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 .