FLUORESCENCE OVERLAY ANTIGEN MAPPING OF THE EPIDERMAL BASEMENT-MEMBRANE ZONE .2. COLOR FIDELITY

In this second report on the fluorescence overlay antigen mapping (FOA M) technique, we highlight some of the errors that may influence faith ful color rendition of slide preparations using triple antigen immunof luorescence staining. Reliable interpretation of multicolor fluorescen ce images requires that the observer can unambiguously assign each col or in these images to the presence of a specific combination of the la beled antigens, This is possible only when the image fidelity meets ce rtain standards, The present study concentrates on color fidelity whic h is easily undermined by spectral matching errors, image contrast err ors, and exposure time errors. Evaluation of these errors, using the p hotomicrographic overlay variant of FOAM, showed the potential unrelia bility of the simultaneous use of multiple fluorophores for immunofluo rescence microscopy. The procedures described here may serve as a soli d starting point in formulating technical conditions that allow reliab le color rendition in multicolor immunofluorescence microscopy. Furthe rmore, these procedures can be adapted to studies other than the analy sis of basement membrane zone antigens, to which they have been first applied.