Particle analysis not count what I need

Hello forum,

I would like to count number of eggs but the software only count the small spot background.

What I did
-Split channels and choose blue channel
-Subtract background
-Treshold
-Particles analysis

eggs size about 50x70 px

Those small spots about 10x10 px

I wonder what did I do wrong?

//Traimate

With 50 px * 70 px, your particles of interest have a size of 3500 pixels^2, so your size threshold is definitely too low.

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I also set to 0-5000 or 0-infinity but it doesn’t recognize my interested particles …

Here is my sample image, if you could show me how to set it.
sample image

I set the size to 1000-3500 and the circularity to 0.2-1 and I get all the eggs. Please check. If it does not work, please upload here your binary image.

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Thank you … I see now, because I didn’t “Make Binary” after I set “Threshold” the picture. Now, it seems to work.

I may ask other question …

Some eggs are quite overlap, for example number 73 in picture, is there anyway to correct this ?

Some spot it’s not the egg, how can I remove it ?

You could apply a watershed transformation (Process -> Binary -> Watershed) to split overlapping eggs.

To fiter non-eggs you could try further shape parameters:

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I would also try smoothing the image before thresholding. Otherwise applying the watershed separation (that is not the 'watershed transform"!) will lead to over-segmentation as it depends on finding ultimate eroded points and those, in turn, depend on the region boundary details.

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You may try the irregular watershed to reduce oversegmentation: http://imagej.net/BioVoxxel_Toolbox

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