Quantification of cytoplasmic foci

cellprofiler

#1

Hello!
I am very interested in using CellProfiler to quantitate the cytoplasmic foci (GW bodies or p-bodies) that our lab are mainly interested in. One
basic question is whether these cytoplasmic foci can be quantitated by CellProfiler.

The following are the images as examples of the cytoplasmic foci that we would like to quantitate:
dental.ufl.edu/Faculty/EChan … 0GWB_0.JPG
dental.ufl.edu/Faculty/EChan … 0GWB_1.JPG

Green staining is anti-GW182, one of the major components of GWBs. The number and size of these cytoplasmic dots are our main focus. Is it fessible to tell the difference in number and size of these foci between the two images?

Please let me know your opinions. Your help is highly appreciated! Thanks a lot!

Shangli


#2

Hi Shangli,

There are two methods of doing this:

  1. Remove the background from the green image, identify, and quantitate

  2. Use the blue channel to identify the objects then quantitate

Here is a pipeline using both methods:
http://jura.wi.mit.edu/cellprofiler/linked_files/2006_04_13_ShangliPIPE.mat

The first method will be better for size measurements, but as you will see using your images and the above pipeline, areas with a high amount of green fluorescence may be identified incorrectly. I would recommend playing with the threshold settings in the module IdentifyPrimAutomatic to refine this.

The second method seems to work very well for a simple “count” of foci. If you have DNA and Actin stains, you can easily relate these two objects and get the number of foci per nuclei or cell. Again, you will notice that this method does not look so good for determining the size of the foci.

Good luck!

Mike


#3

Hi, Mike,

Thanks a lot for making the pipeline for us! We have been playing around with it this week and getting it to work, although we still need more time to make better use of it

Many thanks for your help!

Shangli


#4

Hi Shangli,

No problem, I hope you are able to use CellProfiler to analyze your images. Please feel free to ask more questions if you are unable to figure something out.

Mike