I have confocal images with two markers that I will colocalize, and have 30 micron thick sections, but the stain penetration doesn't always take up 30 microns.
Also would I need to remove the outer few microns of each section in order to have a more accurate estimate of the total volume?
I am able to use the surface function to draw a surface over the ROI (dentate gyrus granular layer and sub granular zone), and to get a statistical calculation of the volume. I would think that this method is just as accurate if not more accurate than Cavalieri's estimator?
Imaris is not suited for stereology. A free and easy to use tool is FreeD. It does slice aligment, surface reconstruction, gives a 3D model and , if set up correctly, it also does the math for you: http://free-d.versailles.inra.fr/html/freed.html
However it does not come with a wizard that helps you to find the right parameters for your cavalieri estimation (as in Neurolucida). You should check the math before. Stereology techniques and explanation of the underlying rules are given here: Schmitz, C., & Hof, P. R. (2005). Design-based stereology in neuroscience. Neuroscience, 130(4), 813–831. http://doi.org/10.1016/j.neuroscience.2004.08.050
Imaris is a very powerful program, and can estimate volume based on Z stacks, so I don't see where this would be a problem, especially if I express the cells in number per volume of the DG. I wouldn't need to get the total amount of cells, but if I did I would be able to multiply by the amount of sections in the sample. This is how I can extrapolate from the methods in stereology to be used in Imaris.
First of all, why do you ask if you already have an answer? I don't get it.
Secondly, considering your reply, Imaris is not doing the stereology, YOU are doing stereology. In your case, Imaris is just a tool to do a geometric measurement. But why would you use a 15'000€ software to do this, if ImageJ is for free and also capable of doing it? I would advice you to check the above-mentioned reference, figure out what exactly needs to be measured and then find a suitable tool.
Thank you for posting the useful article with information on how to perform stereology, it was very informative and even clearer than some of the articles that I have come across.
Ironically this is the software that my intitution has. I'm not sure with the other software you mentioned, but most of the stereology software needs to be equipped to a microscope to scan the slices. Maybe I can use my z stack as a measure of tissue shrinkage in the Z direction, but I'm not sure if that is the right way to go about it. I went through the article that you kindly posted as well as other publications on stereology to extrapolate the factors that are taken into account in the math. Thickness, area, antibody penetration, and the point cavalieri estimator, are used. It seems that the cavalieri estimator also takes into account tissue shrinkage. At this point, I would be lacking in that measurement.
Actually, I was wrong, FreeD does not require a microscope to function and just uses Z stacks. If I can figure out how to make it stop freezing on my computer, I will give it a try. Thanks for the advice.
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