Methods to determine the size of an object in microns

(Difference between revisions)
 Revision as of 13:42, 11 August 2005 (view source)← Previous diff Revision as of 13:54, 11 August 2005 (view source)Next diff → Line 5: Line 5: $\mbox{Pixel size} = \frac{6.45um}{\mbox{total magnification}}$ $\mbox{Pixel size} = \frac{6.45um}{\mbox{total magnification}}$ - Assuming that the magnifier is at position 1x (not 1.5x), this formula gives us: + This formula gives us: *60x  :  0.1075 um/pixel  or  9.30 pixels/um *60x  :  0.1075 um/pixel  or  9.30 pixels/um Line 11: Line 11: *20x  :  0.3225 um/pixel  or  3.10 pixels/um *20x  :  0.3225 um/pixel  or  3.10 pixels/um *10x  :  0.645  um/pixel  or  1.55 pixel/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.

Revision as of 13:54, 11 August 2005

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.