Figure 2.

Z-projection artifacts and incompletely included cells can lead to false positive and false negative colocalization. Cartoons of tissue sections, on the left viewed from the side, and on the right viewed from the top, as indicated by the X, Y and Z axes. Green ovals represent cell bodies with a cytoplasmic label, and magenta circles represent a labeled subcellular structure, e.g. a cell nucleus. (A) There is no colocalization of the two labels on the right when viewed in the ZX plane, but in the commonly used XY-plane a Z-projection artifact arises, presenting as a double-labeled cell. (B) The cell to the right is only partly included, with only the green cytoplasmic signal present in the section. Here, a false negative would arise irrespective of the angle of view. False observations can also occur when both labels are distributed throughout the cell if one signal is weak and goes undetected in cells only partially contained within the section.