To calibrate an existing image, use the Image > Calibrate Image... command. The correct calibration is extremely important for many functions. For instance, only when the image's X/Y-calibration is correct, does the scale bar for an image show correct values.
See also
Performing an X/Y-calibration on the basis of a known linear distance
The following step-by-step instruction describes the subsequent X/Y-calibration of an image that has not been, or has not been correctly, calibrated.
Prerequisite
The image you want to calibrate must contain a linear distance that you know the exact length of.
1.Load the image.
2.Use the Image > Calibrate Image... command.
3.Select the Interactive calibration option.
4.The Calibrate Image > XY-Calibration dialog box will then contain the Set reference distance group.
4.Select the fixed check box.
5.In this case you only have to define one linear distance on the image. From the length of this distance the calibration data for the X- and Y-directions can be unambiguously calculated.
6.If the fixed check box isn't selected, you have to enter two different linear distances on the image.
5.Select the appropriate X/Y-ratio for the camera with which the image is to be acquired. A camera normally has square pixels. In this case, enter the value 1 in the X/Y ratio field.
6.Click the Set Reference Distance button.
7.The mouse pointer will appear in the image window.
7.Use your mouse pointer to determine the beginning and end point of the known reference distance.
8.Click the right mouse button and select Confirm Input 
.
9.The Set Reference Distance dialog box opens.
9.Select an appropriate unit. To do so, click this button 
and select the basic unit and the magnitude you want from the menu.
You can here also select the basis unit in (inch) for the image calibration.
10.In the Distance field, enter the linear distance that you want to use for the calibration.
11.Click the OK button to confirm the calibration.
12.You will now see the calibration data resulting from the calibration you have just performed in the Calibration group. The calibration data responds to the actual size of a pixel when using the current microscope magnification.
12.Close the Calibrate Image dialog box with OK.
13.To double-check, use the [Shift + F4] shortcut to show the scale bar with the new X/Y-calibration.
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