Introduction of a ImageJ plugin called 'Difference Tracker' what we use to measure motility of actin filaments

Difference tracker was originally developed to analyse the movement of mitochondria through axons. This type of data features a large number of moving particles which often exhibit low contrast against the background and can move behind stationary objects.

The aim for this kind of data was not to get the most complete track possible for any individual particle, but to collect relevant aggregate statistics for the movie as a whole.

Difference tracker contains two ImageJ plugins. Difference filter extracts the moving parts of an image to enhance the contrast of the motile particles. From there the Mass Particle Tracker tracks this large set of particles and works out a set of statistics for the number, size and rate and direction of movement for the particles.

Difference Filter Options:

Minimum Difference

This is the minimum absolute intensity difference which must be observed for a pixel to be counted as moving. Increasing this value will cut down on the noise of misidentified moving pixels, at the expense of a reduction in sensitivity for moving particles with low signal to noise.

Difference frame offset

This value says how many frames apart from the source frame the pluin will look to compare images to indentify moving pixels. You should aim to specify the minimum number of frames needed for a particle to move a distance greater than its length.

Mass Particle Tracker Options

Produce full output

If you want to do manual analysis of the tracked regions identified then selecting this option will cause the program to write out a file alongside your image which contains the full tracked positions for every particle.

Minimum tracked intensity

Sets a lower cutoff for the amount of signal which the program will use when tracking.

Minimum feature size

Sets the minimum number of adjacent pixels which will count as a particle to be tracked.

Initial flexibility

Says how far apart two particles can be in adjacent frames to be connected to start a new track. You should set this to the maximum distance which could be covered by a particle in one frame.

Subsequent flexibility

Says how far from its predicted position a particle can be to allow it to still be associated with an existing track.

Min Track Length

Particles which have been tracked for fewer frames than this value will not be included in the aggregate statistics calculated for the movie.