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Abstract. High voltage electron microscopy and computer axial tomography have been used to study the 3-D structure of trans-Golgi cisternae and trans-Golgi networks (TGNs) in NRK cells. Both structures were specifically labeled by photoconversion of a fluorescent analogue of ceramide using a modification of the technique of Pagano et al. (J. Cell Biol. 1991. 113: 1267-1279. Regions of the Golgi ribbon in fixed, stained cells were cut in 250-nm sections and analyzed by tilt series microscopy and subsequent tomographic reconstruction. Resolution of the reconstructions ranged from 6 to 10 nm. The size and structure of the TGN varied considerably throughout the Golgi ribbon; all reconstructions were made from regions with pronounced TGN. Most regions analyzed contained multipie (2--4) Golgi cisternae that stain with ceramide. These "peel off" from the closely stacked cisternae and are continuous at their ends with tubules that contribute to the TGN. Most vesicular profiles visualized in the TGN are connected to TGN tubules. The budding of vesicles appears to occur synchronously along the length of a TGN tubule. Two distinct coats were visualized on budding vesicles: clathrin cages and a novel, lace-like structure. Individual TGN tubules produce vesicles of only one coat type. These observations lead to the following predictions: (a) sorting of molecules must occur prior to the formation of TGN tubules; (b) vesicle formation takes place almost synchronously along a given TGN tubule; and (c) lacelike coats form on exocytic vesicles.

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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.