I'm analyzing images from confocal measurements of the a cell filled with fluorescent calcein, as it is exposed to osmotic change. I've made a 4D-recording: 3D stacks taken after each other.

I mark a cell, and for each stack calculate its volume (by counting the number of voxels whose intensity is greater than a threshold), and for each slice the intensity.

I correct for bleaching (about 0.35% / frame=5 seconds?) or Calcein degradation.

The following things are strange:

• The average fluorescent intensity in a slice through the middle of the cell changes less than the cell volume (about half)
• I've verified that the total amount of intensity in the cell is approximately constant for each stack.
• So where does the intensity go when the cell shrinks?

Example: z=7, t=10:

• Cell volume (normalized)=0.96
• unbleached intensity=690 000
• area=476
• average intensity = 1449

t=29

• Cell volume (normalized)=1.28 (+33%)
  
• unbleached intensity in slice=568000 (-18%)
• area=457 (-4%)
  
• average intensity=1247 (-14%)
  

Found the problem! When I increased the intensity threshold for considering voxels to be in the cell, I got very good matches.