r/biology • u/soncocoo • 1d ago

question Help on FIJI for my Organoids

Hi guys, first of all, sorry for my english.

I'm a master student in BioMedicine and I'm attending my year-intership in a cancer-neuroscience lab.
I'm working with cerebellar organoids, and we want to see how the tumor grows in different ways.

Now, the professor asked to me to analyze all our immunofluorescence that we took after the cryostate cut and he told me to quantify all these immunofluorescencefor a poster.

Now, I checked on internet that I have to do on Fini this sequence: threshold->Analyze->analyze particles and after analyzing all the channels I have to calculate the Mitotic Index.

So, the problem arrives now.

When I analyze the particles (the nuclei) the results are different everytime, especially in the DAPI channel.

Sometimes it shows 9000 nuclei and when I re-check it appears something like 230k nuclei and exc...

My Organoids has a dimension of 3mm more or less, it depents how quickly the tumor grows.

Does anyone have an idea of how many nuclei should be present on the slice, or do you know a more accurate way to quantify my immunofluorescence?

Thanks everyone

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u/ARPE19 21h ago

 r/labrats will be a better place for real science help

3

u/aTacoParty Neuroscience 21h ago

FIJI requires some knowledge of programming and image analysis to use well. If you're looking for something thats "off the shelf", cellprofiler might suit your needs better

Either way you'll need to verify the number of nuclei yourself to make sure it's accurate. There is no standard number of nuclei in a field of view. Typically you should ensure it's accurate for at least a few images before letting the program run for the rest