Size Filters for Neuro- slices

neurons
neuroanatomy
nucleus
3d-objects-counter
fiji
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#1

Hi everyone,

Sorry to be a complete newbie here, but I am just getting my feet wet with Fiji. I have two 3D sections (six .oib files total) that I am analyzing, trying to determine the number of neurons (NeuN), nuclei (DAPI), and oligodendrocytes (ASPA). The 3D Object Counter is incredibly simple and seemingly plug-and-play, but I have no basis of comparison to trust the counts I get from simply manually adjusting the threshold and going for it. Does anyone have any insight into what I should set the “size filter” minimum and maximum parameters to? These size parameters are in voxels, right? Images were collected at 40x


#2

Welcome to the forum, @wessolem!

This really depends on your biological specimen and the way of acquisition. Cell size can vary a lot with cell type and tissue of origin.

If your images are uncalibrated, yes. If ImageJ reads to the calibration from the file metadata (if present), or you specify calibration via Image > Properties…, the measurements will be in {µm}^3 or whatever unit you specified.

This information doesn’t really help much. Depending on the size of your camera sensor, whether you use binning or not, or the zoom setting of your laser settings (in case of confocal laser scanning microscopy), the actual pixel size can vary a lot. You should either calibrate the microscope and set it up to write the correct pixel size into the file metadata, or measure with a known scale and the exact same settings you use for acquiring your experimental images.


Have a look at the Principles page for a general overview.


#3

Great, thanks for the info! These are mouse brain slices that were used, but I don’t know much deeper detail about the microscope used, etc. I realize that’s a big part of the puzzle. As far as I know, the images are uncalibrated. I had the thought of potentially counting these cells manually, then running the 3D Objects Counter at varying parameters until I could closely match my manual number (to establish some sort of baseline / manual calibration), but that seems like maybe a backward way of going about it. I will keep digging and try to sort this out. Thanks again!