Multi-target immunofluorescence using spectral FLIM-FRET for separation of undesirable antibody cross-labeling

Sumeet Rohilla; Benedikt Krämer; Felix Koberling; Ingo Gregor; Andreas C. Hocke

doi:10.1117/12.2526240

22 July 2019 Multi-target immunofluorescence using spectral FLIM-FRET for separation of undesirable antibody cross-labeling

Sumeet Rohilla, Benedikt Krämer, Felix Koberling, Ingo Gregor, Andreas C. Hocke

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Proceedings Volume 11076, Advances in Microscopic Imaging II; 1107618 (2019) https://doi.org/10.1117/12.2526240
Event: European Conferences on Biomedical Optics, 2019, Munich, Germany

Abstract

In this research work, we present a novel indirect immunolabeling method for labeling up to three different antigens using just two primary and fluorophore tagged secondary antibodies. We propose a viable solution to overcome the limitations imposed by limited variability in primary and secondary antibody type by leveraging cross-labeling phenomenon. Cross-labeling among two fluorophore conjugated secondary antibodies leads to FRET effects, resulting in changed spectral and fluorescence lifetime properties of donor molecule. To detect and quantify these changes in photophysical properties of immunolabeled species, we developed an eight-channel spectrally resolved fluorescence lifetime imaging (sFLIM) system. We demonstrate the capabilities of our approach in case of multi-targeted immunostaining in A549 cells. Efficient excitation of samples is achieved using two pulsed laser of wavelengths 485 nm and 561 nm operating in alternating/interleaved manner. Acquired multi-dimensional sFLIM data was pre-processed and analysed using state-of-the-art pattern-matching algorithm¹ which takes into account the information of fluorescence emission spectra as well as lifetime. The sFLIM detection system together with pattern-matching analysis enables separation of cross-linked labels from single labeled species.

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Sumeet Rohilla, Benedikt Krämer, Felix Koberling, Ingo Gregor, and Andreas C. Hocke "Multi-target immunofluorescence using spectral FLIM-FRET for separation of undesirable antibody cross-labeling", Proc. SPIE 11076, Advances in Microscopic Imaging II, 1107618 (22 July 2019); https://doi.org/10.1117/12.2526240

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KEYWORDS

Fluorescence resonance energy transfer

Luminescence

Control systems

Data acquisition

Fluorescence lifetime imaging

Pulsed laser operation

Molecules

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