Hi Ken, thanks for this informative video. How would you analyze the calcium signaling intensity of the growing cells like root hair or root cells? Because the ROI doesn't stay the the initial position due to the growing nature of the cell.
Hi Vyankatesh, what you can do is to coregister images offline and restructure the video so that the ROIs sit within you desired position, and then perform intensity analysis.
@@vyankateshzambare1440 For automatic coregistration, you can use SIFT (ruclips.net/video/n6SsQhyvYCA/видео.html). For manual coregistration, you can use TrackEM2 (imagej.net/plugins/trakem2/).
To be honest, I am not familiar with ionoptix file. However, if there is a series of images that can be loaded to imageJ, then the fluorescence intensity analysis can be done.
I loved your presentation of the analysis. I wonder though in calcium imaging of neurons they show output results as deltaF/F0. my confusion is the max F, and F0 values from an intensity table. since both F0 and maxF change from peak to peak, how would we calculate that, is it to be done manually from peak to peak
Thank you for your comment. In the situation that both F0 and maxF change from peak to peak, we have to calculate the deltaF/F0 manually as you suggest. Maybe macro or coding (in imageJ or Excell) will help.
Hello Tevin. The time interval of video frames depends on the video recording system. In the case of this video, the interval was determined by the exposure time of excitation light. If the fluorescence signal you are interested in is weak, you may need longer interval such as a few seconds. If the signal is strong enough, you may only need microseconds. Hope this answer will be helpful!
I liked your video. It is very informative. I downloaded Time series analyzer. jar format and saved it in ImageJ plugin section. However, when I am trying to run this, I get the message that the file name must end in java or class. Could you please help? Thank you.
Thank you so much for this video, I learned how to track the calcium signals successfully. However, I have one problem that if the video format is not '.AVI', then I could not analyze through imageJ, (even though I transferred the format from MP4 to AVI, it didn't work), could you please give me some advise on this issue?
Hi Yuyan, Format conversion of a video file can be often tricky. As you mentioned, even though one converted .mp4 to .avi, ImageJ may not accept the .avi. This is probably due to the different type of the .avi file (en.wikipedia.org/wiki/Audio_Video_Interleave). Using another file converter (or another conversion option) may help. Hope it helps!
It depends on the type of analysis you are going to perform. If the small ROI you are using is representing the change of whole-cell calcium, it should be OK.
Thank you for teaching me this. Not as complicated as I thought.
Thank you for uploading this video. It was very helpful
This is a useful video for beginners like me. Thank you for uploading it.
You are welcome!
Hi Ken, thanks for this informative video. How would you analyze the calcium signaling intensity of the growing cells like root hair or root cells? Because the ROI doesn't stay the the initial position due to the growing nature of the cell.
Hi Vyankatesh, what you can do is to coregister images offline and restructure the video so that the ROIs sit within you desired position, and then perform intensity analysis.
@@ken-takahashi Co register yourself what plugin?
@@vyankateshzambare1440 For automatic coregistration, you can use SIFT (ruclips.net/video/n6SsQhyvYCA/видео.html). For manual coregistration, you can use TrackEM2 (imagej.net/plugins/trakem2/).
Hi. Can you also make a video to analyze calcium transient using Fluo-4 with ionoptix files?
To be honest, I am not familiar with ionoptix file. However, if there is a series of images that can be loaded to imageJ, then the fluorescence intensity analysis can be done.
I loved your presentation of the analysis. I wonder though in calcium imaging of neurons they show output results as deltaF/F0. my confusion is the max F, and F0 values from an intensity table. since both F0 and maxF change from peak to peak, how would we calculate that, is it to be done manually from peak to peak
Thank you for your comment. In the situation that both F0 and maxF change from peak to peak, we have to calculate the deltaF/F0 manually as you suggest. Maybe macro or coding (in imageJ or Excell) will help.
Mohammed did you solve the problem to show results as deltaF/F0. Because I have the same problem too.
Thanks for the presentation. I would like to ask how did you get the time interval of the video for 0.034s?
Hello Tevin.
The time interval of video frames depends on the video recording system. In the case of this video, the interval was determined by the exposure time of excitation light.
If the fluorescence signal you are interested in is weak, you may need longer interval such as a few seconds. If the signal is strong enough, you may only need microseconds.
Hope this answer will be helpful!
I liked your video. It is very informative. I downloaded Time series analyzer. jar format and saved it in ImageJ plugin section. However, when I am trying to run this, I get the message that the file name must end in java or class. Could you please help? Thank you.
It could be due to the system you are using.
May I ask what OS and what version of java you are using?
suuupoer useful for beginners like me!!!
Glad to know it's useful!
Dr. Takashi, thanks for the video. For some reason I am not able to download the plugin, is there a new website for it?
Hmm, it seems that the download link is invalid, for some reason...
I managed to download it but when opened in Fiji (Windows) is labelled as Version 2 @@ken-takahashi
the object in my video was moving during imaging. how to perform the pre-adjustment to fix the target object in one place using imagej?
Thank you so much for this video, I learned how to track the calcium signals successfully. However, I have one problem that if the video format is not '.AVI', then I could not analyze through imageJ, (even though I transferred the format from MP4 to AVI, it didn't work), could you please give me some advise on this issue?
Hi Yuyan,
Format conversion of a video file can be often tricky. As you mentioned, even though one converted .mp4 to .avi, ImageJ may not accept the .avi. This is probably due to the different type of the .avi file (en.wikipedia.org/wiki/Audio_Video_Interleave).
Using another file converter (or another conversion option) may help.
Hope it helps!
Thanks!
Thank you for your comment! Hope the video is useful for you.
how to copy those ROI size and positions to another imaging sample of the same test group?
is it OK ROI is round shaped and much smaller than the signal region?
It depends on the type of analysis you are going to perform. If the small ROI you are using is representing the change of whole-cell calcium, it should be OK.