DIAMONDS ARE A CRYOSECTIONERS BEST FRIEND

High-pressure frozen Golden Delicious apple leaves were cryosectioned at low temperature with diamond knives. Good cryosections were obtained by optimizing the cutting parameters, i.e. sectioning temperature, mechanical stability of the sample, and sectioning velocity. Cutting artefacts were minimized by reducing the electrostatic interactions between the knife surface and the cryosection. This was accomplished by sectioning the sample in the presence of an ionization electrode. The ionization device, with a primary voltage of 7-8 kV, produces positively and negatively charged nitrogen ions which neutralize the surface charges of the knife and the section. This minimizes the friction on the knife surface and results in ultrathin sections without crevasses or knife marks. Compression of the sections could be minimized, but not avoided, by reducing the knife angle to 30-degrees. Improved contrast of the frozen-hydrated sections was obtained with the Zeiss EM 902 energy-filter microscope operated in the zero-loss mode.