Reconstructing Plant Cells in 3D by Serial Section Electron Tomography - Plant Cell Morphogenesis: Methods and Protocols - page 164

5. Choose approximately 20-30 fi ducial particles from both the
top and bottom surfaces in the zero tilt image. The selected
fi ducial particles should be spread evenly over the image. Mark
the center of each particle with a point. Each point should
belong to a different contour.
6. To view the trajectories of fi ducials in the image tilt series,
open the “model view” window. The trajectories should be
smooth. After shifting deviant points, press the “Track with
Fiducial Model as Seed” button to generate a new model.
7. If the section does not fi t into the sample tomogram, increase
the thickness of the sample tomogram and start over.
8. If combination stops at the Matchorwarp program, examine
the patch vector model. If the model has abnormally long vec-
tors, simply delete them, and save the model (Fig. 2b ). Click
“Replace Patch Vectors” and “Restart at Matchorwarp.” If the
cellular volume in the tomogram has large empty spaces, such
as large vacuoles, the eTomo program cannot fi nd accurate
matches, and the patch vector model will have a lot of large
false vectors. In such cases, create a patch region model to
exclude the empty spaces when the axis A and B tomograms
are compared for combination. If the residual error is slightly
larger than the threshold values, simply increase the threshold
values, and restart Matchorwarp.
9. Because TEM sections cannot be imaged at high angles near
90°, image resolution is not uniform from all directions. This
artifact can be observed in the reconstruction of fi ducials.
Fiducials have haloes that are uneven in the x - y plane (Fig. 2c ,
left panel), and they are elongated in the z direction (Fig. 1f ).
By combining the two orthogonal tomograms, the artifact can
be alleviated (Fig. 2c , right panel; ref. [ 7 ]).

Acknowledgments

This work was supported by NSF (No. MCB-0958107) and USDA
(No. AFRI 2010-04196) to B. -H. K, and the JSPS Institutional
Program for Young Researcher Overseas Visits to K. T.

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