FIJI (ImageJ): Tracking Cells, Single Particles or Spot-like Objects with TrackMate and MTrackJ

[@周晓天-p7s]

Thank you so much for the tutorial, it is really helpful.🥳

[@johanna.m.dela-cruz]

I’m glad to help. Thanks for watching.

[@oldyoungboy0227]

Thank you for your effort in making this video and sharing the analysis methods. I Liked and subscribed your youtube channel.

[@johanna.m.dela-cruz]

Thanks for your support, @oldyoungboy0227.

[@hamzasaoud7236]

Thank you very much for your great efforts 🌹🌹🌹🌹

[@johanna.m.dela-cruz]

Hi Hamza. Thanks for dropping by.

[@charlesbarker5900]

Thank you!!

[@kottapallivp6115]

You are a life saver

[@johanna.m.dela-cruz]

Happy to help. Thanks for watching.

[@IAmBarbieee]

Thank you - really useful!!!

[@johanna.m.dela-cruz]

Thanks for watching and following my tutorial series, ELM.

[@matiasgonzalez1492]

[@johanna.m.dela-cruz]

@Matias Gonzalez. The pleasure is all mine. Thanks for watching.

[@joyye2316]

thank you for your wonderful work, can you make a video to show how to track the rod-shaped cells? I tried trackmate but failed to track the rod-shaped cells. I am very appreciate that if you could help me.

[@johanna.m.dela-cruz]

Hi @joy ye. I’ll see what I can do. I would love to be able to help you.

[@joyye2316]

@@johanna.m.dela-cruz if you need, I could offer the videos for you to try. In my videos, there are two species , one is rod-shaped cells with bigger size and moving with high speed, another is spot-like immobile cells like clustering.

[@johanna.m.dela-cruz]

@@joyye2316 that would be helpful.

[@joyye2316]

@@johanna.m.dela-cruz Cool, then can i have your email please? I will send the videos to your email.

[@johanna.m.dela-cruz]

@joy ye, you can find it in my channel (“About” section).

[@shruthib8176]

Hi, Thank you for the videos they are resourceful. I am trying to track cells in 3D to calculate their angular velocity. I would appreciate some help with this.

[@johanna.m.dela-cruz]

Hi @shruthib8176. I don't think TrackMate has analyzers that measure angular velocity. It does have features that track motility type and mean directional change.

[@shruthib8176]

@@johanna.m.dela-cruz Thank you very much for getting back to me. I used the xy coordinates from the TrackMate to calculate the angular velocity.

[@jamilaiqbal202]

How to batch analyze files in a folder using Trackmate

[@johanna.m.dela-cruz]

Hi Jamila. I think that you will have to rely on scripting to do batch analysis with TrackMate. You might have to adapt the script according to what your first image needs. More details can be found here: imagej.net/plugins/trackmate/scripting/scripting

[@kevinTRQ]

hello, thanks for the video. my DoG calculator is asking me for the estimated size in pixels, while yours says microns. is there way to change this? also can we measure the velocity of the particles through the Manual tracking method using MTrack J plugin?

[@johanna.m.dela-cruz]

Hi @Kevin TRQ. If your image is spatially calibrated (for example, in microns, nm or mm), the DoG detector will know what units your image has. If it is not calibrated, then you will have to set the scaling of your images. For manual tracking with MTrackJ, after you have created the tracks, click on Measure on the GUI. One of the measurements in the Results table is velocity.

[@kevinTRQ]

@@johanna.m.dela-cruz thanks a lot!🙏💯

[@andyevans2519]

Ive been trying to capture an overlay of selected tracks only, which works however when hide image is selected the overlay appears blank with no tracks visible?

[@johanna.m.dela-cruz]

Hi @andyevans2519. Are you using the updated version of Fiji? If your display settings have tracks enabled, "Capture overlay" should capture these tracks even if you hide the image.

[@pac0mp67]

Hi Thank you for this awesome video. I have a question how can I calculate my x/y calibration.? and z calibartion

[@johanna.m.dela-cruz]

Hi @pac0 mp. I appreciate your nice feedback. If your image is not calibrated (in pixels), you would need to get information about pixel size from whatever microscope you used. If your files are saved in the microscope manufacturer's format, it is more likely that this information is saved in the metadata of your image. Have you checked out this video on calibrating your images? kzbin.info/www/bejne/g3TEpIqDnJ2ta6c

[@mrp3248]

hello, when i go in the plugin like you did at 0:45 it doesn't show me a tracking list do i have to download something else to be able to do that please

[@johanna.m.dela-cruz]

Hi @mrp3248. You will need Fiji, rather than the original version of ImageJ. You can download Fiji at fiji.sc/.

