Basic calibrating procedures

BakingTray performs a tile scan over the sample. The individual tiles are later assembled into a single image by StitchIt. In order to place the tiles accurately and produce a good quality stitched image you will need to ensure that the following are true.

• The X and Y scanners must displace the beam along orthogonal directions and the beam must leave the scanhead at 90 degrees to the angle with which it entered. This alignment may need to be done with the scanhead removed and sitting on an optical table. The grid lines on a uniform grid should be orthogonal when imaged. If not, the scanner axes aren't orthogonal. If you can't correct for this, it can be done in software, later. If you're in this position, just align one motion axes with one scan axis and leave the other to be what it is.
• The X and Y stages must displace the sample along orthogonal axes.
• The X scanner should move the beam along the direction of motion of the X stage and the Y scanner along the direction of motion of the Y stage. Rotating the stage assembly about its centre should acheive this.
• The scanning plane (imaging plane) should be parallel with the plane of motion of the X and Y stages. You will need to mount the stages on three points of contact with at least two being height adjustable. Image a thin fluorescein layer under a coverslip. Assuming the image plane is not curved, the whole FOV fluorescein should snap into view at once as you focus through it. If there is field curvature you will likely see the centre appear first and as you move the objective further down you will see a donut. If the stages are aligned with the image plane, the donut should be symmetric.
• You should calibrate the number of microns per pixel.
• The blade must be parallel to the Y stage such that the surface of the sample remains the same distance from the objective as it is translated in Y. Adjust the blade angle to achieve this. This adjustment may need to be repeated periodically depending on your vibrotome.

The above alignments should be made in the order listed above. Once done, **ensure that no out of bounds motions are possible **by setting the min/max position values for each axis. This is important for ensuring the hardware can't be damaged.

See also

• Setting up stages
• Fine tuning positioning accuracy