FIJI (ImageJ): Kymographs [Tracking Moving Objects]

[@pierremangeol4387]

I stepped upon your nice channel and discovered by chance that you present the tool that I wrote (KymographClear). Just a major thank you for this and big kudos to your very useful channel :)

[@johanna.m.dela-cruz]

@@pierremangeol4387 thank you Pierre for a great toolset. I hope I was able to demonstrate what KymographClear can do, albeit a short overview.

[@pierremangeol4387]

@@johanna.m.dela-cruz Your presentation is perfect 🙂 I wish I did it when we published it. I try to do it on our new tools. Your format with text only comments works very well.

[@tvc8199]

Thank you, It’s very help. May I ask a question, how to put scale bar for the time and displacement on the kymograph image.

[@johanna.m.dela-cruz]

Hi! Thanks for your comment. You have to make sure that your image is calibrated correctly. For your original time series, under Image Properties, check that the Frame interval is correct. This should be in sec or msec (or whatever time scale you used when the time series was acquired). In the Kymograph image, pixel width should be in microns (for example) and pixel height should be in time units (ex. sec). When you draw the scale bar, (Analyze-Tools-Scale bar), check horizontal for scale in x and vertical for time.

[@tvc8199]

​@@johanna.m.dela-cruz Thank you very much for your precious time. Now I understand much more after you teach me. May I ask another question, should I take care of these two values of Pixel width/Pixel height or just as the default put 1 of the value; or do as you say, change the unit like microns and sec but the value of Pixel width/Pixel height as the default, I may misunderstand what you say😁. Thank you very much!

[@johanna.m.dela-cruz]

@@tvc8199 Unless you want to express displacement in pixels, then yes, the pixel width and height are important. Have you checked out my tutorial on image scaling? It shows you how you can calibrate your images correctly: kzbin.info/www/bejne/g3TEpIqDnJ2ta6c

[@francescabonato1287]

Hi, thank you for the tutorial, it's very informative! I am wondering if it's possible to automatically select the tracks (in your example the ones generated by z-projection of the time series) and ask one of the plugins to generate all related kymographs. I have an image with constricted tracks (motor proteins walking on fluorescent microtubules) and I would like to know if there is an alternative to selecting each microtubule track one by one... Thanks again!

[@johanna.m.dela-cruz]

Hi @Francesca Bonato. Thanks for watching. Have you tried TrackMate?

[@예예-y5u]

Hi! I am also trying to download and use this method to detect my grip movement. However, I cannot find where to download the Multi kymogrpah plug-in. Also, my ImageJ does not read the video.. Can you give me any feedback? Thanks!!

[@johanna.m.dela-cruz]

Hi. Multi Kymograph now ships with Fiji. Unfortunately, this particular plugin has not been supported for a while. I would advise you to just download FIJI instead. You might also want to try the other plugins to build a kymograph. You can download Fiji from fiji.sc/. What format is your video?

[@Cnia9]

Hello, Thank you for the video, it is very helpful! I would like to ask something about the KymographClear segment of the video. Since the object is moving backwards, why we end up with the same Kymograph forwards and backwards? Thank you!

[@johanna.m.dela-cruz]

Hi @Cnia9. Perhaps the image series I used wasn’t ideal to show the difference because of the not-so-linear movement of the e. Coli, but the backward motion was quite clear (I didn’t have to adjust brightness for this) up to about half of the way. Then other e. Coli came up on the second half of the same route that were moving in varying directions….hence both forward and backward directions were recorded. I suggest that you try KymographClear with your image series and see if it works for you. Thanks for following my channel. I appreciate it a lot.

[@Cnia9]

@@johanna.m.dela-cruz Thank you very much for your reply!

[@noxiousophidian9634]

Thanks for this :) I have huge kymographs with several parallel straight lines. Is there a way to automatically detect all trajectories and give me the slope of each? Thanks again.

[@johanna.m.dela-cruz]

Hi @Noxious Ophidian. I realize that the current way of measuring the slope may be rather inconvenient for a large number of kymograph trajectories. If you know how to write macros, you could generate a line selection tool that writes dx and dy values and the slope to a table. There might be a macro out there that I am unaware of. You could also try to request assistance from macro experts at the image.sc forum (forum.image.sc/). Hope this helps.