dHCR RNA Imaging
Single-Molecule Fluorescence In Situ Hybridization (smFISH)
dHCR RNA imaging using 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.
dHCR 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
dHCR imaging 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 dHCR imaging, we detect a target mRNA using two probe sets that trigger different HCR amplifiers carrying spectrally distinct fluorophores.
dHCR Imaging Pointers
Fidelity Increases with Probe Set Size
Fidelity increases with probe set size due to the benefits of automatic background suppression. For dHCR RNA imaging, we recommend using 30+ split-initiator probe pairs per target RNA (maximize probe set size given target length).
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., thick brain slices).
Compatible with Tissue Clearing
HCR imaging protocols are compatible with tissue clearing to reduce background in highly autofluorescent tissues.
dHCR and qHCR Quantitative Imaging Modes
HCR RNA-FISH supports two quantitative RNA imaging modes (see comparison): dHCR imaging (digital RNA absolute quantitation with single-molecule resolution) and qHCR imaging (analog RNA relative quantitation with subcellular resolution).
Comparison of dHCR to dPCR
While digital PCR (dPCR) enables digital RNA absolute quantitation in vitro without anatomical context, dHCR imaging enables digital RNA absolute quantitation in situ with anatomical context.
✓ Digital RNA absolute quantitation
✓ Single-molecule resolution and sensitivity
✓ Thick autofluorescent samples
✓ Compatible with tissue clearing
✓ Automatic background suppression throughout the protocol