Biology Reference
In-Depth Information
modern medicine to produce image slices of specifi c body areas in
a patient. Electron tomography (ET) is an extension of TEM in
which the resolution of the TEM is enhanced by incorporating
tomography methods [ 5 ]. Images of a 200-300 nm thick resin sec-
tion are captured from a wide range of directions in a transmission
electron microscope in ET, and the images are subsequently pro-
jected back into a virtual 3D volume [ 6 , 7 ]. Images representing
1-4 nm thin slices, termed tomographic slices, can be extracted
from the virtual volume. Because tomographic slices are far thinner
than the resin sections, organelles and macromolecular complexes
in the slices could be examined at resolutions far better than by
conventional TEM especially in the z-direction. 3D views of sub-
cellular components can be constructed from the tomographic
slices. Furthermore, by joining tomograms of a cell from serial sec-
tions, ET can visualize relative large volumes of a cell in 3D [ 8 ].
With its improved resolution and 3D capability, ET has con-
tributed to upgrading our understanding of cellular structures. ET
is especially useful for investigating organelles with complicated
3D architectures. Examples of the published electron tomography
analyses include studies on the trans -Golgi network [ 9 ], the cell
plate [ 10 - 12 ], thylakoids in the chloroplast [ 13 , 14 ], endoplasmic
reticulum membranes [ 15 ], and multivesicular bodies [ 16 ]. It has
also enabled detailed structural characterizations of entities smaller
than the thickness of TEM sections, including transport vesicles
and ribosomes [ 17 - 19 ]. To overcome artifacts of conventional
chemical fi xation, cryofi xation methods for TEM, such as high-
pressure freezing, have been practiced since the 1980s [ 20 ].
Combined with high-pressure freezing, ET can capture short-lived
events in the cell in 3D at nanometer-level resolutions [ 21 ].
ET was historically a technique utilized by a handful of labs
with sophisticated transmission electron microscopes and high-
performance computers. With the rapid increase in computer
power and easy access to intermediate voltage transmission elec-
tron microscopes, ET has become more available to plant biolo-
gists, whose research has benefi tted from incorporating
high-resolution 3D imaging. In this chapter, we will describe how
we prepare TEM sections for ET and calculate tomograms from
tilt image stacks. We have used SerialEM for acquiring raw data
and IMOD for generating tomograms. Both programs are open-
source software freely available from The Boulder Laboratory for
3D Electron Microscopy of Cells at the University of Colorado
( http://bio3d.colorado.edu/SerialEM/ and http://bio3d.
colorado.edu/imod/ ) . We will describe operation of the eTomo
module of the IMOD package. An excellent tutorial is included in
the IMOD package (version 4.5.7), and we recommend that read-
ers consult the tutorial for detailed information. The Boulder
Laboratory for 3D Electron Microscopy of Cells has a YouTube
channel containing tutorial videos, including videos for eTomo
( http://www.youtube.com/bl3demc ) . Due to the page
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