I have recorded a video using fluorescence signaling from live (and unfortunately moving) cells and saved it as a stack in ImageJ. I am looking for a way to select a region of interest that is moving throughout a stack (over time) to measure the fluorescence intensity.
Megha, can you share the video, and post two frames with the region of interest manually labeled?
Megha, you may want to look into what is known as a time-space-plot or kymograph, it is a method to analyse moving objects. There are several implementations for imageJ. For example http://fiji.sc/Multi_Kymograph. In brief, you can color code the z-dimension and make a projections so you reduce by one dimension. By image subtraction along the time axis, you generate tracks of the moving objects. These tracks can then be selected using ROIs. Finally the ROIs are read from every timepoint and re-assembled into a new image for each track. Movement distance along the track is encoded on the x-axis, movement in time on the y-axis and color for depth. It preserves the intensity information of the original images, so should be easy to measure intensity changes along the track. Hope that helps.
You might also consider trying the image registration methods, e.g. StackReg (http://bigwww.epfl.ch/thevenaz/stackreg/), in ImageJ/FIJI as this can modify each frame of the movie in such a way that it puts the cells back into the same location in every frame. This can be very useful for analysing cells that are on the move.
Hi I Think that your rea looking for something like this
https://github.com/ekatrukha/KymoResliceWide
but first you can try to correct the drift is there is some with
https://sites.google.com/site/qingzongtseng/template-matching-ij-plugin
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