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. Author manuscript; available in PMC: 2026 May 17.
Published in final edited form as: ACS Sens. 2026 Feb 6;11(2):909–922. doi: 10.1021/acssensors.5c01707

Figure 1.

Figure 1.

Multiplexed dark FRET (MDF) eliminates spectral crosstalk and enables independent resolution of multiple biosensors. (A) Schematic representation of multiplexing challenges using conventional fluorescent acceptors. When an mNeonGreen/mCherry (mNg/mCh) FRET biosensor is combined with any red-shifted biosensor (e.g., mScarlet-I3/ShadowR; mScI3/ShR), the strong mCh emission overlaps the mScI3 donor detection window, conflating the two fluorophore FLTs. In the case of mCh, its short 1.6 ns FLT results in an artificially shortening the apparent mScI3 donor FLT and creates a false-positive FRET signature. (B) In contrast, multiplexing two MDF biosensors (mNg/ShadowY and mScI3/ShadowR) eliminates acceptor emission due to the ShadowY/R low quantum yield (QY) and prevents spectral bleed-through, allowing clean, independent FLT measurements for each channel. Emission spectra for mNg (green/red donor), mCh (green/red acceptor), and mScI3 (red/far-red donor), along with corresponding emission filter sets, highlight the spectral overlap concern that leads to artifacts when multiplexing FRET systems with conventional acceptor (right panels of A and B). Control linker biosensors consist of an acceptor–linker (L)–donor fusion using a 23-aa G4S-based linker. FRET efficiency was determined by comparing donor FLT in the presence versus absence of acceptor under 473 nm excitation for mNg (C) or 532 nm excitation for mScI3 (D). As shown in panel D, the mCh-L-mNg construct produces a short-lifetime component in the red channel due to mCh emission, complicating interpretation of any red-donor FLT measurement. The change in FLT (ΔFLT) for the donor+acceptor system relative to donor-only system indicates FRET. However, the acceptor emission (red channel) from mCh-L-mNg has a short FLT, compounding direct measure of FLT for a red donor biosensor. (E) Monitoring red channel FLT for mixed biosensor samples where cells are expressing either ShR-L-mScI3 or mCh-L-mNg constructs results in a significantly reduced observed FLT (albeit not an actual FRET signal). (F, G) In contrast, when two MDF shadow acceptor biosensors are used (ShY-L-mNg and ShR-L-mScI3), this artifact is not present, allowing the recovery of each individual population’s FLT. Panels A, B generated in Biorender. Data represent mean ± SD of n = 3 biological replicates; significance determined by one-way ANOVA (ns = p > 0.05; **** = p < 0.0001).