Using TrackMate to track single cell migration

[Brittany Boribong]

Hello everyone,

I am attempting to track cell migration using the TrackMate plugin in Fiji
and I have a couple questions. Here is a link to a dropbox with sample
movies:
https://www.dropbox.com/sh/a1k56hm4xkt1tyt/AABQ-mMSR_1pLj4jHfod7357a?dl=0

I want to measure velocity and directionality of the single cells in my
movies. I first tried to calibrate my image to 0.645 uM/pixel and 3
min/frame. However, it doesn't seem like changing the calibration settings
in the beginning has any effect on the statistics output. Is there a way to
correct this?

I detech the cells using the LoG Detector with parameters 15 pixel diameter
and threshold equal to 1. I track the cells using the LAP Tracker
(parameters Frame to Frame Linking = 190 and Track Segment Gap Closing =
50, Max Gap Closing = 2). This isn't consistent in tracking the cells
across all frames. How can I optimize my parameters to ensure the tracks
are tracking each individual cell throughout the entire movie?

How is TrackMate calculating its statistics? I am unsure of the output's
units. The values I am receiving also don't seem as if they would align
with what I am expecting, which is around 15 - 30 uM/min for a single cell
migrating across the entire frame.

When using the "Capture Overlay" function to record the tracks of cell
migration, the movie cuts off at the bottom so the entire movie is not
captured. An example can be seen in the dropbox file
"Trackmate-Capture.AVI". Is there a way to correct this?

I'm open to any suggestions on how to carry out my analysis.

Thank you for your help,
Brittany Boribong

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[ctrueden]

Hi Brittany,

> I am attempting to track cell migration using the TrackMate plugin in
> Fiji and I have a couple questions.

You might have better luck posting on the ImageJ Forum [1]. Alternately,
you could post a new issue in the TrackMate GitHub repository [2]. The best
person to answer your questions is probably the author of TrackMate,
Jean-Yves Tinevez [3]; he does support TrackMate on the forum and on GitHub.

Regards,
Curtis

[1] http://forum.imagej.net/
[2] https://github.com/fiji/TrackMate/issues
[3] http://forum.imagej.net/users/tinevez/activity

--
Curtis Rueden
LOCI software architect - http://loci.wisc.edu/software
ImageJ2 lead, Fiji maintainer - http://imagej.net/User:Rueden
Did you know ImageJ has a forum? http://forum.imagej.net/


On Mon, Oct 10, 2016 at 10:59 PM, Brittany Boribong <[hidden email]> wrote:

> Hello everyone,
>
> I am attempting to track cell migration using the TrackMate plugin in Fiji
> and I have a couple questions. Here is a link to a dropbox with sample
> movies:
> https://www.dropbox.com/sh/a1k56hm4xkt1tyt/AABQ-mMSR_1pLj4jHfod7357a?dl=0
>
> I want to measure velocity and directionality of the single cells in my
> movies. I first tried to calibrate my image to 0.645 uM/pixel and 3
> min/frame. However, it doesn't seem like changing the calibration settings
> in the beginning has any effect on the statistics output. Is there a way to
> correct this?
>
> I detech the cells using the LoG Detector with parameters 15 pixel diameter
> and threshold equal to 1. I track the cells using the LAP Tracker
> (parameters Frame to Frame Linking = 190 and Track Segment Gap Closing =
> 50, Max Gap Closing = 2). This isn't consistent in tracking the cells
> across all frames. How can I optimize my parameters to ensure the tracks
> are tracking each individual cell throughout the entire movie?
>
> How is TrackMate calculating its statistics? I am unsure of the output's
> units. The values I am receiving also don't seem as if they would align
> with what I am expecting, which is around 15 - 30 uM/min for a single cell
> migrating across the entire frame.
>
> When using the "Capture Overlay" function to record the tracks of cell
> migration, the movie cuts off at the bottom so the entire movie is not
> captured. An example can be seen in the dropbox file
> "Trackmate-Capture.AVI". Is there a way to correct this?
>
> I'm open to any suggestions on how to carry out my analysis.
>
> Thank you for your help,
> Brittany Boribong
>
> --
> ImageJ mailing list: http://imagej.nih.gov/ij/list.html
>

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