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

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. Competing Interest Statement The authors have declared no competing interest. Footnotes ↵6 Lead contact Funder Information Declared National Institutes of Health, 1R35GM134963, S10OD024998 Copyright The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-NC-ND 4.0 International license.