Single-Dye, Transfection-Free FLIM Multiplexing via Bioorthogonal Chemistry

Fluorescence microscopy is a foundational tool in cell biology research. Its power shines brightest when used to visualize multiple independent species simultaneously, a strategy known as multiplexing. However, the compressed nature of the visible spectrum effectively limits the number of independent species that can be multiplexed, especially in live cells and using long wavelength light to limit phototoxicity. Fluorescence lifetime imaging microscopy (FLIM) can overcome this limitation, allowing the simultaneous visualization of multiple independent species in a single spectral window. Most multiplexing strategies that rely on FLIM make use of self-labeling tags to localize a fluorophore to a specific organelle or cell structure. Although simple to execute, this strategy restricts experiments to cell types that can be transfected or engineered and does not easily support long time-lapse imaging. Here we show that cell-permeant 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 fluorescence lifetimes that are distinct for each partner and each organelle. The lifetime differences are sufficiently separable to support FLIM multiplexing. Using this strategy, we selectively image three cell structures in live cells without genetic manipulations using a single FLIM-active dye and one spectral channel.