Freezing of tissue-limits for the autoradiographic localization of diffusible substances

Frozen thin sections and sections from freeze-dried and embedded tissue are used for the autoradiographic localization of diffusible substances at the electron microscope level. The presence of ice crystals in such sections may limit the autoradiographic resolution. Ice crystals are formed during freezing and may grow during subsequent processing of tissue. The contribution of ice crystal growth to the final image was estimated by measuring the distribution of the ice crystal sizes in freeze-etch replicas and in sections from freeze-dried and embedded tissues. A surface layer (10-15 mu) without visible ice crystals was present in both preparations. Beneath this surface layer the diameter of ice crystals increased towards the interior with the same relationship between crystal size and distance from the surface in the freeze-etch preparation as in the freeze-dry preparation. Ice crystal growth occurring during a much longer time during freeze-drying compared to freeze-etching does not significantly contribute to the final image in the electron microscope. The formation of ice crystals during freezing determines to a large extent the image (and therefore the autoradiographic resolution) of freeze-dry preparations and this probably holds also for thin cryosections of which examples are given.