Grids for Calibration¶

The grid used for calibration should have reasonable contrast and features at multiple
scale. A frozen grid is generally not convenient for the purpose since the contrast is low and
can drift easily.

Commercial cross grating replica with latex beads are pretty good for calibration purpose except that sometimes no area is free of support film, making bright image collection difficult.

Large protein complex negatively stained on a 200-400 mesh em grid with Quantifoil, C-falt, or home-made holey carbon support is ideal. You can even use one that is abandoned after a cryo run, just negatively stain it to improve the contrast. From our experience, small protein negatively stained on continuous carbon support often gives low contrast at intermediate magnification and therefore not ideal. If your Quantifoil or C-flat grid is too clean, there may be a problem of false peaks from the lattice. In this case, you
can add some gold clusters such as Nanogold from Nanoprobe.

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