Traditional single-molecule fluorescence in situ hybridization
(smFISH) methods for RNA detection often face sensitivity challenges
due to the low fluorescence intensity of the probe. Also, short-lived
autofluorescence complicates obtaining clear signals from tissue sections.
In response, we have developed an smFISH probe using highly grafted
lanthanide complexes to address both concentration quenching and autofluorescence
background. Our approach involves an oligo PCR incorporating azide-dUTP,
enabling conjugation with lanthanide complexes. This method has proven
to be stable, convenient, and cost-effective. Notably, for the mRNA
detection in SKBR3 cells, the lanthanide probe group exhibited 2.5
times higher luminescence intensity and detected 3 times more signal
points in cells compared with the Cy3 group. Furthermore, we successfully
applied the probe to image HER2 mRNA molecules in breast cancer FFPE
tissue sections, achieving a 2.7-fold improvement in sensitivity compared
to Cy3-based probes. These results emphasize the potential of time-resolved
smFISH as a highly sensitive method for nucleic acid detection, free
of background fluorescence interference.