m biophys's List: electron tomography

Sections were first imaged in a Philips CM10 TEM (Philips Electronic Instruments, Mahwah, NJ) operating at 100 kV to identify cells in various stages of SPB duplication and early spindle formation. Low-magnification overview maps were taken to facilitate locating these same cells in the high-voltage electron microscope (HVEM), where contrast is greatly reduced. For tomography, the grids were placed in a tilt-rotate specimen holder (model 650; Gatan, Pleasanton, CA) and imaged in a JEM-1000 HVEM (JEOL USA) operating at 1 MeV. Images were taken every 1.5° over a ±60° range on Agfa (Mortsel, Belgium) Scientia film. The grids were then rotated 90°, and a second tilt series was imaged. The film was developed in D19 (Eastman Kodak, Rochester, NY) diluted 1:1 in water for 8 min. The negatives were digitized using a cooled charge-coupled device camera (STAR-1; Photometrix, Tuscon, AZ) into a Silicon Graphics (Mountain View, CA) INDY computer at a pixel size of 2.3 nm. The tilted views were aligned using the positions of the gold particles, and tomograms were computed for each tilt axis using the R-weighted back-projection algorithm (Gilbert, 1972). The two tomograms were aligned to each other and combined using the methods described by Mastronarde (1997)