Pixel size calibration

First get the fluorescent calibration slide from the Sorger Lab. Then follow the following protocol which I got from Applied Precision (www.api.com), the makers of the calibration slide. The protocol was edited to fit our microscope setup. Obviously, the pixel-um correspondance will vary from one objective to the next.

1. Put the slide, with the metal piece containing the calibration grid facing downwards.
2. Using white light, find the grid.
3. Switch to IPLab and focus. Then switch to the lamp for fluorescence and move the filter to the FITC/GFP filter (as of 6/15/2005, filter #2)
4. Max image size, best camera speed, magnification slider at 1X.
5. Focus on the silicon target grid (9.9 μm/square).
6. Align the grid image to vertical/horizontal. There are potentially three ways of doing this:
   1. Rotate the camera. I'll try to find out if there is a way of rotating it by very small angles;
   2. Manually move the slide. I'm not sure if this is precise enough. Also, there might be a danger of scratching the optics.
   3. Rotate the image using the software. This is the equivalent of measuring along a diagonal.
7. Draw a line across the screen from a point on the top left square to the same point on the top right square. If a delta of more than four (4) in the vertical is measured re-align the slide.
8. Repeat this process at the middle and bottom.
9. Count and record the number of grid elements and record distance in Pixels.
10. Repeat the above three steps in the vertical direction.
11. Calculate Size:

Pixel Size (um) =

___________________9.9um/box__________________

Σ (Measured pixels)/(Number of grids per measurement)