dHCR imaging enables RNA absolute quantitation with single-molecule resolution in the anatomical context of thick autofluorescent samples.
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).
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).
HCR imaging protocols are compatible with tissue clearing to reduce background in highly autofluorescent tissues.
Orthogonal HCR amplifiers enable 1-step signal amplification for up to 5 target RNAs simultaneously. Straightforward multiplexing is achieved using the same 1-day protocol independent of the number of target RNAs. Learn more
Custom Probe Set Design
Custom probe set design is available for any target mRNA in any organism across the tree of life. Learn more
Read-Out/Read-In Quantitative Discovery
By quantifying RNA within intact specimens, dHCR imaging enables bidirectional quantitative discovery in an anatomical context. Learn more
dHCR and qHCR Quantitative Imaging Modes
HCR supports two quantitative imaging modes: digital RNA absolute quantitation with single-molecule resolution (dHCR mode), and analog RNA relative quantitation with subcellular resolution (qHCR mode). See comparison