2.4 Cryosectioning
of Plant Tissues
Cryosectioning consists of two steps: (a) trimming, where the
block-face is carved out [35], and (b) cryosection collection in
which the block-face is sectioned and the resulting sections
and/or ribbons are collected on EM grids. The required material
in order to perform cryosectioning followed by Cryo-Electron
Microscopy of Frozen Hydrated Sections (CEMOVIS) has been
previously addressed thoroughly [35, 36]. In general, the following
materials are required for a cryosectioning session of plant tissues:
1. 200 mesh carbon-coated copper finder grids (Ted Pella Inc) or
copper 200 mesh R2/2 NH2 Quantifoil grids.
2. Trimming knives: Trim 20 and Trim 45 (Diatom).
3. Section collection knives: Cryo-dry 25
, 35
, or 45
(see Note
13) (Diatom).
4. Hair (eyelash, blonde baby, or Dalmatian) glued to the tip of a
wooden stick for cryosection manipulation.
5. Static line ionizer (Diatom).
6. Leica Ultracut UCT with Leica EMFCS cryo-chamber (Leica
Microsystems) or equivalent cryo-ultramicrotome and associated microtools (pen-like pressing tool, freezing carrier
holders, etc.).
7. Micromanipulator (for more stable ribbon collection).
8. Large amounts of liquid nitrogen.
9. Hair dryer or heating bench to warm and dry the tools.
10. Various EM grade precision forceps (EMS style 2, 5, 5Â, and
Dumont style 7).
After preparing the above-mentioned materials, the process of cryosectioning can be performed as follows:
2.4.1 Preparation
1. Cool down the cryo-ultramicrotome to À165
C (see Note
14).
2. Mount the carrier in the cryo-ultramicrotome (see Note 15).
3. Decapsulate the lid of the carrier using a home-made tool
(Fig. 3c).
4. Install and cool the knives, setting them as far as possible from
the specimen holder to prevent accidental heat transfer.
2.4.2 Trimming
1. Light the system from below in order to highlight the knife-tosample distance.
2. Bring the knife close to the sample, using the command panel
and/or sliding the knife platform forward. Use the command
panel only in the final stages of approach.
96
Mohammed Kaplan et al.
of Plant Tissues
Cryosectioning consists of two steps: (a) trimming, where the
block-face is carved out [35], and (b) cryosection collection in
which the block-face is sectioned and the resulting sections
and/or ribbons are collected on EM grids. The required material
in order to perform cryosectioning followed by Cryo-Electron
Microscopy of Frozen Hydrated Sections (CEMOVIS) has been
previously addressed thoroughly [35, 36]. In general, the following
materials are required for a cryosectioning session of plant tissues:
1. 200 mesh carbon-coated copper finder grids (Ted Pella Inc) or
copper 200 mesh R2/2 NH2 Quantifoil grids.
2. Trimming knives: Trim 20 and Trim 45 (Diatom).
3. Section collection knives: Cryo-dry 25
, 35
, or 45
(see Note
13) (Diatom).
4. Hair (eyelash, blonde baby, or Dalmatian) glued to the tip of a
wooden stick for cryosection manipulation.
5. Static line ionizer (Diatom).
6. Leica Ultracut UCT with Leica EMFCS cryo-chamber (Leica
Microsystems) or equivalent cryo-ultramicrotome and associated microtools (pen-like pressing tool, freezing carrier
holders, etc.).
7. Micromanipulator (for more stable ribbon collection).
8. Large amounts of liquid nitrogen.
9. Hair dryer or heating bench to warm and dry the tools.
10. Various EM grade precision forceps (EMS style 2, 5, 5Â, and
Dumont style 7).
After preparing the above-mentioned materials, the process of cryosectioning can be performed as follows:
2.4.1 Preparation
1. Cool down the cryo-ultramicrotome to À165
C (see Note
14).
2. Mount the carrier in the cryo-ultramicrotome (see Note 15).
3. Decapsulate the lid of the carrier using a home-made tool
(Fig. 3c).
4. Install and cool the knives, setting them as far as possible from
the specimen holder to prevent accidental heat transfer.
2.4.2 Trimming
1. Light the system from below in order to highlight the knife-tosample distance.
2. Bring the knife close to the sample, using the command panel
and/or sliding the knife platform forward. Use the command
panel only in the final stages of approach.
96
Mohammed Kaplan et al.