Grid prep

This page will walk you through the equipment and methodology to create plastic-coated grids suitable for carbon coating grids to be used in transmission electron microscopy. Sample links are provided for the more esoteric components. the Cianfrocco lab does not endorse any particular vendor, this is just what we have on hand. Reasonable substitutions will work just fine.

Required equipment and materials

You will need

1) A liquid-tight vessel with a surface area ~ 175 cm² (pictured 75 x 150 mm Pyrex crystallizing dish)

2) Colloidion in 2% amyl acetate

3) 400 Mesh copper grids (or similar)

4) Whatman filter paper or other thick, pliable sheet (for manipulating grids)

5) High precision self-closing tweezers for EM (or similar, to preference)

6) Pasteur pipette, broken off near the funnel, with bulb

7) Printer paper

8) Standard glass or plastic petri dish with lid

Not pictured - Milli-Q water, kimwipes, razor blade/scalpel/sharp tweezers.

Methodology

Find an out-of-the-way spot in your lab space to perform this procedure. You want to minimize exposure to air flow, which will deposit dust and other contaminants on your grids as they incubate. Perform this procedure the afternoon prior to carbon coating.

1) Fill the retention vessel (1) with milli-Q water, all the way full until there is a convex liquid surface on top

2) Using strong light, check the surface reflection for dust particles. Fold up a kimwipe lengthwise and use it like a windhield wiper blade to drag across the surface, removing any dust particles. Top off the vessel if necessary.

3) Using a pasteur pipette with a thick opening (6), deposit one drop of Colloidion on the center of the water surface. If it touches the edge of the vessel, scrape it off and reapply

4) Dump untreated grids (3) onto Whatman paper (4), as many as you intend to treat (one dish can hold up to 100, or 1 full pack of standard size)

5) Using microtweezers, pick up grids by their edge and place them on top of the Colloidion film.

    • The grids are not directional. Either side can be treated, but the same side should be used to ensure consistency. The Cianfrocco lab prefer to place the darker side down for carbon coating.
    • Be very careful not to puncture the film. If the grids touch water, they are ruined.
    • If you put a grid down in the wrong orientation, do not try to rescue it. Leave it as-is and either discard it later or make a note which side is "active"
    • Place them close together, but not touching. The pattern and size will need to fit into a standard petri dish (8)

6) Cut the printer paper to the size and shape of your grid pattern, but smaller than the area of the Colloidion. Gently place it on top of the grids. Do not let the paper touch the water edge, and be very careful not to puncture the film. If you do, your grids are ruined. Go to 1, do not pass Go.

7) Let the paper sit on top of the grids/Colloidion overnight (room temperature, away from traffic).

8) Using a blade or sharp tweezers, "saw" through the Colloidion, close to the paper. Gently lift it out (grids should adhere to paper), and place it grid-side-up in a petri dish

9) Dry grids (overnight on benchtop, or put them in a dessicator/vaccuum chamber until dry).


These grids are now ready to undergo carbon coating.