Single-Molecule Quantitative RNA Imaging
Single-Molecule Fluorescence In Situ Hybridization (smFISH)
HCR™ Gold RNA-FISH enables RNA absolute quantitation with single-molecule resolution in the anatomical context of thick autofluorescent samples, exploiting automatic background suppression troughout the protocol for dramatically enhanced performance and ease-of-use


Quantitative Signal
Single-molecule signal provides digital quantitation in the form of diffraction-limited dots representing individual RNA target molecules
High-Fidelity smFISH in a Whole-Mount Chicken Embryo
Redundant 2-Channel Detection
To illustrate the quantitative nature of single-molecule HCR™ Gold RNA-FISH, we detect a target mRNA using two probe sets that trigger different amplifiers carrying spectrally distinct fluorophores.


High-Fidelity Single-Molecule RNA Imaging
HCR™ Gold RNA-FISH enables high-fidelity imaging of single mRNA molecules even in thick autofluorescent samples (e.g., whole-mount vertebrate embryos and thick brain slices).
Single-Molecule RNA Imaging Pointers
Fidelity Increases with Number of Binding Sites
Fidelity increases with number of target-binding sites due to the benefits of automatic background suppression. For single-molecule HCR™ Gold RNA-FISH, we recommend using 30+ binding sites per target RNA (maximize probe set size given target length).
Single-Molecule Resolution and Sensitivity
For single-molecule HCR™ Gold RNA-FISH, limiting signal amplification to 45-90 minutes will lead to growth of amplification polymers short enough to retain diffraction-limited single-molecule resolution, but long enough to provide single-molecule sensitivity even in thick autofluorescent samples (e.g., thick brain slices).
Compatible with Tissue Clearing
HCR™ Gold RNA-FISH protocols are compatible with tissue clearing to reduce background in highly autofluorescent tissues.
Single-Molecule and Subcellular Quantitative Imaging Modes
HCR™ Gold RNA-FISH supports two quantitative RNA imaging modes (see comparison): single-molecule imaging (digital RNA absolute quantitation with single-molecule resolution) and subcellular imaging (analog RNA relative quantitation with subcellular resolution).
Comparison of Single-Molecule HCR™ Gold RNA-FISH to dPCR
While digital PCR (dPCR) enables digital RNA absolute quantitation in vitro without anatomical context, single-molecule HCR™ Gold RNA-FISH enables digital RNA absolute quantitation in situ with anatomical context.