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Kymograph analysis using Image J

Page history last edited by peiwenchn@gmail.com 6 years, 1 month ago

Image J Kymograph                         by Pei-Wen Chen

  •          To install ImageJ, go to



  •          To install related plugins (you only need "MultipleKymograph.class" for this analysis), go to


and follow instructions on the webpage (below)

Kymograph (time space plot) Plugin for ImageJ



J. Rietdorf FMI Basel + A. Seitz EMBL Heidelberg


2004/11/11: Macro: some updates / bugfixes
2005/06/07: Macro: bleach correction integrated
2008/10/31: Update of Plugins




Download MultipleKymograph_.class, MultipleOverlay_.class, StackDifference_.class, WalkingAverage_.class to the ImageJ plugins folder and restart ImageJ

Download tsp050706.txt (e.g. click with right mouse button and select �save target link as�) to the plugins folder and install it:
ImageJ Menu...Plugins...Macro...Install...


The kymograph plugin for ImageJ is intended to measure velocities of moving structures in an image time series. Please read the detailed plugin description (including installation intructions)!


  •          Start Image J


  •          Load a time lapse movie


  •          Image>Stacks>Z Projection

Choose the start and stop slice, usually the first and the last time point of the movie

Projection Type:choose “Max Intensity”


  •          Save the Z projection image as a new Tiff file


  •          Line tool>right click & choose “Segmented Line”


  •          Mark the track of interest on the Z projection image


  •          Click on the original time lapse movie file


  •          Edit>Selection>Restore Selection

You should see a line appear on the time lapse movie file


  •          Plugins>MultipleKymograph

Line width: choose 5


  •          Save the Kymograph image as a new Tiff file

The Y axis is time and the unit is the time intervals of the movie

The X axis is the distance the object of interest travels along the chosen segmented line and the unit is the pixel size of the movie


  •          Analyze>Set Measurements

Choose “Bounding rectangle”


  •          Line tool>right click & choose “Straight Line”


  •          Trace the track of interest


  •          Analyze>Measure (or Ctrl+M)


  •          Use “Width” and “Height” to calculate velocity





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