A target-triggered self-assembly QFRP strategy for in situ single molecule imaging of intracellular mRNAs

Precise visualization of scarce mRNA transcripts is hindered by the limited performance of conventional probes and the complexity of the intracellular milieu. Here, we present a target-triggered self-assembly-based single quantum dot (QD) fluorescence resonance energy transfer (FRET) probe system (QFRP) for high-resolution mRNA imaging in diverse living cell lines. Compared with conventional single-fluorophore probes, QFRP exhibits substantially enhanced sensitivity and quantitative accuracy, achieving a detection limit as low as 35 fM while markedly suppressing false positives through dualsignal colocalization. By leveraging QDs as photostable donors and assembled Cy5 acceptors, QFRP enables effective visualization of subtle differences in mRNA expression between cancerous and normal cells, revealing essential biological heterogeneity in complex intracellular environments. These findings demonstrate the robustness, specificity, and versatility of this single-entity RET-based nanosystem, underscoring its potential for advanced molecular imaging and precision medicine, particularly in the visualized analysis of low-abundance targets within complex microenvironments related to tumor progression.