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Principles & Protocols

Protocols - Grid Preparation

Principles

Grid selection

Deciding the grids required for a particular EM application is an important but easy decision. There seems to be an overwhelming variety of possibilities. The camp can be divided in two: tissue samples for tomography or imaging of isolated proteins. Usually, proteins are imaged using 300 or 400 square mesh grids, while tissue sections can be placed on slotted grids for improved tilting capabilities.

Shiny vs. Dull side

There is no solid evidence that using either side is better, therefore as long as you keep track of which side you use, everything should be fine. The shiny side tends to be smoother, while the dull side is more like sand paper. There are grids that have one side flashed with Rh, allowing a very easy identification of the side where the sample is placed.

Mesh Size (lines per inch)

There is a great variety of mesh shapes and sizes for grids. The most common selections are as follows:

400 mesh - recommended for support films. Protein imaging applications, for example.
200 mesh - A good compromise for tomography.
Slotted grids - For tomography and serial sections. Need to be coated with a plastic film.

Material

Grids can be made from a wide variety of materials. Most grids are metallic, but one can also find grids made of nylon, silicone or some other materials. We only use metallic grids.

Common

Copper - Most common - inexpensive, conductive, beam stable. Relatively thin grid bars.
Copper/Palladium - Palladium adds rigidity to the grid, but makes it more expensive.
Copper/Rhodium - non-tarnishing, reduction in bar thickness. Recognizable sides.

Less common

Gold or Platinum - non-reactive, expensive, beam stable. Easily bent (especially gold)
Molybdenum - Expensive, Coefficient of contraction similar to carbon. Quite rigid.
Nickel - Used mainly for silver enhancement.
Beryllium - Minimize background radiation. Mainly used for material science.
Titanium - Extra stiff.

Mesh types

Square Mesh. These are by far the most common. There are many brands with different peculiarities each.
Finder Grids - These are particularly useful when one needs to go back to collect data from a known area.
Hexagonal Mesh - Used for tomography more than anything, but can be used for the same applications as the square grids.
Slot Grids - For tomography. More fragile than others since there is less support

Support films

The support film is probably the most important consideration for the grid selection. Desired properties of the support film are integrity, stiffness and transparency to electrons. The most common support film used in biological electron microscopy is carbon, due to the fact that it is somewhat conductive, produces low scattering and is very stable under the electron beam. However, carbon films also tend to be fragile and for large areas, something extra has to be done. The most common support films are made as follows:

With CarbonCoating

Formvar and Carbon - A formvar film is made on a glass slide. Then the film is floated in water and grids placed on it. The grids are then picked up with paper from the water surface and carbon is evaporated on top of the formvar.
Carbon only - In this case, carbon is evaporated on a freshly cleaved mica sheet. The carbon film is then floated off of the mica and lowered onto submerged grids placed on a piece of filter paper.
Nitrocellulose and Carbon - Similar to the formvar-carbon film. A drop of nitrocellulose is placed on a crystallization dish, forming thus a thin plastic film. Grids are placed on the surface, picked up with paper and coated with carbon.

Carbon with holes - These films are used commonly for frozen hydrated samples. There are several ways to prepare them, or they can be purchased from vendors (Quantifoil, C-flat). Commercially available grids have regular holes.
- Quantifoil - commercially available. Carbon is ~200 A thick, sturdy, but generally not suitable for imaging. A thin coating of carbon can be floated on these grids if the sample always settles on carbon and not the holes.
- C-flat - commercially available. Thinner than Quantifoil, but more fragile. May be able to image over this carbon
- Lacey Carbon - Home-made plastic films which are carbon coated, then plastic is dissolved away. These grids can be used to support tissue sections, or can serve as support for a very thin carbon film and used also for single particle analysis.

Uncommon coatings

- Beryllium
- Silicone monoxide - low background contrast, stable under beam, more hydrophilic than carbon

-- KdDerr - 28 Aug 2007