[@mrp3248]

@@johanna.m.dela-cruz thanks, it worked, thank you very much , i'm saved lol thanks for responding so fast

[@linandu-s8q]

Can we calculate the number/rate of mitochondrial fission？ THANK YOU!

[@johanna.m.dela-cruz]

Hello. TrackMate can be set to detect and deal with splitting events like fission (or merging events like fusion). These events are mapped in TrackScheme. I believe the Track Analysis does include speed and the number of split events.

[@linandu-s8q]

Thank you very much for your reply, I have difficulty with the operation, I use TrackMate's Mask detector, but I can't get the results I want, can you share how to set it up.

[@johanna.m.dela-cruz]

@@linandu-s8q Perhaps you can send me an email?

[@linandu-s8q]

@@johanna.m.dela-cruz Thank you very much！I'll email you.

[@AbdulSalam-ds3qh]

Can we calculate the velocity of these moving objects from the data which we obtained from Tackmate?

[@johanna.m.dela-cruz]

Hi. Among the track analyzers built into Trackmate are velocities generated from link speeds between spots (distance between 2 spots divided by the time difference). Mean velocity is the mean of the link velocity over all the links of the track. Maximal, mimimal and median velocity, and Velocity standard deviation are feature values that take the link velocities of a track as a distribution they summarize. As for velocity of particles, TrackMate does not directly give this. You might have to import the tracking results and compute this yourself.

[@AbdulSalam-ds3qh]

@@johanna.m.dela-cruz Thank you for your suggestion. I'm not getting things clearly. More clearly, I would like to tell you my problem is that I want to calculate the speed/velocity of lysosomes inside the cell. If you have any suggestions please do let me know to solve this issue.

[@johanna.m.dela-cruz]

@@AbdulSalam-ds3qh On the TrackMate window, under Display Options, click the Tracks button (at the bottom part). On the left side, you will find Spots, Edges and Tracks. Click on Tracks. One of the columns will give you the mean speed of each of the tracks found.

[@SangeetaKumari-z1i]

using mtrackj...i need time data also(showing 0 sec)...can u please help me to add time in excel sheet itself?

[@johanna.m.dela-cruz]

Hello Sangeeta. Time would depend on your acquisition parameters. When you look at the Image Properties, this should be indicated under Frame Interval. You can then just put this interval in Excel, starting from time 0.

[@SangeetaKumari-z1i]

@@johanna.m.dela-cruz thank you for your response

[@milernalain]

Hello. Thank you for this video is very helpful. I was wondering what format do you use to analyse using TrackMate? Thanks

[@johanna.m.dela-cruz]

Hi @Marisol. Thanks for your kind feedback. I don't quite understand what you mean by format...is it the detector (e.g. DoG, LoG, etc.)?

[@milernalain]

@@johanna.m.dela-cruz sorry, I should have phrased my question better. When you import the file to analyse to Fiji, do you import it as an .avi file or as a stack of .tiff files? Thank you!

[@johanna.m.dela-cruz]

@Marisol Pérez Toledo You should load a stack of images. Tiffs are fine.

[@sarahbeauvais8573]

Hi I do not understand where you read the object's size as 5 microns. I am looking at the imageJ bar and I see X, Y, and Z coordinates but not in microns. I see where is says length=4.8 but no unit. Is that the object size microns?

[@johanna.m.dela-cruz]

Hi @sarahbeauvais8573. 5 um is an estimated object size. The length of 4.8 is in microns, since the image is calibrated. I just rounded up 4.8.

[@lorenzocozzolino3020]

Hello Johanna, Thank you very much for your video. I have a photo lapse as samples but I don't know how to import it. I read you said to import it as a stack of images in format .tiffs but I don't know how to create a stack of images. Could you help me please? Thank youu very much! Best, Lorenzo

[@johanna.m.dela-cruz]

Hi @Lorenzo Cozzolino. You can try opening your file as a virtual stack. There are several built-in commands in Fiji/ImageJ that you could use: File>Import>Image Sequence; File>Import>Raw; File>Import>Stack From List; File>Import>AVI. Hope this helps.

[@lorenzocozzolino3020]

@@johanna.m.dela-cruz Thank you so much for the quick reply. Gonna try this way :)

[@johanna.m.dela-cruz]

@@lorenzocozzolino3020 I got a notification of another question from you, but I can't seem to see it anymore. I'm wondering what kind of file you have that you can't open it as a stack.

[@rubalsingh7885]

can we use this for cell migrantion timelapse image stacks?

[@johanna.m.dela-cruz]

Hi @Rubal Singh. Thanks for your interest. Yes, my image sample is actually a time-lapse stack. You will probably have to track your cells (maybe the nuclei) using a different tracking detector (perhaps LoG). Let me know how it goes with your images.

[@rubalsingh7885]

@@johanna.m.dela-cruz OK THANKS