Is there a setting in Fiji when you do a max. int. projection of a time course so that the settings are applied the same way to every frame? I start with very dim cells and during my time course bright spots appear. I don’t think the images are being treated the same way in every frame.
Do you mean that the maximum across the stack is not the maximum?
The maximum of the stack at each time point looks like it is the maximum for that stack. It seems like at every time point they maximum is different. I want to the same parameters applied to every time point in my time course.
I feel there is some confusion about what the “max z projection of a stack” does.
What “parameters” are you referring to? The only parameter in the z projection is the type of projection (max, min, average, etc), there are no other parameters to this operation.
After a max projection, you should get the bright spots all in the same result image regardless of when they appear in the time sequence.
I might be using the wrong processing technique. I want to show a projection of the whole cell through out my time course. I want to make sure that the signal min/max is the same in every frame. When I start imaging, my cells have a dim GFP background, during, my time course I get the appearance of bright spots. Is there a better way to collapse the z-stacks and have the signal constant?
could it be possible to upload the stack you want to process?
maybe it will be clearer the goal you are trying to achieve.
have a nice day,
Here is the full file of stacks and what I get when I use the maximum intensity projection.
the dots you are seeing are present in the z planes.
This is an example:
Indeed, when you perform the Max Projections those dots are highligthed since they are the pixels with the maximum values at those coordinates (x,y, along z planes) for that time.
So the Maximum Projection over time it’s well evaluated.
The problem so it’s not in the minimum/maximum settings (they are not considered for the Z-Max projection calculation).
It could be interesting, maybe, understand what are those dots, and if they are considered “artifacts” you can try to remove them after the acquisition with some filters (maybe median?).
It will change also the cells’ pixels values themselves, of course.
If it woulb be possible, it will be better try to avoid them during acquisition.
Another idea is to not consider cells with the dots.
But all of the above suggestions really depend on the experiment and biological meaning itself (that you know better of us)
Just an add,
this is a plot of image intensities over time of the MAX projection of a point put in the cell (in a place without the bright dots):
and this in a place where there is a bright dot:
maybe it can help to understand better
Thank you for your help Emanuele. The bright spots are biologically relevant. I was having trouble telling if the cells were photobleaching or if is was just the projection. It does look like I am getting photobleaching in the cell according to the plot you made over the background in the cytoplasm. That is something I would not have thought to do. Thank you again!