We are working to improve methods for the study of cellular fine structure. Our approach is to advance each of the key steps in the preparation of specimens for EM: high quality fixation that will preserve both structure and antigenicity; methods for specific labeling; efficient acquisition of 3-D electron microscopic data; and software for 3-D reconstruction and display.
Our work on high quality structure preservation has focused on methods for fast freezing and freeze substitution. Both plunge freezing of specimens grown on coated gold grids and high pressure freezing of either cultured cells or tissue specimens have yielded well preserved material. These samples are suitable for freeze substitution fixation with either anhydrous aldehydes in acetone at -90°C, for the preservation of antigens, or aldehydes, tannic acid, OsO4, and uranyl acetate for optimal preservation the structure.
We have used a JEOL JEM-1,000 high voltage microscope to image sections about 250nm thick, employing a goniometer stage to perform dual axis tomography for 3-D reconstruction with approximately isotropic resolution at ∼7nm.