In the PMT set up dialog in ScanImage you should name the channels appropriately. This matters because the autoROI algorithm that finds the samples works best using the Red PMT. The far-red PMT tends to have too little autofluoresence and in the blue the agar autofluoresces a lot at longer excitation wavelengths. You should name your PMTs "Red", "Green", and "Blue". If you have "Far Red" also, that is fine: the system will ignore it. The names are case-insensitive. If you do this makae sure the colon is present. Just don't name the PMTs anything else, like "Low", "Med", "Short" or "<490 nm", etc. If you do not name the PMTs the autoROI algorithm will choose the brightest channel upon which to work.