Fluorescence Multiplexing with Spectral Imaging and Combinatorics

Ultraviolet-to-infrared fluorescence is a versatile and accessible assay modality but is notoriously hard to multiplex due to overlap of wide emission spectra. We present an approach for fluorescence called multiplexing using spectral imaging and combinatorics (MuSIC). MuSIC consists of creating new independent probes from covalently linked combinations of individual fluorophores, leveraging the wide palette of currently available probes with the mathematical power of combinatorics. Probe levels in a mixture can be inferred from spectral emission scanning data. Theory and simulations suggest MuSIC can increase fluorescence multiplexing ∼4–5 fold using currently available dyes and measurement tools. Experimental proof-of-principle demonstrates robust demultiplexing of nine solution-based probes using ∼25% of the available excitation wavelength window (380–480 nm), consistent with theory. The increasing prevalence of white lasers, angle filter-based wavelength scanning, and large, sensitive multianode photomultiplier tubes make acquisition of such MuSIC-compatible data sets increasingly attainable.