Hi everyone, We are trying to count the number of cells within a ROI in the Dentate Gyrus from fluorescently labeled mouse brain tissue (sectioned at 40 microns). We have samples with cells that express low levels of fluorescence and others with a mix of high and low level expressing cells.
We’ve tried using segmentation by using a preset threshold given by ImageJ (ex. Otsu). The sections with low fluorescence yield a binary image that when analyzed gives higher counts than those with higher fluorescence, even though by eye it is clear that the sections with higher fluorescence have more visible cells. This is possibly due to the dimmer cells blending with the dim background.
I've already tried creating a binary image by using subtract background before hand, and using find maxima as a threshold. Those both did not pick up on dimmer cells. If anyone has advice I would really appreciate it. Thank you! :)
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