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    • CommentAuthorjuruble
    • CommentTimeJul 13th 2007 edited
     
    This protocol explains how to: measure neurons with ImageJ.

    Protocol revised: 7/21/2006

    Protocol written by: Barbara O'Donnell

     

    1. Open Image J on the computer.
    2. Click File – then Open. Open your images to analyze from wherever you have them located.
    3. Open one of your images (doesn’t matter which one – you have to have one open in order to install the macros program you will use for analysis).
    4. Make sure that you are using images of your neurons that have had the cell body removed. If not, the program will measure the cell body as well and your results will be useless.
    5. Set your macros program for analysis according to the following:
      - click plug ins >> macros >> record (a box pops up; leave it there)
      - click process
      >> binary >> threshold
      - click process
      >> binary >> skeletonize
      - click analyze
      >> set scale
      Change "distance in pixels" to 402 and change "known distance" to 100 -- the scale should say 4.020 pixels/inch (measurement does not have to be in um). Click OK. NOTE: this is only for pictures taken at 100X.
      - click analyze
      >> set measurements >> unclick perimeter >> click OK
      - click analyze
      >> analyze particles >> set minimum size to 5 pixels AND bins to 20 >> click OK

     

    A set of results will pop up for the image you opened to program the macros – close it out; no need to save it.

    Your recorder pop up box should look like this:

    recorder

    1. Name your macros (whatever name you want) by changing the info in the name box in the recorder pop up.
    2. Click create >> macros >> install macros
    3. Close install pop up AND recorder pop up. You should now be able to click plug ins >> macros >> your named macros
    4. Open your image (WITHOUT cell body) and click plug ins >> macros >> your named macros
    5. A results box will pop up with either one number OR a series of numbers (depending on the number of pieces in your image).
    6. Add up those numbers and multiply by 4.02 – record your result. There is a template for recording your Image J results available on Public >> Biology >> Biologylom >> Image J Results Form Template (Add or delete rows as needed).
    7. Close the results box and the image. You DON’T need to save either of them.
    8. Repeat steps 9 – 11 for each of your images.

    WARNINGThe macros that you made will NOT save when you close Image J. You will need to remake them each time.


    **After you have recorded your data, you will need to normalize it. This is done by dividing each result entry by the average of your control.**


    Once your data has been normalized, you can use "chart wizard" to graph your results in Excel.