This macro and plugins suite for ImageJ (and Fiji) serves to measure the velocity of moving structures and visualize them, from image time series (2D over time).

The module can be installed in ImageJ as a Macro Menu and each function/component can be called separately. The full workflow consists in calling some, or all, the functions sequentially in order to get from the image preparation (e.g. filtering and visualization of tracks) to the production of the kymographs (time vs. distance plot) and their analysis (retrieving the velocities).

Here is the full workflow sequence:

  • Load image sequence
  • Crop and time-filter the image sequence ("Walking average" plugin)
  • Generate tracks by z-projection ("Stack difference" plugin)
  • Select tracks and restore them in the original stack.
  • execute plugin "multiple kymograph"
  • Analyse: select edges of moving tracks graphically and quantify movement in a table.

input: 8-bit, 16-bit stacks, 2D in time. Calibrated is better for meaningful velocity measurements.

ouput: the kymograph image, the velocity measurements tables.

Requires ImageJ version: 1.33.n minimum.

Example of applications:

  • velocity of moving objects/ structures with sharp edges, incl. the velocity of microtubules (and their plus ends),
  • the velocity of vesicles or particles along a 2D path
  • the velocity of migration of the edge of a cell or a multicellular group
  • retraction velocity of contractile bundles (e.g. actin fibers) or multicellular tissues after mechanical disruption (e.g. laser surgery)