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    • CommentAuthorjuruble
    • CommentTimeJan 31st 2008
     

    This protocol explains how to: analyze a previously-acquired time-lapse growth cone movie using ImagePro.

    Protocol revised: 1/30/2007

    Protocol written by: Barbara Lom

     

    1. Open your saved movie sequence.  Check that ImagePro+ is set to the appropriate objective.  Go to the “Measure” menu and select “Calibration” and then “Set System.”  If you’ve calibrated before you should chose the appropriate objective (usually 40X).  Click on the “...” button and choose the magnification (40X again usually) and the units of measurement (um usually) in the box labeled image as well.  Click apply.  If you have not yet calibrated ImagePro, see this protocol for instructions.
       
    2. Go to the “Measure” menu and select the “Measurements” option and then and select the tool that is a circle with crosshairs.
       
    3. Use the magnifying glass to zoom in on the image – magnification usually helps you do better measurments.
       
    4. Click on the image to mark the position of something (usually it’s going to be the leading edge of your growth cone lamellipodia) in movie frame 1.   Advance the movie to frame 2 and click to delineate the end of the lamellipodia in the frame.  Continue through all the frames.
       
    5. Now select the line distances tool (looks like a low/wide H) from the Measurements tool bar and make sure that “Ctr-to-ctr Distance” is selected on the pull-down menu in the lower left.  Hold down the control button and then click on point 1 and point 2 and a red line (D1) will be drawn.  Click on points 2-3, 3-4, 4-5, etc. to get the distances between all points.  Make a note if the
      distance is backwards (as ImagePro+ only calculates absolute distances)
    6. Export your data to Excel by going to the Input/Output tab of the Measurements window and clicking on the “DDE to Excel” button and then pressing “Export Now”.  You should notice (in the background) that a new workbook will open in Excel with your data.  
    7. There will be many blank/unnecessary columns in the Excel file – the ctr-ctr distance is the most important measure for you.  Make sure you know what distances belong with what times.  Frame 1 should be time zero.