Light-Scattering In Situ Imaging for Intracellular Gene Fusion Transcript Analysis at Single-Molecule Resolution

Imaging of intracellular fusion gene transcripts at single-molecule resolution is crucial for elucidating their pathophysiological roles in diseases, particularly hematological malignancies such as leukemias and lymphomas, and for advancing molecular diagnostics. While fluorescence in situ hybridization (FISH)-based methods enable intracellular detection of single gene fusion transcripts, their utility is limited by labor-intensive washing steps for removing excess fluorophore-labeled probes and the inherent photobleaching susceptibility of organic fluorophores. To overcome these limitations, we developed a washing-free imaging platform that leverages the resonance light-scattering properties of gold nanoparticles (AuNPs) for photostable, low-background detection of fusion transcripts at single-molecule resolution in situ. The assay begins with fusion-transcript-triggered in situ ligation, followed by rolling circle amplification (RCA) to generate tandem-repeat DNA sequences. AuNP-DNA probes specifically target and bind these RCA-amplified sequences, forming assembled plasmonic nanostructures that produce enhanced light-scattering signals that can be detected at single-transcript resolution. Critically, unbound AuNP-DNA probes remain dispersed, generating negligible background signals without washing steps. Furthermore, the intrinsic optical stability of AuNPs eliminates the photobleaching effect inherent to fluorescence-based approaches. This robust methodology allows reliable imaging and precise enumeration of intracellular fusion transcripts, offering transformative potential for investigating gene fusion and advancing clinical diagnostics in malignancies.