# Methods to determine the size of an object in microns

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- | + | ==Mathematically== | |

- | + | 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: | ||

- | |||

- | + | ::'''Cell size (per pixel) = Physical length of a pixel on the CCD / total magnification''' | |

- | 60x : 0.1075 um/pixel or 9.30 pixels/um | + | |

- | 40x : 0.1613 um/pixel or 6.21 pixels/um | + | The physical length of a pixel on our CCD is 6.45um. So for the following magnification, this formula gives us: |

- | 20x : 0.3225 um/pixel or 3.10 pixels/um | + | |

- | 10x : 0.645 | + | *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? | ||

+ | |||

+ | [[Category:Protocol]] [[Category:Microscopy]] |

## Current revision

## Mathematically

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:

**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

- 13 squares gave 1204.26 pixels in length. 1204.26 pixels/(13 square * 9.9um/squares) =

- 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.

- Horizontal : 3 measurements of 8 squares gave 1106.2, 1105.2 and 1106.2 pixels in length. -->

## 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)

- Based on three samples I got

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?