Multiplexing FRET by PIE-FastFLIM and the phasor plots

Yuansheng Sun; Richard N. Day; Shih-Chu Liao; Beniamino Barbieri

doi:10.1117/12.2587523

5 March 2021 Multiplexing FRET by PIE-FastFLIM and the phasor plots

Yuansheng Sun, Richard N. Day, Shih-Chu Liao, Beniamino Barbieri

Proceedings Volume 11648, Multiphoton Microscopy in the Biomedical Sciences XXI; 116480B (2021) https://doi.org/10.1117/12.2587523
Event: SPIE BiOS, 2021, Online Only

Abstract

Förster resonance energy transfer (FRET) microscopy is a technique that provides the spatial and temporal resolution necessary to investigate protein-protein interactions in live specimens. One of the most robust ways of quantifying FRET is to measure the fluorescence lifetime of the donor fluorophore. Fluorescence lifetime imaging microscopy (FLIM) is routinely used in the biological sciences to monitor dynamic signaling events inside living cells. However, the demands of studying complex signaling networks in living cells using FRET-based biosensor probes requires the ability to track more than one donor fluorophore at the same time. Here, we demonstrate a novel (Pulsed Interleave Excitation) PIE-FastFLIM technique that can be used to simultaneously measure two FRET-based biosensor probes at the same pixel. The PIE-FastFLIM configuration uses tunable wavelengths and synchronized gating detection, which can be tailored and optimized for each biosensor probe. When combined with the model-free phasor plots analysis, the PIEFastFLIM approach enables quantitative multiplexed FRET measurements for tracking multiple signaling events simultaneously in live cells.

Conference Presentation

Citation Download Citation

Yuansheng Sun, Richard N. Day, Shih-Chu Liao, and Beniamino Barbieri "Multiplexing FRET by PIE-FastFLIM and the phasor plots", Proc. SPIE 11648, Multiphoton Microscopy in the Biomedical Sciences XXI, 116480B (5 March 2021); https://doi.org/10.1117/12.2587523

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