Cryoultramicrotomy: evidence against melting and the use of a low temperature cement for specimen orientation.

A technique is presented showing the use of toluene as a low temperature cement to attach portions of frozen tissues in the desired orientation on to the cryoultramicrotome chuck. Examples of transverse sections of skeletal and vascular smooth muscle are illustrated. Electron probe analysis of these sections indicates that the distribution of elements is maintained. Evidence is presented against through section melting based on sectioning toluene at a temperature just below its melting point (178K). Toluene was sectioned (about 100 nm thickness) successfully at three block temperatures (177, 163 and 113 K) when the knife temperature was by 1 degree or mor colder than the m.p. of toluene. In all cases the toluene sections melted when the knife temperature reached 178K. We conclude that sufficient heat is not generated and/or transferred to the toluene sections during cryosectioning under our conditions to raise the temperature of a 100 nm section by 1 K or more.