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    • CommentAuthorjuruble
    • CommentTimeJan 31st 2008

    This protocol explains how to: calibrate ImagePro before using it to measure features.

    Protocol revised: 8/5/2007

    Protocol written by: Mike Neri


    NOTE:  This only needs to be done once when you open the program at a computer, unless it doesn’t come up the next time you open ImagePro.

    You must first have taken a picture of the scale bar using the correct objective (use the same magnification to photograph the scalebar that you used to photograph the image you want to measure) and saved it as a .tif file.


    1) Go to “File” -> “Open” and open the .tif picture file that you took of the scale bar

    2) Go to “Measure” -> “Calibration” -> “Spatial Calibration Wizard”

    3) In the “Spatial Calibration Wizard” Box that pops up, select the “Calibrate the Active Image” under the “I would like to:” prompt and select “Next >”

    4) Create a name for your calibration, set the units to microns (ums), and put a check in the “Create a Reference Calibration” box and the “Set as System Calibration” box (which will pop up when you check the first box) and click “Next >”

    5) In the next window, click on the “Draw Reference Line” button

    6) A green line will pop up on your picture as well as a “Scaling” box.  In the scaling box, make sure the number under the “Represents how many units?” question is 200.  Then draw a reference line as closely as you can to cover 20 units on the scale bar.

    7) You can zoom in and out using the scroll button on your mouse to get it more exact.  Once you have a line drawn that you are satisfied with, click “OK” in the scaling box.

    8) Under the “Draw Reference Line” button, take a look at the “Calibration Report.”  This tells you how many microns will be represented per pixel.  I always made sure that my calibrations were the same value, and that was 0.261053 um/pixel.  You may want to try for this number, or at least keep it constant throughout your data as well as it will allow for more consistent results.

    9) Once you are satisfied with you calibration, click “Finish” (you can redraw your calibration line if you are not happy with how it turned out).  You can now close out of your calibration picture and you are ready to measure.


    NOTE:  When you open an image to measure, be sure to select the calibration you just completed from the "System Spatial Calibration” box and click "Apply."