# Methods to determine the size of an object in microns

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 Revision as of 13:54, 11 August 2005 (view source)← Previous diff Current revision (13:38, 5 August 2008) (view source)m (→Mathematically) (20 intermediate revisions not shown.) Line 1: Line 1: - ===Mathematically=== + ==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: + Th number of pixels the image of a cell takes on the CCD camera depends on: + # the magnification used, and on + # the physical size of the pixels on the CCD camera (in our case, it is 6.45um) + In order to relate the size of a cell in pixels to its size in um, use the following formula: - $\mbox{Pixel size} = \frac{6.45um}{\mbox{total magnification}}$ - This formula gives us: + ::'''Cell size (per pixel) = Physical length of a pixel on the CCD / total magnification''' + + + The physical length of a pixel on our CCD is 6.45um. So for the following magnification, this formula gives us: + + *100x :  0.0645 um/pixel  or  '''15.50''' pixels/um + *90x  :  0.0717 um/pixel  or  '''13.95''' pixels/um + *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 - *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: 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 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. * We assumed here that the bin size is 1x1. If it is, say, 2x2, the size of a pixel will be double. + * 90x corresponds to 60x objective with 1.5x intermediate magnification + + ==Calibration slide== + + Use a calibration slide which has a grid with known line-to-line spacing (9.9um). Align the slide so that the grid lines are parallel/perpendicular to the x and y axes. + I borrowed such a slide from Peter Sorger's lab. + + * 60x DIC Plan Apo + ** 13 squares gave 1204.26 pixels in length. 1204.26 pixels/(13 square * 9.9um/squares) =  '''9.35''' pixels/um + ** 14 squares gave 1297.30 pixels in length. 1297.30 pixels/(14 squares * 9.9um/squares) = '''9.36''' pixels/um + + * 60x DIC Plan Apo with 1.5x intermediate magnification: + ** Horizontal : 3 measurements of 8 squares gave 1106.2, 1105.2 and 1106.2 pixels in length.  --> ''13.96''' pixels/um  (Std error = 0.00) + ** Vertical: 3 measurements of 7 squares gave 968.2, 968.2, 968.2 pixels in length --> ''13.97'' pixels/um (Std error = 0.00) + ** 60x Ph Plan Apo with 1.5x intermediate magnification gave virtually the same results. + + ==xy-motorized stage== + + Put a sample on a slide or pad (cells, sphere) or find a grain of dust on the slide or pad. 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. + + * 60x + **Based on three samples I got '''9.22''' +/- 0.13 pixels/um  (error represents the standard deviation) - ===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=== + To test the calculated um/pixel for a given objective, one could do the following: Get the calibration slide. Take a picture. Knowing how wide (in pixels) our CCD camera is, determine the width (in um) of the CCD image. Move the stage by that distance. Take another picture. Put the two pictures next to one-another. Do the grids line-up? - 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. + [[Category:Protocol]] [[Category:Microscopy]]

## Mathematically

Th number of pixels the image of a cell takes on the CCD camera depends on:

1. the magnification used, and on
2. the physical size of the pixels on the CCD camera (in our case, it is 6.45um)

In order to relate the size of a cell in pixels to its size in um, use the following formula:

Cell size (per pixel) = Physical length of a pixel on the CCD / total magnification

The physical length of a pixel on our CCD is 6.45um. So for the following magnification, this formula gives us:

• 100x : 0.0645 um/pixel or 15.50 pixels/um
• 90x  : 0.0717 um/pixel or 13.95 pixels/um
• 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.
• 90x corresponds to 60x objective with 1.5x intermediate magnification

## Calibration slide

Use a calibration slide which has a grid with known line-to-line spacing (9.9um). Align the slide so that the grid lines are parallel/perpendicular to the x and y axes. I borrowed such a slide from Peter Sorger's lab.

• 60x DIC Plan Apo
• 13 squares gave 1204.26 pixels in length. 1204.26 pixels/(13 square * 9.9um/squares) = 9.35 pixels/um
• 14 squares gave 1297.30 pixels in length. 1297.30 pixels/(14 squares * 9.9um/squares) = 9.36 pixels/um
• 60x DIC Plan Apo with 1.5x intermediate magnification:
• Horizontal : 3 measurements of 8 squares gave 1106.2, 1105.2 and 1106.2 pixels in length. --> 13.96' pixels/um (Std error = 0.00)
• Vertical: 3 measurements of 7 squares gave 968.2, 968.2, 968.2 pixels in length --> 13.97 pixels/um (Std error = 0.00)
• 60x Ph Plan Apo with 1.5x intermediate magnification gave virtually the same results.

## xy-motorized stage

Put a sample on a slide or pad (cells, sphere) or find a grain of dust on the slide or pad. 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.

• 60x
• Based on three samples I got 9.22 +/- 0.13 pixels/um (error represents the standard deviation)

To test the calculated um/pixel for a given objective, one could do the following: Get the calibration slide. Take a picture. Knowing how wide (in pixels) our CCD camera is, determine the width (in um) of the CCD image. Move the stage by that distance. Take another picture. Put the two pictures next to one-another. Do the grids line-up?