Biology Reference
In-Depth Information
Chapter 12
High-Pressure Freezing and Low-Temperature Processing
of Plant Tissue Samples for Electron Microscopy
Ichirou Karahara and Byung-Ho Kang
Abstract
Use of electron tomography methods improves image resolution of transmission electron microscopy
especially in the
z
-direction, enabling determination of complicated 3D structures of organelles and cyto-
skeleton arrays. The increase in resolution necessitates preservation of cellular structures close to the native
states with minimum artifacts. High-pressure freezing (HPF) that immobilizes molecules in the cell instan-
taneously has been used to avoid damages caused by convention chemical fi xation. Despite the advantages
of HPF, cells could still be damaged during dissection prior to HPF. Therefore, it is critical to isolate cells/
tissues of interest quickly and carefully. The samples frozen by HPF are often processed by freeze substitu-
tion (FS), and FS should be carried out under appropriate conditions. Here we describe dissection, HPF,
and FS methods that we have utilized to prepare plant samples for electron tomography/immuno-electron
microscopy.
Key words
High-pressure freezing, Freeze substitution, Microfi lament, Microtubule, Coated pit and
vesicle, Onion, Maize endosperm
1
Introduction
Slow penetration of chemical fi xatives has been shown to cause
artifactual morphological changes at the cellular level that are read-
ily detected in electron micrographs [
1
]. To overcome this prob-
lem, cryofi xation techniques, such as cold metal block freezing,
plunge freezing, spray freezing, propane jet freezing, and high-
pressure freezing [
2
], have been developed to immobilize cellular
components at rates much faster than those of chemical fi xatives
[
3
]. Among these cryofi xation techniques, high-pressure freezing
(HPF) is especially effective for thicker samples, such as plant tis-
sues. HPF can freeze samples as thick as 100-300
m, which is far
thicker than the other techniques can freeze [
3
-
5
], without the
tissue-damaging formation of ice crystals. A combination of cryo-
fi xation and freeze substitution (FS) has been used to visualize sub-
cellular structures in plant cells by electron microscopy (EM;
6
,
7
).
μ
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