Hi @iarganda & @tibuch
Thanks for the replies. I suppose my problem with subtracting an average of the whole stack is that the cell moves quite a lot over the course of the movie. And in fact, this is just 73 frames of a 1,000+ frame movie - so subtracting the average or any z-projection results in a large ‘smear’ across the image. I’m really interested in seeing the change in cell shape with the highest temporal resolution possible (i.e. frame by frame)
I have written a macro to calculate the ‘difference’ frame by frame using the Image Calculator, however just working out the difference isn’t quite right either. If the cell spreads in one direction, and retracts from another, both show up as high values.
So for example, I’ve created this image in which:
Red = Frame 0
Blue = Frame 15
Green = Average projection of 73 frame stack
Here, blue means we have the cell advancing, red means it has retracted, and grey indicates no change. The background of the plastic dish shows up nicely as grey, as I have corrected for drift. This gives me really nice contrast in the red, blue, and green channel - so my question is, how can I segment the image using these channels? Assuming I expand my macro to generate a stack of images like this?