Hello everyone,
I am working with pericyte-like cells whic have long processes. I imagine Ca2+ in these cells using Oregon green and LSM800 laser scanning confocal micsroscopy. I add an agonist and make a movie of fluorescence over time. So far I only know how to see mean fluorescence over time in a ROI, but I need to find a way to see the fluorescence as function of radial distance and time and the speed of the fluorescence spreading. I am sure there is some software which can do it, even ImageJ maybe, but I am not aware what I should look for,
Thank you in advance, Olga
In ImageJ, if you have 2d images in a stack as a function of time, then you can do a reslice to get at the information you are interested in.
Use the Line tool (though if you click you can switch it to freehand or segmented, which should work as well) and draw a line over the area you want to examine, for example a radial direction from the nucleus to the perimeter of the cell.
Next, under the menu Image, select Stack and Reslice. The default settings, 1 pixel spacing and 1 slice, are fine. Confirm the dialog.
You will get a new image that is 2D. On one axis, the vertical one, y, you will have your stack, so time. On the other, x will be the pixels along the line you drew, so distance. Thus a given horizontal line gives you the values of all of the pixels along the line at a moment in time. So you can see how far the signal has propagated, for example. A given vertical line, on the other hand, gives you the intensity of a given pixel, or location, as a function of time.
Peter is correct. this is the way to do it. The final output is called a kymograph if you would like to learn more or see other examples.
Thank you!!! I did the kymographs, but I could not straightfowardly see the propagation pattern. I know it is possible to analyze velocity of the changes in the kymograph? I know it is possible by drawing a line through it, but I am not sure where and in which direction the line should be drawn,
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