Counting foci in bacterial cells

Hello, I’m just wondering if someone could give some advice on using Cell Profiler to count the number of fluorescent foci present per bacterial cell in an image.

I need to be able to identify cells, and count how many GFP-tagged protein ‘spots’ are present in each cell, and having looked at the Speckle Counting example on this website, it seems possible. Up until now, I have been doing this both manually (very time consuming for hundreds of images!) and by writing scripts in imageJ but so far this has proven difficult and unreliable. If Cell Profiler can do this for me, could someone give me some pointers on how best to adapt the Speckle Count script to suit the images and cells I am working with?

I have attached a bright field and its respective GFP example images showing what I need to count, which I hope will make things clear (they may need to be dowloaded and opened in imageJ as for some reason they’re appearing as black when uploaded)

Thanks to anyone who can provide assistance!

Martin




Centre for Bacterial Cell Biology
Institute of Cell and Molecular Biosciences
Newcastle University
United Kingdom

Hi,

Attached is a pipeline which does what I think you’re asking: identifies the cells from the bright field (I called them nuclei by accident), then counts the foci in each. You would definitely get better results if the signal to noise were improved- the background is rather speckled which requires some filtering before the identification step, and I have trouble perfectly segmenting each cell (right now I actually don’t try to separate those that are touching, which leads to slightly inaccurate counts but better than the alternative where each cell got chopped up into pieces because the intensity is so variable).

The GFP channel also has some signal to noise ratio issues- I think the foci identification is rather good in this pipeline, but could be even better with slightly better imaging conditions.

If you run this pipeline and open up the Nuclei.xls file it creates, look at Children_foci_Count, that will be the number of foci per each identified cell.
count_foci_per_bacteriaPIPE.mat (1.31 KB)