ArticleCell reports methods2026
Development of far-red fluorescent proteins for temporal domain multiplexing and super-resolution imaging.
Article in Cell reports methods, 2026. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Not yet cited in PubMed.
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Abstract
The diverse spectral and photochemical properties of fluorescent proteins enable imaging applications ranging from organelle labeling to super-resolution and multiplexed live-cell microscopy. Here, we report three far-red fluorescent proteins, named mfRFP, mfRFP-A, and mCardinal-A, that share similar fluorescence spectra (excitation/emission ∼600/660 nm) but exhibit distinct photobleaching rates. Exploiting differential photostability, we performed per-pixel unmixing of three proteins simultaneously using temporal domain multiplexing (TDM), acquiring BrainBow-like images of cellular populations and resolving subcellular structures in 3D within a single imaging channel, without hardware modifications. We established quantitative criteria for selecting FP pairs that support efficient TDM unmixing and benchmarked TDM against fluorescence lifetime- and photobleaching kinetics-based alternatives. The most photostable variant, mfRFP, was further validated for STED super-resolution imaging of structural proteins in mammalian cells and for neuroimaging in mice, zebrafish, and C. elegans.
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