Analysis of Ca2+ waves dynamics with ImageJ

[Olga_K]

Hello everyone,
I have an image time series from an LSM800 confocal microscope in .czi format. I image Ca2+ signal in cells with long processes. My task is to see the direction and the velocity of the Ca2+ wave movement over the distance of the cell process over time. Is there some way ImageJ can help me to do it? So far I only know how to import a .czi file in ImageJ and see the mean fluorescence in a ROI over time, but no idea how to track the movement of the fluorescent signal.
Thank you in advance, Olfa

[Pedro J CamelloDr Pedro J Camello]

Hi Olga,

two possible ways (I assume you have stack files):
1- if you have clear the path which speed you want to calculate, simply mark two ROIs, plot their Ca2+ traces and calculate the time needed for the signal to reach a given threshold (50 of maximal response is typical, but I prefer the real initiation of the local (ROI) response -for example the response point reaching x2 standard deviation of preceding resting trace-). You divide space/time and that´s all.

2- you can also draw a line or polyline and use Stack--Reslice along the pathway you want to investigate. You´ll get a kymograph (Y will be time and X will be space) and each horizontal line is the fluorescence along the path -the line you drew- for each frame  -each time point- of your stack. Response will show as gradients ane slope would be speed.

Hope this helps

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[Olga_K]

Hi Pedro,
Thank you so much for your help! The first method is very easy to understand. I also tried to do the kymograph, but I have some concerns. I do not have only one wave travelling along the process during the recording, it looks there are 2-3 waves per recording time (see attached), in the kymograph won't they just overlay on each other? How can I be sure some fluorescence point in space and time still reflects the propagation of the first wave and not initiation of the second wave?