The Observation of Frozen Hydrated Sections by CTEM and STEM

Abstract

With the ultimate aim of studying biological structures close to their native state, we are attempting to prepare and examine thin frozen hydrated sections using catalase crystals and skeletal muscle as test objects. There have been a few observations under these conditions.small droplet of catalase crystals stained in 2% aqueous uranyl acetate and re-suspended in water was placed on a copper specimen holder and plunged into liquid nitrogen. Small pieces of rat femoral muscle were fixed in 2% gluter- aldehyde in 0.05 M cacodylate buffer, and frozen as above after washing in buffer with or without the addition of 1 M sucrose.Frozen sections for examination in the CTEM were cut with glass knives in a Sorvall MT2B microtome with the LTC2 cryo-attachment operating at ca. -120°C. For examination in the STEM, frozen sections were cut in a Reichert Ultracut with FC4 cryo-attachment operating with the knife and the specimen at -150°C and the chamber at -170°C. Sections that appeared transparent were manipulated onto carbon coated grids with a pre-cooled eyelash probe and then sandwiched in place with a second carbon coated grid.