Reconstructing Plant Cells in 3D by Serial Section Electron Tomography - Plant Cell Morphogenesis: Methods and Protocols - page 162

from each axis. By excluding the empty regions, tomogram
combination is performed more effi ciently. Leave the
“Volcombine Controls” and “Intermediate Data Storage”
boxes unchanged. Press the “Create Combine Script” button
and then the “Start Combine” button ( see Notes 8 and 9 ).
13. In the post-processing step, the noncellular volume is trimmed,
and the tomogram is reoriented so that images of the XY plane
are loaded when 3dmod opens the tomogram. The combined
tomogram is always named sum.rec. Open the sum.rec fi le by
pressing “3dmod Full Volume.” Scan through the tomogram
and determine the X, Y, and Z ranges to be included in the fi nal
tomogram. Type in the X, Y, and Z ranges, and choose either
“Swap Y and Z dimensions” or “Rotate around X axis.” As
long as the selection is consistent for all tomograms from serial
sections, either selection will suffi ce. Press “Trim Volume.”

14. The “Clean Up” step is used to delete large temporary fi les. If
the fi nal tomogram is satisfactory, highlight all the intermediate
fi les displayed in the window and press “Delete Selected.”

1. First collect all tomograms and place in a new folder. Determine
the order of the serial tomograms, from the bottom to the top.
The lowest Z end of the bottom tomogram will be the lowest
Z end of the fi nal tomogram, and the top Z end of the top
tomogram will be the top Z end of the fi nal tomogram. It is
helpful to include a number when naming the tomograms to
denote their order, such as example1.rec, example2.rec, and
example3.rec. Make a working directory in the folder for tem-
porary fi les and for fi les produced during joining.

3.4 Joining
Tomograms from
Serial Sections

2. Open eTomo and choose “New Join” in the “Front Page -
eTomo” window. The setup panel will open in the “New Join -
eTomo” window. Give the path and name of the working
directory and the root name for the output fi le. Press the “Add”
button in the section table to choose tomograms. Start from the
bottom tomogram (Z Order 1) to the top tomogram. In the
“Final” column, enter the ranges of tomograms to be included
in the joined tomogram by typing the range (start and end slice
numbers). To prepare sample slices for aligning tomograms,
enter 5-10 extra slices on both sides of each tomogram. Press
the “Make Samples” button to prepare an image stack showing
boundaries between tomograms. The image stack will be used
for aligning tomograms. When the process is done, click the
“Align” tab to open the Align panel.

3. Click “Midas” to open the sample slices in the Midas program
(Fig. 3 ). This program is useful for adjusting alignment between
pairs of images by manipulating translation, rotation, magnifi -
cation, and stretching of the images. For more detail, see the
Midas page in the 3dmod package manual. After completing
alignment at each boundary, save the transformation fi le.