Cryo-SEM as a suitable tool to study vitrification in cryopreserved tissue.

Biological samples are successfully preserved for indefinite periods after vitrification, providing ice formation is avoided. Glass is difficult to characterize, especially at the extremely low temperatures required for biological tissues vitrification. Glass transition is traditionally observed by differential scanning calorimetry (DSC), in spite of its reduced sensitivity and lack of spatial information. In this work, a novel procedure, based on low-temperature scanning electron microscopy (cryo-SEM), is explored to locate vitrified areas and ice crystals in tissues and cells. Cryo-SEM observation of biological samples requires an etching phase to create contrast: a temperature rise, allowing ice partial sublimation. Glassy water, differently from crystallized water (ice), has a neglectable sublimation rate. Consequently, while the dark image of sublimated crystals and the resulting structural details can be observed in ice-containing samples, vitrified tissues show a smooth landscape, without dark areas or visible structure elements.