What are problems that can occur in (multiplex) immunofluorescent imaging and how can they be solved?

Autofluorescence; by placing the tissue in a dark, UV-lit box, which removes the autofluorescence. spectral overlap (the overlap between the emissions of the different fluorophores); this is solved by using multispectral microscopy, in which you stepwise scan the visible spectrum. this generates separate pixels for each fluorophore per scan. these pixels can be separated and you can determine from which fluorophore the pixel comes from.