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
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,