Biology Reference
In-Depth Information
Chapter 13
Reconstructing Plant Cells in 3D by Serial
Section Electron Tomography
Kiminori Toyooka and Byung-Ho Kang
Abstract
In micrographs acquired with a transmission electron microscope, 3-dimensional (3D) objects are superim-
posed onto a 2D screen. This reduction in dimension necessarily leads to a degradation of image resolution.
To overcome this problem, 3D microscopy techniques, such as tomography and single particle analysis,
have been developed. Tomography has been used to visualize cells in 3D, and single particle analysis has
been used to investigate macromolecules and viral particles. In this chapter we will describe how we have
collected tilting series micrographs from plant cells and how we have reconstructed the cellular volumes
using dual axis electron tomography.
Key words
Electron tomography, IMOD software package, Serial section, eTomo, 3D reconstruction
1
Introduction
Micrographs acquired by transmission electron microscopy (TEM)
are projections of 3D volumes onto a 2D screen. The reduction of
dimensionality inherently causes a loss of spatial information
because varying densities from cellular components are superim-
posed along the direction of projection [
1
]. TEM samples are
sliced into 60-80 nm thin sections. By preparing serial thin sec-
tions, it is possible to obtain 3D information about cellular struc-
tures [
2
-
4
]. However, the resolution of the TEM in the direction
of the electron beam is, at best, limited to the section thickness. In
theory, image resolution could be improved by preparing thinner
sections, but reliably acquiring sections thinner than 40 nm from
resin embedded samples is challenging, even for skilled ultrami-
crotomy specialists.
Employing a tomography algorithm can partially retrieve the
information by reduction in dimension. Tomography is a technique
for generating images representing 3D structures of an object from
a set of 2D-projected images of the object taken with penetrating
radiation at many angles. Tomography is most commonly used in
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