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.