is found. This observation is explained by the higher specific mass of high-pressure ice compared to Ice I, an interpretation which requires a higher specific mass for high-pressure vitreous water compared to vitreous water produced at ambient pressure. The larger diffuse first ring of the diffraction pattern from high-pressure vitreous water confirms this argument.