Methods to determine the size of an object in microns

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Mathematically

Pixels on our CCD are 6.45um. The size (in microns) of one pixel in an image will depend on how much the image was magnified before reaching the CCD:

\mbox{Pixel size} = \frac{6.45um}{\mbox{total magnification}}

This formula gives us:

  • 60x  : 0.1075 um/pixel or 9.30 pixels/um
  • 40x  : 0.1613 um/pixel or 6.21 pixels/um
  • 20x  : 0.3225 um/pixel or 3.10 pixels/um
  • 10x  : 0.645 um/pixel or 1.55 pixel/um

Important notes:

  • We assumed here that the magnifier is at position 1x (not 1.5x). If it is at 1.5x, total magnification must be multiplied by 1.5.
  • We assumed here that the bin size is 1x1. If it is, say, 2x2, the size of a pixel will be double.

Calibration slide

Use a calibration slide which has a grid with known line-to-line spacing. I borrowed such a slide from Peter Sorger's lab. Results will be posted here soon.

xy-motorized stage

Put a sample on a slide or pad (grain of dust, cells, sphere). Record the position of the sample. Use IPLab to tell the stage to move a certain distance in um. Determine the distance (in pixels) between the sample's former position and its new position.

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