Quantitative models of Förster resonance energy transfer (FRET)—pioneered by Förster—define our understanding of FRET and underpin its widespread use. However, multicolour FRET (mFRET), which arises between multiple, stochastically distributed fluorophores, lacks a mechanistic model and remains intractable. mFRET notably arises in fluorescently barcoded microparticles, resulting in a complex, non-orthogonal fluorescence response that impedes their encoding and decoding. Here, we introduce an ensemble mFRET (emFRET) model, and apply it to guide barcoding into regimes with extreme FRET. We further introduce a facile, proportional multicolour labelling method using oligonucleotides as homogeneous linkers. A total of 580 barcodes were rapidly designed and validated using four dyes—with FRET efficiencies reaching 76%—and used for multiplexed immunoassays with cytometric readout and fully automated decoding. The emFRET model helps to expand the barcoding capacity of barcoded microparticles using common organic dyes and will benefit other applications subject to stochastic mFRET.

由Förster率先提出的Förster共振能量转移（FRET）的定量模型定义了我们对FRET的理解，并支持了FRET的广泛使用。但是，出现在多个随机分布的荧光团之间的多色FRET（mFRET）缺乏机理模型，并且仍然难以处理。mFRET明显出现在荧光条形码的微粒中，导致复杂，非正交的荧光响应，从而阻碍了它们的编码和解码。在这里，我们介绍了一个集成的mFRET（emFRET）模型，并将其用于指导条形码进入具有极端FRET的状态。我们进一步介绍了一种使用寡核苷酸作为均质接头的简便，成比例的多色标记方法。使用四种染料快速设计和验证了总共580条条形码，FRET效率达到76％，并用于带有细胞计数读数和全自动解码的多重免疫分析。emFRET模型有助于使用常见的有机染料扩展条形码条形码的条形码编码能力，并将使其他受随机mFRET限制的应用受益。