ImageJ Kymograph Makro

ImageJ Kymograph (written by J. Rietdorf (rietdorf@embl.de) and A. Seitz)
The purpose of this collection of macros and plugins is to measure the velocity of moving
structures in image time series (2D over time).
For each frame of a time series, grey values along a line type Region Of Interest (ROI) that you
specify by hand drawing are read out.
From these lines of grey values, a new image (the kymograph or time-space-plot) is assembled: The
line read from the first frame of the time series is put down as the first line in the kymograph, the
line from the second frame the second line of the kymograph, and so forth.
In this way, the y axis of the kymograph becomes a time axis and the unit is the time interval of the
sequence. The x axis is the distance along the line ROI and the unit is the pixel size of your
sequence.
If a structure characterized by a certain intensity and contrast is moving along the ROI, it will
appear as a contrast edge in the kymograph.
Velocity (displacement per time interval) can be measured by drawing a line type ROI along this
edge. The angle of this line in the kymograph is proportional to the velocity. For example, if the
line in the kymograph is parallel to the y (time) axis, this means there is no movement (no
displacement over time).
Installation
Workflow
Copy plugins ’WalkingAverage_.class, StackDifference_.class,
MultipleOverlay_.class, MultipleKymograph_.class’
to ImageJ plugin folder,
install macro tsp040421.txt (Plugins…Macro…Install…)
Load image sequence
Crop and filter image sequence, Plugin ‘WalkingAverage’
Generate tracks by z-projection or Plugin ‘StackDifference’
Select tracks using the segmented line tool
Restore the selection onto the original sequence,
Or use plugin ‘MultipleOverlay’ to select and store more than one track
Execute plugin ‘Multiple Kymograph’
Select edges to read velocities using the segmented line tool
Quantify movement using macro ‘read velocities from tsp’

Step by step procedure:
• For the installation of ImageJ please refer to the instructions at
http://rsb.info.nih.gov/ij/download.html
• Copy ’WalkingAverage_.class, StackDifference_.class, MultipleOverlay_.class,
MultipleKymograph_.class’ to plugin folder. Install ‘tsp040421.txt’ (ImageJ Menu
Plugins…Macro…Install…)
• Start ImageJ
• Load a time series
• Optional: Smooth the sequence by Median filtering or using the plugin
‘WalkingAverage’.
• Generate tracks by maximum or average projection or by using the
StackDifference plugin which generates a stack of subtraction images and a
projection of this stack.

maximum Z-projection of the sample image sequence
average Z-projection of the sample image sequence
• Select a segmented line ROI along one of the tracks in the image (example image
inverted for better visualization of the ROI).
inverted Z-projection ROI selection (yellow line)

• After selection using the segmented line tool, select the image window containing the
original time sequence and restore the segmented line ROI.
• Alternatively, use the plugin ‘MultipleOverlay’ to store the selected ROI in an overlay
image and select further segmented lines in case you want to read out multiple tracks from
one sequence.
• Start the ‘MultipleKymograph’ plugin. It will generate kymographs for all selections.

• Select a (segmented) line ROI along the structure of interest.
• To read the velocities from the kymographs use either the LineAnalyzer plugin from the
ImageJ homepage or use the ‘read velocities from tsp’ macro in tsp040221.txt. (This txtfile
contains a selection of macros that offer a functionality similar to the plugins described
above, installation: ImageJ Menu Plugins…Macro…Install…).
• For each line interval of the segmented line selection the actual displacement in x
(space) and y (time) direction, the sum of displacement in x (space) and y (time) direction,
the actual and the average speed are plotted.