We have developed a computer software package, IMOD, as a tool for analyzing and viewing three-dimensional biological image data. IMOD is useful for studying and modeling data from tomographic, serial section, and optical section reconstructions. The software allows image data to be visualized by several different methods. Models of the image data can be visualized by volume or contour surface rendering and can yield quantitative information.

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 techique 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 multiple (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) lace-like coats form an exocytic vesicles.

The Golgi apparatus is a membranous organelle that plays central roles in the secretory processes of eukaryotic cells. The apparatus consists of multiple convoluted cisternae, some of which are connected to a tubulo-vesicular system known as the trans-Golgi network (TGN). The TGN is thought to be the primary site for sorting and targeting of lipids and proteins to other cellular locations. Only a few studies have addressed the 3-D structure of the Golgi apparatus, and fewer still have focused on the structure of the TGN.We have employed high voltage electron microscopy (HVEM) and computer axial tomography to study the TGN in 3-D. For these experiments, the trans-most cisternae and TGN of normal rat kidney (NRK) cells were labeled by photoconversion of internalized BODIPY-ceramide. Cells were then postfixed and embedded in Epon-Araldite. Semithick (250nm) sections were cut, post-stained with uranyl acetate and lead citrate, and surface-labeled with 15nm colloidal gold to provide fiducial markers for image alignment. Sections were viewed at 18,300x in a JEM-1000 HVEM operating at 1MeV.

Since the early 1980’s, there have been several software packages available for no or low cost that have been developed for image processing of 3-dimensional (3-D) biological images as well as several viewing packages that can be used for viewing 3-D data sets. Work has been done to develop a modeling program that can make interactive 3-D models in stereo, but there is a present a gap between the image processing and model viewing steps for complex 3-D biological data. Although packages available for no or low cost have only rudimentary model building tools the biologist would like to have as accurate a 3-D model as possible. Here we describe a new image modeling software package for the 3-D analyses of biological EM data.We have developed a useful software package (IMOD, Image Modeler) that allows the user to draw two dimensional contours in 3-dimensional image data. The power of IMOD is the ability to see the 3-dimensional image data in several different views at the same time.