Reconstructing Plant Cells in 3D by Serial Section Electron Tomography - Plant Cell Morphogenesis: Methods and Protocols - page 156

limitations of this chapter, the details for modeling organelles and
for 3D quantitative analysis of cellular volumes will be discussed
elsewhere. There are also many great reviews about 3D analysis
using ET [ 22 - 25 ].

2 Materials and Equipment

1. UC-7 (Leica Microsystems, Buffalo Grove, IL) or a similar
ultramicrotome.
2. Formvar-coated EM grids (copper or gold slot grids,
1 mm × 2 mm slot size).
3. Diamond knife.
4. 2 % (w/v) uranyl acetate (UA) in 70 % methanol or 2 % aque-
ous UA and Reynolds' lead citrate solutions.
5. Unconjugated 15 nm gold particles (Catalog number: 15704-
20, Ted Pella, Redding, CA; see Note 1 ).

2.1 Ultramicrotomy
and Grid Preparation

1. Intermediate voltage transmission electron microscopes, such
as TF20/TF30 (FEI, Hillsboro, OR) or JEM-2200FS/FEM-
3100F (JEOL, Peabody, MA), equipped with a motorized
eucentric goniometer for collecting tilting series.
2. Grid holder for dual axis tomography, like the Fischione Model
2040 holder (Fischione, Export, PA).

2.2 Acquisition
of Tilting Series

1. Personal computer running a Linux or Unix operating system
or a Windows computer with a Linux emulator such as Cygwin
( see Note 2 ).
2. Monitor resolution at least 1,600 × 1,200.
3. Mouse with 3 buttons (left, middle, and right).

2.3 3D
Reconstructions

3 Methods

1. Trim blocks into trapezoids which contain the tissues/cells of
interest. Ensure that the top and bottom sides of the trape-
zoids are parallel to keep the serial section ribbon straight.
2. Collect thin sections (60-70 nm) from the samples to locate
the desired regions for further analysis by ET and to assess
preservation quality in those regions.
3. Once regions for ET have been chosen, collect ribbons of
200-300 nm thick serial sections on Formvar-coated slot grids
(2 mm × 1 mm; see Note 3 ).
4. Stain the thick section ribbons with UA and Reynolds' lead
citrate solutions. If needed, perform immunogold labeling
prior to heavy metal staining.

3.1 Serial Sectioning
of Resin Blocks and
Preparation of Grids
for ET