Fluorescence overlay antigen mapping of the epidermal basement membrane zone: II. Color fidelity

In this second report on the fluorescence overlay antigen mapping (FOAM) technique, we highlight some of the errors that may influence faithful color rendition of slide preparations using triple antigen immunofluorescence staining. Reliable interpretation of multicolor fluorescence images requires that the observer can unambiguously assign each color in these images to the presence of a specific combination of the labeled antigens. This is possible only when the image fidelity meets certain standards. The present study concentrates on color fidelity which is easily undermined by spectral matching errors, image contrast errors, and exposure time errors. Evaluation of these errors, using the photomicrographic overlay variant of FOAM, showed the potential unreliability of the simultaneous use of multiple fluorophores for immunofluorescence microscopy. The procedures described here may serve as a solid starting point in formulating technical conditions that allow reliable color rendition in multicolor immunofluorescence microscopy. Furthermore, these procedures can be adapted to studies other than the analysis of basement membrane zone antigens, to which they have been first applied.