I’m teaching myself to do colocalization quantification for revision to a manuscript, but I’m very much out of my depth. After reading the Colocalization Analysis and Colo2 wiki as well as “A practical guide to evaluating colocalization in biological microscopy”, I have a very basic question on what pre-processing/thresholding is needed for my particular data in order to calculate MCC values. In case this is of interest, the two channels I’m using for coloc2 are (1) nanoparticle fluorescence and (2) cav1 protein fluorescence.
I have super-resolution images of cells acquired on a DeltaVision OMX microscope. The images were processed using the scope’s software (softWoRx) to perform structured illumination reconstruction and camera alignment. The result is a 32-bit image stack that I have saved as a tif file. I’ll be honest, I don’t understand this preprocessing outside of the “big picture”, but it is my understanding that this is comparable to deconvolution processing.
There is very little background after the SI reconstruction, and so I am wondering if I need to do any further processing, such as Costes autothresholding, when using Coloc2, or if I should only look at the original Mander’s coefficients?
I appreciate any help or advice you can give.