I’m a research assistant in a lab that currently primarily deals with 35 micron thick tissue slices, stained using antibodies (it’s pretty standard immunohistochemistry procedure). One unexpected question we were posed during a recent presentation we did was, “How do you (we) account for differences in tissue thickness when generating max projections from Z-stacks?” We took a look at some of our .lif files (generated using a Leica confocal microscope) and realized that the number of “slices” containing signal differed between regions of interest, and the first slice containing signal differed as well. For example, ROI#1 may contain signal in slices 4 through 24, but ROI#2 had signal in slices 1 through 25. These stacks would represent different volumes of tissue, and as such different numbers of cells, and the whole purpose of my project is to develop an automated image processing/cell counting procedure. The grad student and PI of my lab agreed it would be best to set the slice acquisition range very broadly; i.e., we’d have “blank” slices at the beginning and end of every stack. What I’m trying to figure out is:
Using FIJI, what is the best strategy to exclude blank slices from a max projection, while making note of how many slices were included so we know what the volume of the tissue is? Is there an automated option, or will I have to manually decide the slices range for every max projection?
If I do have to manually decide what range of slices to use, should I keep that range the same for all ROI in a given sample? Following the example given above, maybe I only use slices 5-23 for ROI#1 and #2, because I know every slice in that range will contain signal.
If possible, I’d appreciate if anyone can provide me with some examples in the literature where Z-slice range decisions were made, so I can learn from other’s example, rather than begging to be spoon-fed on an image analysis forum! Any other useful references would be much appreciated as well; the more I learn about automated image analysis, the more I respect its utility in the modern lab, so the more I want to learn
Thanks so much!