Fluorescence Multiplexing with Spectral Imaging and Combinatorics.

Hadassa Y Holzapfel,Mehdi Bouhaddou,Danielle Putur,Sarah Comer,Marc R Birtwistle,Caitlin M Anglin,Alan D Stern

doi:10.1021/acscombsci.8b00101

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 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.

Highlights

Fluorescence in the UV to infrared range is one of the most widely-used and accessible quantitative and qualitative assay modalities across the life and physical sciences1
We present an approach for fluorescence multiplexing using spectral imaging and combinatorics (MuSIC)
The emission spectra of a mixture of fluorophores can be cast as the sum of its component parts with a matrix equation (Fig. 1A)

Summary

INTRODUCTION

Fluorescence in the UV to infrared range is one of the most widely-used and accessible quantitative and qualitative assay modalities across the life and physical sciences. One of the arguably most multiplexed and data dense experimental modalities—Illumina “next-generation” deep DNA sequencing, relies on such four-color imaging; one for each DNA base. Alternatives to fluorescence are many; for example mass cytometry which measures levels of ~30 specific isotope tags as opposed to fluorophores12,13 Despite these advances, there remains yet to be reported, to our knowledge, a fluorescence-based technology that simultaneously can demultiplex more than 4-7 analytes within a mixture. There remains yet to be reported, to our knowledge, a fluorescence-based technology that simultaneously can demultiplex more than 4-7 analytes within a mixture Such an ability may have widespread impact, due to the prevalence, sensitivity and versatility of fluorescence as a measurement tool. The advent and accessibility of white lasers, angle filterbased emission wavelength scanning, and large, sensitive multi-anode photo-multiplier tubes make acquisition of such MuSIC-compatible datasets increasingly attainable

RESULTS AND DISCUSSION

METHODS

16 Fluorescent Proteins