Figure 5

Spectral-FLIM-FRET for separation of cross-labelling in triple antigen indirect immunofluorescence. A549 cells were immunolabelled for three different cellular antigens with just two primary (mouse, rabbit) and secondary (“goat-anti-rabbit Alexa488”; “rabbit-anti-mouse Alexa546”) antibody (AB) species types. Following a sequential labelling scheme, single labelling of TOM20 (“goat-anti-rabbit Alexa488”), golgin (“rabbit-anti-mouse Alexa546”) and cross-labelling of pan-cytokeratin (“goat-anti-rabbit Alexa488”; “rabbit-anti-mouse Alexa546”) was achieved. (A) Conventional channel mode imaging was insufficient for eliminating false-positive pan-cytokeratin (closed arrowhead) in the TOM20 channel and golgin channel. (B) The spectral-FLIM data acquisition and pattern-matching analysis allows to separate the fluorescence contributions of all three antigens into independent analysis channels: Different structures (TOM20 (open arrowhead), pan-cytokeratin (closed arrowhead), golgin (open circle)) are clearly visualized with a notable false-positive suppression (compare merged images of A and B). (C) Reference patterns, combining fluorescence decay (left panel, 485 nm and 561 nm excitation laser wavelength were used) and emission spectra (right panel) information, of Alexa488 labelled TOM20, Alexa546 labelled golgin as well as FRET AB pair labelled pan-cytokeratin used by pattern-matching algorithm for unmixing of their fluorescence contribution per pixel. Representative images from three independent experiments are shown; scale bars 5 µm.