As with Sterecon, the first step in a tomographic study is to clarify what the investigator hopes to learn from a reconstruction. We may find that a simple tilt-series animation, or the use of Sterecon, would be more appropriate. Once we agree that tomography is the approach of choice, specimen thickness and magnification must be carefully considered. The user is then trained in recording the tilt-series in the HVEM (usually by K. Buttle) or the IVEM (usually by M. Marko). In some cases, our staff does the microscopy. Protection of the specimen from contamination and excessive irradiation are important. The recording of a single-axis tilt series, typically takes 1-2 hours, but we normally do double-tilt recording, which takes twice as long. If images are recorded on film, we assist the user in setting up the scanning camera for image digitization and pre-alignment. The user then completes the scanning in about 2 hours for a double-tilt series (122 images). The final alignment uses gold markers as fudicial marks, and must be done even if the images are recorded digitally using the IVEM. The locations of the gold markers must be indexed on each image. This procedure takes about 3 hours, and only requires a few minutes of training. The reconstruction can be completed in about 3 hours for a double-tilt volume of about 512x512x200 pixels, or half an hour for a single-tilt volume of the same size. Visualization of the reconstruction is the most time-consuming portion of the project. In many cases, we do this with the investigator looking on. If the investigator is to do this independently, there must be training in the use of our workstations, the SPIDER software system, and VoxelView, Iris Explorer, or another visualization package. In most cases Sterecon is used for segmentation, the first step in visualization. Visualization training takes about a week. Some investigators have visualization capabilities in their own labs, and if they use s oftware we are familiar with (or supply), we also provide assistance, usually over the Internet. We take every opportunity to promote the unique benefits of HVEM tomography at meetings via presentations and informal discussions. Full documentation on the tomographic reconstruction methods we use was written in html, with links to sample data. This was put on the SPIDER www homepage. In conjunction with a book chapter, all of the visualization methods used at Albany for tomographic volumes were applied to a test volume of a kinetochore, and illustrations were made. The examples of visualization from this chapter will also be used in html documentation to be written later. McEwen, B. and Marko, M. (1998) Three-dimensional transmission electron microscopy and its application to mitosis research. Methods in Cell Biology, 61:82-113.
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