Low-temperature scanning electron microscopy of frozen hydrated mouse lung

A method is presented by which water is preserved as ice during examination of the lung in the scanning electron microscope (SEM). The lung need only be inflated, frozen, transferred to the microscope and examined with the electron beam. Chemical fixation, solvent dehydration, and drying are not necessary. The low-temperature SEM of Pawley and Norton [11] maintains lung at −180° C, nearly liquid nitrogen temperature, for extended periods with a Joule-Thomson refrigerator built into the stage. It has an integral high-vacuum preparation chamber attached to the microscope column which allows serial fracture, low-magnification stereo light microscopy, radiant etching, and evaporative coating with gold or carbon. The stage can be tilted from 0° to 45° and rotated a full 360°. It is demonstrated that the air-liquid interface in the lung can be examined and that low-temperature SEM can be used to investigate the shape of alveoli, the patency of the pores of Kohn in the hydrated state, and the shrinkage and distortion of lung with drying.