![]() (EDIT: for the record, that’s not on you. I know this has been discussed ad nauseam in other circles, but for those who don’t know, Pearson was a vocal antisemite, eugenics advocate, and social darwinist: Karl Pearson - Wikipedia. Probably also worth it to generate scrambled control images as well.įinal thought: It bugs me to no end that we continue to use that bigot’s name. 2 in image B to get the PCC for the rest of the cell. 2 in image A to get the PCC for the inner circle, and channel 1 v. You can now run your colocalization tests on channel 1 v. Next just do the same process of converting to 32 bit and converting those zeros to Nan. If you then hit clear outside it’s only one step. Or you can make a ROI that is specifically the cytoplasm minus the box by selecting your two ROIs in the roi manager and then going to the “more” button on the ROI manager and selecting XOR and then selecting “Add” to create a new selection that is just the cytoplasm minus the inner circle. You can do this in two steps by selecting your cell boundary and running clear outside, then selecting the inner circle and running clear. Do the same thing for Image B, but you want to clear the inner circle and the area around the cell boundary.Then in the very last step you can use math>Substract…>1.0. Note: Depending on whether you have true zero value pixels within that circle it might make sense to use math>Add…> 1.0 to your image before running “clear outside” so that those “real” zeros don’t get converted to NaN. To do so, you can convert your image to a 32 bit (image>type>32-bit) and then threshold (image>adjust>threshold), set your lower bound to 0.01 and your upper bound to the highest value, then hit apply and “set to NaN”. ![]() Since you understandably don’t want those zeros to skew your Pearson’s* correlation coefficient, you need to then convert them to NaNs. I think the easiest way to do this is to first select the ROI corresponding to the inner circle and “clear outside.” This will make all pixels outside of your selection equal to zero. Duplicate the two color image so Image A will be used for the colocalization of red and green pixels in the internal circle (presumably pericentrosomal signal?) and image B will be used for the colocalization of red and green pixels in the rest of the cytoplasm.įor Image A you will then set the pixels outside of your ROI of interest to NaN. ![]()
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