Reliable use of resistance evaporation of Pt and C for high resolution freeze-fracturing and a crystal surface image complementary to the E-face of yeast plasma membranes.

Abstract

Freeze-fracture specimens of bakers yeast plasma membranes faces were prepared in both a modified Denton DFE-2-freeze-etch module and a modified Balzers BAF-301 freeze-etch unit. Each unit was equipped with a liquid nitrogen cooled shroud, resistance evaporators with PT-C and C sources 7 cm from the specimens and with a resistance monitor to control PT-C shadow film thickness. Optical diffraction patterns of specimens prepared in these units have fourth, fifth or sixth order spots. Therefore, on the basis of optical diffraction patterns, resolution of yeast plasma membrane specimens prepared in these units is equivalent to or better than that obtained by others with an ultrahigh vacuum system equipped with specially redesigned electron guns. A new image with tube-like particles in hexagonal arrays, each surrounded by six substructure particles, nearly perfect high-resolution complement to the hexagonal array of ring-like depressions and the six surrounding subunit depressions of the E-face, has been revealed on the surfaces of cubic crystals (presumably ice) which formed in the gap between the P- and E-faces within fissures that occurred when the samples were frozen in liquid Freon 22. When the samples were subsequently freeze-fractured at 77 K at a chamber vacuum of 13 microPa in which the specimens were protected from surface contamination by a liquid nitrogen cooled shroud, these crystals remained attached to the P-face but pulled away from the E-face against which they had apparently made molecular contact.