Freeze-drying from tertiary butanol in the preparation of endocardium for scanning electron microscopy

The scanning electron microscope appearances and shrinkage of blocks of canine endocardium prepared by freeze-drying directly, by freeze-drying after replacing tissue water with tertiary butanol (2-methyl propan-2-ol) and by critical point drying were compared. All three methods demonstrated endothelial cells which showed nuclear prominences, microvilli and intercellular boundaries. The microvilli varied in size and number from dog to dog but were generally less well defined in specimens freeze-dried from water. Shrinkage due to t-butanol dehydration was significantly less than that which occurred in ethanol in the critical point drying method. Overall the reduction in surface area was significantly less in specimens freeze-dried directly at -65 C (6.8%) than in those dried from t-butanol at -20 C (15.4%) and those prepared bly critical point drying (22.1%). However the amount of shrinkage observed in t-butanol treated tissue was not significantly different from that which was critical point dried. It was not possible to distinguish between comparable samples prepared by these two methods on the basis of their scanning electron microscopic appearances. Thus the relative simplicity and convenience of the t-butanol method, together with its saving of time, its use of standard freeze-drying equipment and the avoidance of ice-crystal artefact justify its consideration as an alternative method of preparing wet biological tissue for scanning electron microscopy.