To overcome limitations of indirect immunofluorescence, a new method is presented to employ the ostensible disadvantage of cross-labeling secondary antibodies by separation of the fluorescence signals via spectral FLIM-FRET. The undesirable cross-labeling among secondary antibodies leads to the generation of new characteristic FRET emission spectra including a change in the donor lifetime. We used a spectrally resolved FLIM detection system with pulse interleaved excitation. The combined spectral FLIM-FRET and pattern-matching analysis forms an excellent tool for use in indirect immunofluorescence that overcomes the undesirable effect of secondary antibody cross-labeling by assigning separate color channels to cross-labeled fluorescent antibodies.
Sumeet Rohilla, Benedikt Kraemer, Felix Koberling, Uwe Ortmann, Ingo Gregor, Andreas C. Hocke, and Rainer Erdmann, "Multi-target immunofluorescence using spectral FLIM-FRET for separation of undesirable antibody cross-labeling and autofluorescence (Conference Presentation)," Proc. SPIE 11244, Multiphoton Microscopy in the Biomedical Sciences XX, 1124418 (Presented at SPIE BiOS: February 04, 2020; Published: 9 March 2020); https://doi.org/10.1117/12.2542413.6138713608001.
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