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.

中文翻译：

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荧光多路复用与光谱成像和组合

紫外到红外荧光是一种通用且易于使用的测定方式，但众所周知，由于宽发射光谱的重叠而难以复用。我们提出了一种称为多路复用的荧光方法，使用光谱成像和c组合学（音乐）。MuSIC 包括从单个荧光团的共价连接组合创建新的独立探针，利用当前可用探针的广泛调色板和组合学的数学能力。混合物中的探针水平可以从光谱发射扫描数据中推断出来。理论和模拟表明，使用目前可用的染料和测量工具，MuSIC 可以将荧光复用增加 ~ 4-5 倍。实验性原理验证证明了使用约 25% 的可用激发波长窗口 (380–480 nm) 对九种基于溶液的探针进行了稳健的解复用，这与理论一致。白色激光、基于角滤光片的波长扫描以及大型、