Reconstructing Plant Cells in 3D by Serial Section Electron Tomography - Plant Cell Morphogenesis: Methods and Protocols - page 159

fi le with the newly prepared fi xed stack fi le. Click “Done” to
go to the next step.
4. The “Coarse Alignment” step is for adjusting rotations and
scales of images in the stack, using a cross-correlation method,
to prepare a roughly aligned image stack. Click the “Calculate
Cross-Correlation” button to determine adjustments between
sequential images. Once the calculation is completed, press
“Generate Coarse Aligned Stack” to create a coarsely aligned
image stack. The pre-aligned stack (example_a.preali) can be
viewed in the 3dmod interface by pressing “View Aligned
stack in 3dmod.” If cross-correlation cannot fi x large shifts in
images, the images should be aligned in the Midas program.
Press “Fix Alignment With Midas” to open the program. Click
“Done” to go to the Fiducial Model Generation step.
5. “Fiducial Model Generation” is where fi ducial models are cre-
ated using the gold particles deposited on the top and bottom
surfaces of the sections. After checking the “Make seed and
track box,” press the “Seed Fiducial Model” button. Two tool
dialog boxes “3dmod: example_a.preali” and “Bead Fixer”
will open. Choose 20-40 gold particles in the image at the
center of the tilting series (Fig. 1c , see Note 5 ). Each point
should belong to one contour in a single model. After saving
the model, click the “Track Seed Model” button. This will
produce a fi ducial model from the seed model by tracking the
gold particles in all images in the stack. Once the tracking is
done, click the “Fix Fiducial Model” button. Several gaps will
appear on the “Bead Fixer” window. These gaps represent
images in which the tracking program failed to fi nd the posi-
tions of fi ducial particles. Connect the trajectories of fi ducial
particles by adding new point(s) in the image(s). Pressing the
“Go to Next Gap” button will move to the next image with a
missing point. Repeat until the message, “No more gaps
found,” shows up in the main window after pressing the “Go
to Next Gap” button. Save the fi le, and press “Done” ( see
Note 6 ).
6. Click “Compute Alignment,” with the default parameters.
Press the “View Residual Vectors” button to examine residuals,
the distances between actual positions of the gold fi ducial
markers and their predicted positions (Fig. 1d ). If the residuals
are too large, fi x them in the “bead fi xer” manually or by using
the “Move Point” feature. To view trajectories of fi ducials in
the tilting series, press “View 3D model” (Fig. 1e ).
7. “Tomogram Positioning” is used to locate the section in the
reconstructed volume to make the fi nal tomogram fl at and to
minimize blank slices in it. This can be achieved by determin-
ing tilting and shift of the section in 3 sampled regions. First