Property

Signal 

Probe set size

Amplification time

Magnification

Target expression level

dHCR RNA imaging
Digital single-molecule dots
30+ split-initiator probe pairs
45-90 min
Higher (63× to 100×)
Low or medium

qHCR RNA Imaging

Analog subcellular voxel intensities

20+ split-initiator probe pairs

Overnight

Lower (20× to 40×)

Medium or high

Property

Signal 

Probe set size

Amplification time

Magnification

Target expression level

Comparison of qHCR and dHCR Quantitative RNA Imaging Modes

HCR RNA-FISH probe sets, amplifiers, and buffers support two quantitative imaging modes with automatic background suppression throughout the protocol:

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qHCR RNA Imaging

qHCR RNA imaging enables analog RNA relative quantitation with subcellular resolution in the anatomical context of thick autofluorescent samples. 

dHCR RNA Imaging

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

dHCR RNA imaging
Digital single-molecule dots
30+ split-initiator probe pairs
45-90 min
Higher (63× to 100×)
Low or medium

qHCR RNA Imaging

Analog subcellular voxel intensities

20+ split-initiator probe pairs

Overnight

Lower (20× to 40×)

Medium or high

Property

Signal 

Probe set size

Amplification time

Magnification

Target expression level

Same Reagents For Both Imaging Modes
The same HCR probes, amplifiers, and buffers are used for both imaging modes, so imaging can be performed in qHCR mode (longer amplification time, lower magnification) or dHCR imaging mode (shorter amplification time, higher magnification) depending on the expression level observed in situ. 

​Limiting Signal Behavior
Because the qHCR signal per imaging voxel is analog, it will properly decrease to zero as the number of targets per imaging voxel decreases to zero. However, because the dHCR signal per target molecule is digital, it remains the same as the number of target molecules decreases to zero. 
 
Complementary Imaging Modes​​
qHCR and dHCR quantitative imaging modes are complementary, with qHCR suitable for medium- and high-copy targets (where the quantitative signal dominates autofluorescent background) and dHCR suitable for low-copy and medium-copy targets (where the signal from individual target molecules can be spatially separated).

Comparison of qHCR to qPCR

While quantitative PCR (qPCR) enables analog RNA relative quantitation in vitro without anatomical context, qHCR imaging enables analog RNA relative quantitation in situ with anatomical context.

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. 

Read-Out/Read-In Quantitative Discovery

By quantifying RNA while preserving the sample anatomy, qHCR and dHCR imaging enable bidirectional quantitative discovery using read-out/read-in analyses to move back and forth between expression space and anatomical space. 

qHCR and dHCR RNA Imaging Modes

 HCR RNA-FISH probe sets, amplifiers, and buffers
✓ RNA quantitation in an anatomical context

✓ Analog or digital signal

Relative or absolute quantification

✓ Subcellular or single-molecule resolution

Thick autofluorescent samples

Automatic background suppression throughout the protocol