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Single-Molecule Quantitative RNA Imaging

Single-Molecule Fluorescence In Situ Hybridization (smFISH) 

HCR™ RNA-FISH enables RNA absolute quantitation with single-molecule resolution in the anatomical context of thick autofluorescent samples, providing automatic background suppression throughout 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

High-Fidelity Single-Molecule RNA Imaging

HCR™ 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). 

Redundant 2-Channel Detection

To illustrate the quantitative nature of single-molecule HCR™ RNA-FISH, we detect a target mRNA using two probe sets that trigger different amplifiers carrying spectrally distinct fluorophores.  

Single-Molecule RNA Imaging Pointers

Fidelity Increases with Probe Set Size

Fidelity increases with probe set size due to the benefits of automatic background suppression. For single-molecule HCR™ RNA-FISH, we recommend using 30+ split-initiator probe pairs per target RNA (maximize probe set size given target length). 

Single-Molecule Resolution and Sensitivity

For single-molecule HCR™ RNA-FISH, we recommend signal amplification for 45-90 minutes to grow amplification polymers that are 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™ RNA-FISH protocols are compatible with tissue clearing to reduce background in highly autofluorescent tissues. 

Single-Molecule and Subcellular Quantitative Imaging Modes 

HCR™ 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™ RNA-FISH  to dPCR

While digital PCR (dPCR) enables digital RNA absolute quantitation in vitro without anatomical context, single-molecule HCR™ RNA-FISH enables digital RNA absolute quantitation in situ with anatomical context. 


Reagents will not generate amplified background even if they bind non-specifically within the sample


The same 2-stage enzyme-free protocol is used independent of the number of target RNAs


Custom probe set design for any target mRNA in any organism across the tree of life

Single-Molecule HCR™ RNA-FISH

 Digital RNA absolute quantitation

✓ Single-molecule resolution and sensitivity

✓ Thick autofluorescent samples

✓ Compatible with tissue clearing

✓ Automatic background suppression throughout the protocol

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