Reconstructing Plant Cells in 3D by Serial Section Electron Tomography - Plant Cell Morphogenesis: Methods and Protocols - page 160

enter the sample tomogram thickness. Set it large enough so
that the sample regions can contain the entire tomogram. For
example, for a 300 nm thick section at 15,500× magnifi cation
(pixel size = 1.54 nm), enter 300-400, which is 50-100 %
larger than the 200 expected from the pixel size
(300 nm 1.54 nm × 200). Click the “Create sample tomo-

gram” button. After the sample tomogram is prepared, click
the “Create Boundary model” button. It will open “top.rec”
in the Zap window of the 3dmod interface ( see Note 7 ). Draw
a line along the boundary at each edge of the section as shown
in Fig. 1f . Repeat in “mid.rec” and “low.rec” tomograms. Use
the page up and page down buttons to move between the
sample tomograms. Do not put more than two points in the
line. After drawing six lines, two per sample tomogram, click
“Compute Z shift & Pitch angle.” Finish the step by pressing
the “Create Final Alignment” button.
8. Open the Final Aligned Stack page, and press “Create Full
Aligned Stack” to generate the fi nal image stack for tomogram
calculation. The stack can be examined in the 3dmod program
by pressing “View Full Aligned Stack.”
9. The Tomogram Generation page is used for calculating tomo-
grams from the fi nal aligned stack. Simply press the “Generate
Tomogram” button. Once the tomogram calculation is com-
pleted, examine the tomogram by pressing the “View
Tomogram in 3dmod” button.
10. The next step is to generate the second axis tomogram. In the
eTomo window, press axis B (Fig. 1b ). The procedure for the
second axis (axis B) tomogram is almost the same as for the
fi rst axis (axis A) tomogram, except that fi ducial selection is
transferred over from the fi rst axis image stack instead of being
selected de novo. After doing the pre-processing and coarse
alignment, open the “Fiducial Model Generation” page and
press “Transfer Fiducials From Other Axis” button. eTomo
displays the result of the fi ducial transfer from the fi rst axis after
looking for corresponding fi ducials in the two image series.
Occasionally eTomo fails to fi nd all matching fi ducials in the
two orthogonal image stacks, but a failure in transferring a few
fi ducials does not hinder calculation of the axis B tomogram.

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11. Press “Track Fiducial Seed Model” and proceed through the
rest of the steps as completed for the fi rst axis tomogram
(Fig. 2a ).
12. After generating the axis B tomogram, return to the axis A
page (Fig. 1b ). Press the “Tomogram Combination” button.
The Tomogram Combination page consists of three tabs. As
the combination proceeds, the page moves from the “Setup”
tab to the “Initial Match” tab and to the “Final Match” tab