Hi all, I try to characterise vesicle staining of two channels, one with protein of interest (0) and with a specific marker (1). What I am doing is using a spot detection to get my vesicles recognised in each channel, and then use labelling arithmetic to define, which actually do overlap (Setting AND). However, I realised, that it then also marks those which only partially overlap, which I don’t want. Unfortunately, I can’t use “Congruent”, as the signal is almost never 100% the same in both channels even when both vesicles overlap to 100%. Therefore, I would like to work with the centre of mass (using Segment features and a math code), and then see the distance, and if it is below a certain threshold, count the two vesicles as overlapping. I know there is already a nice example workflow, however, this one calculates from one ROI to all other labels. I would like to use the Labeling Arithmetic to get the ones which overlap somehow, and then use those new generated labels, to take always the two who led to that overlap and only calculate the distance between their centre of mass. Can you give me some idea on how to start this? I guess I need something to iterate through the overlapping labels one by one, and in this loop use Labeling arithmetic with AND to get the corresponding vesicle. Then I would have to define the centre of mass and then append the second vesicle informations next to the first one and then calculate the distance. I created a workflow, up to the point where I am now, based on a workflow once made by stelfrich :slight_smile: and a simplified example picture with some circles (two overlap just by parts, two overlap completely). If you can give me some help would be great, also, if my approach has some issues as well. Best, Chris Count_Overlapping_Vesicles_8.knwf (172.1 KB) a.tif (470.9 KB)

Vesicle characterisation

imagejan January 13, 2021, 7:16pm 3

Maybe not exactly the same issue, but this discussion might help: