Fluorescence Multiplexing with Spectral Imaging and Combinatorics

Abstract

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 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 multi-anode photo-multiplier tubes make acquisition of such MuSIC-compatible datasets increasingly attainable.

Fluorescent Antibody Multiplexing with Oligo-Based Combinatorial Labeling

“…The use of MuSIC probes is in principal compatible with the cyclic methods, which would expand the number of probes that can be used per round of imaging using spectral scanning microscopes. Current cyclic methods on average use 10 rounds of four-color imaging and our previous simulation studies suggested that ~25 MuSIC probes can be accurately unmixed 37 . Therefore, the use of MuSIC probes could allow 10 rounds of 25 color imaging, thus increasing multiplexing capabilities by roughly another six-fold.…”

“…We recently developed an approach called Multiplexing using Spectral Imaging and Combinatorics (MuSIC), which leverages currently available fluorophores along with the power of combinatorics to increase the number of available probes for simultaneous staining 37 . Förster resonance energy transfer (FRET), a phenomenon whereby a higher energy fluorophore donates energy to a lower energy fluorophore 38 , is central to creating MuSIC probes with unique spectral signatures.…”

“…MuSIC probes are created using FRET-producing fluorophore combinations, which results in a probe emission spectrum that is linearly independent from that of the individual fluorophores that make up the combination, enabling unmixing. Our previous work, based on simulation, suggested that MuSIC may increase simultaneous fluorescence multiplexing capabilities ~4-5 fold 37 . Proof-of-principal experimental studies that focused on a small range of excitation wavelength space have shown that nine MuSIC probes can be accurately unmixed, which should increase when the full range is used.…”

Fluorescence Multiplexing with Spectral Imaging and Combinatorics

“…The first aspect of this simulation study was to consider how to combine the individual FPs. Bimolecular FRET is common 29 , and trimolecular FRET less so, but has been reported 30 . We therefore exhaustively considered single FPs, dimers and trimers, but .…”

Intriguing Heteroleptic ZnII bis(dipyrrinato) Emitters in the Far-Red Region With Large Pseudo-Stokes Shift for Bioimaging

“…Fluorophores with large ( pseudo )-Stokes shifts are highly desirable in biochemical experiments so that the label emission is at a significant longer wavelength than excitation (e.g. intracellular imaging enabling multiplexing) ( Rauf et al, 2010 ; Jeong et al, 2011 ; Shcherbakova et al, 2012 ; Holzapfel et al, 2018 ). Our new heteroleptic Zn II bis(dipyrrinato) complexes herein presented have intriguing properties to be used as fluorescent emitters for bioimaging.…”