2.3.2 High Pressure
Freezing of Plant Tissues
The size of plant cells (ranging from 15 to 50 μm), their large
aqueous vacuoles and their pecto-cellulosic cell wall make them
challenging to cryo-preserve. Optimal vitrification of whole plant
cells can only be achieved by HPF and the samples must be less than
200 μm thick. In practice, any plant sample that can fit in the freezer
carriers used for HPF can be vitrified. However, experience shows
that some freeze better than others. In general, the following
materials are required for a HPF session of plant tissues:
1. Dextran cryoprotectant solution: 25% and 40% solutions are
needed for the freezing of Arabidopsis thaliana root tips and
cultured cells. 5 mL of each is sufficient.
2. Benchtop mini-centrifuge.
3. Dissection stereomicroscope.
4. Single edged carbon steel razor blades (EMS).
5. X-Acto knife set (for decapsulating tool) (EMS) or hemostatic
forceps (EMS).
6. Tooth picks.
7. Various EM grade precision forceps (EMS style 2, 5, 5Â, and
Dumont style 7).
8. Freezing carriers: two types can be used for cryosectioning
of plant tissues: the 200 μm deep brass carriers described in
more detail elsewhere [35] or the 100 μm carriers composed
of Type A gold-plated copper dishes (3 Â 0.50 mm with
0.1/0.2 mm cavities, Technotrade International) and Type B
aluminum hats (3 Â 0.50 mm with a 0.3 mm cavity, Technotrade International).
9. Liquid nitrogen.
10. High Pressure Freezer: Bal-Tec HPM 010.
After preparing the above-mentioned materials, the process of HPF can be performed as follows:
2.3.3 Dissection
and Freezing of Plant
Tissues
1. Under a binocular loupe, put a drop of water on a glass slide.
2. Mount the bottom part of the type A carrier onto the Bal-tec
HPM 010 freezing rod.
3. With a sharp razor blade, dissect the plant tissue of interest. The
only requirement is that it is small enough to fit in the freezing
planchette.
4. Quickly transfer the freshly dissected tissue to the type A carrier
using the appropriate tools (forceps or toothpicks) and add a
few μL of 25% Dextran until the liquid forms a shallow dome
over the surface of the carrier.
Methods in Cryo-Electron Tomography
93
Freezing of Plant Tissues
The size of plant cells (ranging from 15 to 50 μm), their large
aqueous vacuoles and their pecto-cellulosic cell wall make them
challenging to cryo-preserve. Optimal vitrification of whole plant
cells can only be achieved by HPF and the samples must be less than
200 μm thick. In practice, any plant sample that can fit in the freezer
carriers used for HPF can be vitrified. However, experience shows
that some freeze better than others. In general, the following
materials are required for a HPF session of plant tissues:
1. Dextran cryoprotectant solution: 25% and 40% solutions are
needed for the freezing of Arabidopsis thaliana root tips and
cultured cells. 5 mL of each is sufficient.
2. Benchtop mini-centrifuge.
3. Dissection stereomicroscope.
4. Single edged carbon steel razor blades (EMS).
5. X-Acto knife set (for decapsulating tool) (EMS) or hemostatic
forceps (EMS).
6. Tooth picks.
7. Various EM grade precision forceps (EMS style 2, 5, 5Â, and
Dumont style 7).
8. Freezing carriers: two types can be used for cryosectioning
of plant tissues: the 200 μm deep brass carriers described in
more detail elsewhere [35] or the 100 μm carriers composed
of Type A gold-plated copper dishes (3 Â 0.50 mm with
0.1/0.2 mm cavities, Technotrade International) and Type B
aluminum hats (3 Â 0.50 mm with a 0.3 mm cavity, Technotrade International).
9. Liquid nitrogen.
10. High Pressure Freezer: Bal-Tec HPM 010.
After preparing the above-mentioned materials, the process of HPF can be performed as follows:
2.3.3 Dissection
and Freezing of Plant
Tissues
1. Under a binocular loupe, put a drop of water on a glass slide.
2. Mount the bottom part of the type A carrier onto the Bal-tec
HPM 010 freezing rod.
3. With a sharp razor blade, dissect the plant tissue of interest. The
only requirement is that it is small enough to fit in the freezing
planchette.
4. Quickly transfer the freshly dissected tissue to the type A carrier
using the appropriate tools (forceps or toothpicks) and add a
few μL of 25% Dextran until the liquid forms a shallow dome
over the surface of the carrier.
Methods in Cryo-Electron Tomography
93