2 Materials
2.1 Grid Specimen
Preparation
1. Crystal sample suspension.
2. Pipettes with tips (1000, 200, 20, and 2 μL).
3. High-grade mica sheets.
4. Graphite rods for carbon evaporation.
5. Carbon evaporator with an oil-free high vacuum.
6. Embedding buffer with sugar only (see Note 1).
7. Sample mother buffer for dilution.
8. Small reservoirs for holding the buffers (see Note 2).
9. Molybdenum EM grids (300 meshes) (see Note 3).
10. Whatman #2 filter paper.
11. A 4–5 mm platinum wire loop.
12. Grid boxes for storing the frozen grids.
13. Anti-capillary self-closing tweezers.
14. Liquid nitrogen.
2.2 Frozen Grid
Specimen Transfer
1. A side-entry cryo-specimen holder for the TEM with a sideentry specimen stage (see Note 4).
2. Liquid nitrogen.
3. Transferring tools (a clip-ring holder and a tweezer).
3 Methods
3.1 Sample
Preparation
Before preparing a frozen specimen, screening good crystal samples
and optimizing their concentrations using negative-stain EM are
strongly recommended. The diffraction spots on the image power
spectrum are a good indication of the crystal packing. A detailed
protocol for preparing negatively stained samples can be found in
the previous study [30]. Once the 2D crystal sample is screened and
ready for electron imaging or diffraction, one will prepare the grid
specimen with a sugar-embedded 2D crystal on a flat carbon support. The specimen can be readily frozen into liquid nitrogen. The
first part of the protocol will describe the procedure for preparing
the carbon films. The support carbon film is required to be flat and
intact in an appropriate thickness. The second part of the protocol
will cover the procedures of back-injection method and carbonsandwich method, which embeds the crystal sample with the chosen sugar on a flat carbon support.
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Ka-Yi Chan et al.
2.1 Grid Specimen
Preparation
1. Crystal sample suspension.
2. Pipettes with tips (1000, 200, 20, and 2 μL).
3. High-grade mica sheets.
4. Graphite rods for carbon evaporation.
5. Carbon evaporator with an oil-free high vacuum.
6. Embedding buffer with sugar only (see Note 1).
7. Sample mother buffer for dilution.
8. Small reservoirs for holding the buffers (see Note 2).
9. Molybdenum EM grids (300 meshes) (see Note 3).
10. Whatman #2 filter paper.
11. A 4–5 mm platinum wire loop.
12. Grid boxes for storing the frozen grids.
13. Anti-capillary self-closing tweezers.
14. Liquid nitrogen.
2.2 Frozen Grid
Specimen Transfer
1. A side-entry cryo-specimen holder for the TEM with a sideentry specimen stage (see Note 4).
2. Liquid nitrogen.
3. Transferring tools (a clip-ring holder and a tweezer).
3 Methods
3.1 Sample
Preparation
Before preparing a frozen specimen, screening good crystal samples
and optimizing their concentrations using negative-stain EM are
strongly recommended. The diffraction spots on the image power
spectrum are a good indication of the crystal packing. A detailed
protocol for preparing negatively stained samples can be found in
the previous study [30]. Once the 2D crystal sample is screened and
ready for electron imaging or diffraction, one will prepare the grid
specimen with a sugar-embedded 2D crystal on a flat carbon support. The specimen can be readily frozen into liquid nitrogen. The
first part of the protocol will describe the procedure for preparing
the carbon films. The support carbon film is required to be flat and
intact in an appropriate thickness. The second part of the protocol
will cover the procedures of back-injection method and carbonsandwich method, which embeds the crystal sample with the chosen sugar on a flat carbon support.
252
Ka-Yi Chan et al.