Cell outline based on transmission channel



I am trying to quantify the fluorescence dots of an PLA assay per cell using the Spot Detection Pipeline of the examples section. As an input I have .tif images which show an overlay of three channels: DAPI, PLA_dots (red fluorescence) and transmission.
Trying to identify the cell outline using the Identifysecondaryobjects module I was not able to generate outlines matching the cell outlines I can see in the transmission channel. Is there any chance to use the transmission channel for identifying the cell outlines so the shape is more proper and includes all PLA signals? (The nucleus does not need to be separated.)
I would be happy about any advice on quantifying a PLA assay properly :slight_smile:
At this Dropbox-Link you will find one of the images mentioned and also all three channels as single images: https://www.dropbox.com/sh/i86zkit9cg2t4hk/AAB-AMo1GONovgO7A95yPzhya?dl=0

Thank you and best regards,


IdentifySecondary (and CP in general) assumes fluorescent images, not brightfield, so I’m not surprised you didn’t get a good outcome here.

For transmitted light images we often suggest a workflow that combines ilastik and CellProfiler, and I definitely think that would work for you. Because your TL images are so textured though, you might be able to get around that by using the EnhanceOrSuppressFeatures->Texture module on your TL images; that’ll turn them into a psuedo-fluorescent image that would act as a good substrate for IDSecondary. Here’s a zoom in from the workspace viewer showing some cells where I identified the nuclei in IDPrimary and used that Enhance image for IDSecondary.

Pipeline attached
EnhanceTL.cppipe (8.4 KB)


Thank you for the fast reply and the pipeline!

I tried using the EnhaceOrSupressFeatures but as not all of the images are that well textured it didn’t work for running the whole batch. Here ilastik seems to get better results - so thank you for that suggestion, too! There seems to be an implementation to CP in progress - so maybe its getting even easier soon :slight_smile: