Hi all, I am performing an exosome quantification assay and I need to normalize my results to total number of cells. I will not be able to detach my cells from the plate, and therefore cannot use a hemocytometer/Countess/etc.
Is it possible to take several brightfield images of each well, stitch them together into one image, and use a FIJI plugin to automate a cell count? Would I need to stain them first? If possible, I would like to avoid fluorescence microscopy.
Hi,
The answer for you is yes, FIJI can do all the things you want.
I fear just if you really want an absolute number, you would need to go for fluorescence microscopy, stain with something like Hoechst and count nuclei. Its an easy to use and cheap stain.
In case confluency is enough to normalize your samples, most of the time it is if the cells don't grow to much on top of each other, you do not need the fluorescence staining. Best would be a Phase Contrast Image, highlights the cell boarders really well. Depending on your setup with a brightfield correction and smoothing of the image might be necessary before threshholding to generate the best result.
There are some algorithms available that try to seperate and count single cells out of a phase contrast image, from my experience it doesnt work too well.
If you need more help with FIJI i can send you some workflows.
Good Luck
Jürgen
Hello,
As you said, you may collect images to analyse by FIJI without staining.
There are many youtube tutorials showing how to use FIJI for counting.
Good luck
Raef
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