Fine tuning positioning accuracy

The procedure for using a grid to derive the number of microns per pixel is described here. This is sufficient to get the system working pretty well. You will likely need to conduct this measurement for each image resolution that you plan on using. The number of microns per pixel can vary by about 1% between different image sizes.

After conducting this procedure (and aligning the stages) you may still see errors in tile placement. These errors should be consistent and larger for larger tiles. One way of eliminating them is to tweak the number of microns per pixel in the recipe file and re-stitch with chessboard stitching. This way it's possible to get near-perfect stitching. Then tweak the objective resolution in ScanImage to reflect this new value.

The following image shows the effect of fine-tuning stitching accuracy. On the left is the stitching accuracy using the number of microns per pixel obtained by measuring with a grid (1.316 microns per pixel) on the right is the with this number manually over-ridden to 1.323 microns per pixel. So that's a tiny difference: only 7 nm per pixel! In this case the images shown have been downsampled such that 1 pixel is 2.632 microns.

Once you have the more accurate number for the microns per pixel, you could try running Grid2MicsPerPixel using a grid pitch that produces this number. For example, our grid will reliably produce 1.316 microns per pixel using a certain set of scan settings. However, we found stitching accuracy was better at 1.323. So we can instead do Grid2MicsPerPixel(ImageData,'GridPitch',25.125), which produces a microns per pixel value of 1.322. From now on, using this tweaked value for the number of microns per pixel (e.g. when measuring for different objectives or image resolutions) should produce a slightly more accurate value.