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

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.