Biology Reference
In-Depth Information
Fig. 3
(
a
) “Midas” window. (
b
) Two images are overlaid to align them.
Top
and
bottom
images are shown in two
colors (
magenta
and
green
). Using 4 operations, translation, rotation, magnifi cation, and stretching, the fl ank-
ing sections can be transformed to maximize overlap between them
4. Go to the “Join” panel and make a trial tomogram to assess
the range and alignment of the joined volume. If the trial
tomogram is satisfactory, click “Finish Join” button.
4
Notes
1. The gold particles serve as markers, termed fi ducials, for accu-
rate image alignment. Positions of each particle in an image
series are automatically tracked to generate its trajectory. In
our experience, 15 nm particles are more easily tracked than
smaller gold particles.
2. The IMOD software package has eTomo, a graphic user inter-
face, for generating tomograms from tilt image series, which
runs on Windows, Mac OS X, and Linux computers. The pro-
gram runs more smoothly on computers with large memory
(8-32 GB) capacity, to which entire tilting series or tomo-
grams can be uploaded.
3. For a detailed explanation about sample preparation and serial
sectioning,
see
ref. [
26
].
4. Image contrast is reduced in the intermediate voltage TEM,
and locating specifi c areas in the ribbon of serial sections can
be diffi cult. The standard TEM previewing also “bakes” the
sections, preventing drift, when tilted images are collected in
the intermediate voltage TEM.
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