How to only count cy3 objects a certain distance from a dapi object


#1

I am counting cilia (which may appear as tiny dots or thin string like objects). The cilia are in Cy3, while the dapi are blue. Unfortunately, the antibody for cilia sometimes binds non-specifically, resulting in random dots that look like cilia, but aren’t cilia (we know they aren’t cilia since they are not near a dapi object). The cilia should be no more than 15 microns from a dapi object, so I am wondering if there is a way to tell the program to only count cy3 objects that are no more than 15 microns from a dapi object.
We have told the program already how to identify cilia objects and dapi objects, but due to the non-specific cilia antibody staining, we want the program to only count the cy3 objects that are 15 microns from dapi.

Thanks for the help!


#2

Hi,

You should be able to expand the DAPI objects by however many pixels 15 microns is in your images using the ExpandOrShrinkObjects module set to “Expand by a specified number of pixels” (call the output objects say, DAPI_Expanded_15microns); once you’ve done that, you can use MaskImage to mask your Cy3 image to only include areas inside the new DAPI_Expanded_15microns objects you just made; you can then identify and count cilia in this masked image instead of the whole Cy3 image.

Good luck!


#3

Hi, thanks for the advice! I shall try this