Tracking cells using imageJ

Hi everyone,

My question is about using Manual Tracking plugin in imageJ but before explaining my problem please let me know if you know any other method to measure cell migration using ImageJ. So, here is my problem:

I need to track cells to quantify their migration in x and y direction. I used “Manual Tracking” plugin which is quit cool but I need help in how to use it. When opening the plugin, first under Parameters section you have to define your Time Interval and x/y calibration. Can anyone explain me what are these two parameters. I ran my experiment for 96 hours using time-lapse microscope for every 20 minutes. Also my cells dimensions are nearly 20 Micrometer. Considering these numbers, what should I put for “Time Interval” and “x/y calibration”?

Also is there any way to separately calculate for x direction and y direction velocities? As I see in the outputs, Manual Tracking only gives the velocity between two frames but how we can calculates velocity components in x and y directions?

Hi @AminHB,

Have you had a look at http://imagej.net/TrackMate.
TrackMate is a remarkable tool to do automatic tracking (mainly in fluorescent image sequences).

If you do brightfield imaging then you might need pre-filtering first to optimize cell detection or you can have a look at https://sites.google.com/site/itrack4usoftware/home

I think this will most likely be advantageous over any manual tracking in precision as well as (and especially) time performance wise.

If you still want to continue doeing the manual tracking (where you actually also have the option to do this in TrackMate) the parameter “time interval” referes to the time inbetween your individual time frames (images) whereas the x/y calibration referes to the “pixel size” (actually pixel distance) to be able calculating a velocity for you in e.g. µm/sec.

Thus, your 20min are the time interval but for the pixel distance <ou need to have a look into the metadata of your image sequences (e.g. by >Image >Show info…) [quote=“AminHB, post:1, topic:595”]
Also is there any way to separately calculate for x direction and y direction velocities? As I see in the outputs, Manual Tracking only gives the velocity between two frames but how we can calculates velocity components in x and y directions?
[/quote]

In TrackMate you can at least have a look on the x- or y-displacement over time and calculate this yourself e.g. in Excel by using the output data table of the individual links with x and y positions.

It is recommendable going through the nice and comprehensive documentation of TrackMate first to see its capabilities and understand the handling. This should also give you a reproducible and comparable tracking output.

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