dHCR Imaging

dHCR imaging enables RNA absolute quantitation with single-molecule resolution in the anatomical context of thick autofluorescent samples.

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Digital Quantitative Signal

dHCR signal is digital in the form of diffraction-limited dots representing individual target molecules. 

High-fidelity single-molecule fluorescence in situ hybridization (FISH) in a whole-mount chicken embryo

Redundant 2-Channel Detection

To illustrate quantitative fidelity with single-molecule resolution within a whole-mount vertebrate embryo, we detect a target mRNA using two probe sets that trigger different HCR amplifiers carrying spectrally distinct fluorophores.  

Fidelity Increases with Probe Set Size

Using HCR v3.0, automatic background suppression ensures that fidelity increases with probe set size. Learn more

As a result, for dHCR imaging, we recommend using 30 or more split-initiator probe pairs per target RNA (maximize probe set size given target length). 

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Single-Molecule Resolution and Sensitivity

For dHCR imaging, we recommend HCR 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., 0.5 mm brain slices). 

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Tissue Clearing

HCR imaging protocols are compatible with tissue clearing to reduce background in highly autofluorescent tissues.