Reconstructing Plant Cells in 3D by Serial Section Electron Tomography - Plant Cell Morphogenesis: Methods and Protocols - page 157

5. Coat the stained grids with 15 nm unconjugated gold particles.
Put 10 l of the gold colloid solution onto one side of the

grids. Wait 10 min, rinse by dipping the grids in distilled water,
and dry the grids. Repeat for the other side of the grids.

μ

1. Examine the serial sections in a standard transmission electron
microscope operated at 100-120 kV to confi rm that the
regions for ET are free of damage in all sections and to capture
low magnifi cation images. Prepare a map to assist in readily
locating regions for ET in the intermediate voltage TEM ( see
Note 4 ).
2. Collect tilted images at 1° intervals, from −60° to +60° (or
from −70° to +70°) in each section. Transmission electron
microscopes for ET are equipped with eucentric tilting stages
and with an automatic image acquisition programs such as
SerialEM. Record images at 15,500× or 20,000× to accom-
modate an approximately 3 micrometer × 3 micrometer or
2 micrometer × 2 micrometer fi eld of view, respectively, in the
tomograms.
3. After acquiring tilting series from all sections on the grid,
rotate the grid 90° and record a tilting series of the same areas
in the sections for dual axis ET analysis. Name the pair with a
root name followed by a and b, e.g., example_a.st and
example_b.st.

3.2 Acquisition of
Tilting Series

1. Create a data folder and copy the pair of tilting series fi les (also
termed image stack fi les) from the dual axis recording to this
folder. Create a subfolder for backup fi les. Start the eTomo
program and choose “New tomogram” in the “Front Page -
eTomo” window to open the “Setup Tomogram - eTomo”
window (Fig. 1a ). In the dataset box, type the name for the
fi rst of the two tilting series (e.g., example_a.st), or press the
“open fi le” button to select a tilting series. Enter the location
of the backup fi le folder in the window.
2. Click the “Scan Header” button to get values for pixel size
(nm) and image rotation (degrees). The two boxes should
autopopulate after clicking the button. Enter the size of the
gold fi ducial in the fi ducial diameter (nm) box (e.g., 15). To
examine the tilting series, click the “View Raw Image Stack”
button. To go to the next step, click on “Create Com Scripts.”
3. The main window has several process control buttons
(Fig. 1b ). “Pre-processing” is for erasing bad pixels resulting
from X-rays and/or removing bad CCD rows from the image
stacks. Press the “Pre-Processing” button and choose “Find
X-rays (Trial Mode).” Wait several seconds for processing, and
click the “Create Fixed Stack” button to write a new image
stack fi le. Press the “Use Fixed Stack” to replace the original

3.3 Reconstruction
of Tomograms with
eTomo