Cryo-Electron Microscopy of Vitreous Sections (CEMOVIS) of Yeast

Application Note for Leica EM HPM100 - Life Science Research

August 12, 2016

Protocol
The sections are of yeast frozen with a Leica HPM100 High Pressure Freezer in the copper tube system, the cell paste was mixed with a pH 6.5 MES/dextran buffer so that a final MES concentration of 50mM and a dextran concentration of 20% was achieved.

The samples were sectioned on a Leica EM UC7/ EM FC7 Ultramicrotome with micromanipulator at -140°C and the section thickness was set to 50nm. The sections were attached to Agar lacey grids.

The sections were imaged using a Tecnai Polara 300KeV (FEI, The Netherlands) microscope fitted with a 4K Gatan CCD camera. The magnification for the sections was 23K, with a defocus of -6um for the tomogram and -8um for the projection image, and the diffraction was done with a camera length of 930mm. The image in panel A is an average of the central 10 slices of a reconstruction done with the IMOD package (Kremer et. al., 1996), image processing software from a tomogram collected using the FEI software.

Fig.1:
Left – optical slice from a tomographic reconstruction
Upper right – micrograph of a vitrified yeast cell
Lower right – diffraction pattern image

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