Multi-target immunofluorescence by separation of antibody cross-labelling via spectral-FLIM-FRET

In biomedical research, indirect immunofluorescence labelling by use of primary and secondary antibodies is central for revealing the spatial distribution of multiple cellular antigens. However, labelling is regularly restricted to few antigens since species variation of primary and corresponding secondary antibodies is limited bearing the risk of unspecific cross-labelling. Here, we introduce a novel microscopic procedure for leveraging undesirable cross-labelling effects among secondary antibodies thereby increasing the number of fluorophore channels. Under cross-labelling conditions, commonly used fluorophores change chemical-physical properties by 'Förster resonance energy transfer' leading to defined changes in spectral emission and lifetime decay. By use of spectral fluorescence lifetime imaging and pattern-matching, we demonstrate precise separation of cross-labelled cellular antigens where conventional imaging completely fails. Consequently, this undesired effect serves for an innovative imaging procedure to separate critical antigens where antibody species variation is limited and allows for multi-target labelling by attribution of new fluorophore cross-labelling channels.