I need to find neuronal density from a 30micron sections. I was told to use the nucleator to get the volume but I’m not sure how I’ll get the density from that, should i use the optical fractionator and cavalieri method instead? how do you get the density from there?
The nucleator will give you individual cell size, but it probably won’t help with volume or density.
When I think of cell density, I think of number of cells per cubic micron. I usually use two probes on the same subject to get this:
- Trace sections and count using the Optical Fractionator (this gives me the total cell population for my ROI)
- Then, in the same data file, usually reusing the same contours, I would run the Cavalieri probe, which gives area (and volume).
- Finally, divide the cell population estimate from the OF by the area or volume from the Cavalieri probe to get density (cells/square or cubic micron). This approach does require thick (15+ micron mounted thickness) tissue, which fortunately you have.
It’s worth noting that you can run the nucleator probe at the same time that you are running the OF, which will give you total cell population and mean cell size. I don’t think the nucleator will really help you with density, but cell size is a valid and extremely useful measurement to have.
But, there is another equally valid approach: If you want the total volume occupied by cells relative to the total volume of the tissue, you can use the Area Fraction Fractionator probe. This approach allows you to place two markers in the same probe run; one marker type on non-cell tissue and a different marker type on cells.
The AFF will NOT give you a cell population estimate, but it will give you the total volume occupied by all cells. It’s the perfect tool to give you ratios of anything - it’s often used in AD research to quantify plaque volume relative to healthy tissue. While it’s typically run on thin tissue, you can use it on thick sections as well, just focus to the middle of your tissue.