# Methods to determine the size of an object in microns

### From OpenWetWare

(Difference between revisions)

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<math>\mbox{Pixel size} = \frac{6.45um}{\mbox{total magnification}}</math> | <math>\mbox{Pixel size} = \frac{6.45um}{\mbox{total magnification}}</math> | ||

- | + | 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:

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.