High-Pressure Frozen Mouse Lung: Cryo Scanning Electron Microscopy

High-pressure facilitates freezing of biological specimens: pressurizing to 2000 bar before cooling retards growth and reduces size of ice crystals in the specimen. Although the lung is 80% air and is generally difficult to freeze, we report here good cellular ultrastructure in high-pressure frozen lung when: 1) the lung is allowed to collapse (empty of air) prior to sampling, or 2) air-filled lung is allowed to compress during pressurization in the high-pressure freezer prior to freezing or 3) airspaces are filled with relatively incompressible fluorocarbon liquid.Small Balb mice (28-30g) were anaesthetized and ventilated via endotracheal tube. The chest was opened and a 2mm diameter disk of lung at the thin anterior margin of the right lung was clamped between two aluminum tophats (200μm wells), sealing in air. In other experiments, the lung was allowed to collapse to 0 cm water transpulmonary pressure prior to clamping. In the third series of experiments, air was gradually replaced with fluorocarbon liquid (FC5312, 3M, Minneapolis MN) and the lung inflated with liquid to functional residual capacity. The top hat assembly containing the sealed lung was transferred to the high-pressure freezer (HPM 010, Bal-Tec, Balzers, Lichtenstein) where they were pressurized and frozen.