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Single-dye, transfection-free FLIM multiplexing via bioorthogonal chemistry

Dadina, N.; Kwon, J. H.; Lesiak, L.; Zheng, S.; Zoltek, M.; Brauer, D.; Schepartz, A.

2025-08-16 biochemistry
10.1101/2025.08.13.670148 bioRxiv
Show abstract

Fluorescence lifetime imaging microscopy (FLIM) can visualize multiple targets in a single spectral window, making it a powerful tool to overcome multiplexing limitations during live cell fluorescence microscopy. Here we show that small molecule probes-which are well-suited for imaging applications due to high specificity, low toxicity, and the elimination of transfection requirements-can be fine-tuned via bioorthogonal chemistry to exhibit predictably different fluorescent lifetimes suitable for FLIM multiplexing.

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