How to make a background image in the presence of the larva

Sometimes I need to put zebrafish larva in the recording chamber from the beginning of a recording and still want a background image for image processing. How can it be possible to obtain a background image from this movie?

If the larva is moving around a lot, there is a simple way. I use Z Project… in ImageJ.


Let’s go through all the options for the Projection type anyway, because the results are interesting to see.


First one is “Average Intensity“.

The second one is “Max Intensity“. It may be useful to see the trajectory of a bright object.

The third one is “Min Intensity“. This may be useful to see the trajectory of a dark object.

The fourth one is “Sum Slices“.

And now, “Standard Deviation“. Because only the pixels that changed appear, the background disappears and the fish movement can be seen.

And finally, “Median“. Only the background is remaining. Of course, this does not work if the fish stayed in the same place longer than half of the time.