This question is ultimately about determining background thresholds when using Coloc2.
Some years ago I determined Manders colocalization coefficients using the Zeiss AIM software for our multiphoton scope. The images were actually acquired with a Leica confocal and then deconvolved with AutoQuant. I found that the user interface for AIM provided the best pathway for determining the coefficients, and a large part of this was by enabling me to make a judgement about threshold values. The AIM software has now been replaced by ZEN, which I have not used, and so I’m looking at alternatives. Coloc2 looks like a great way to go, except that I’m not sure if it will allow me to determine thresholds manually. Can anyone let me know about this? I’m sure that many people will feel that automatic thresholding is the way to go, but I have enough prior information about my proteins of interest and my scanning methodology to prefer do it manually.
Thanks for any and all help.