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Property

Signal 

Probe set size

Amplification time

Magnification

Target expression level

Property

Signal 

Probe set size

Amplification time

Magnification

Target expression level

Comparison of Subcellular and Single-Molecule 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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Subcellular Quantitative RNA Imaging

Subcellular HCR™ RNA-FISH enables analog RNA relative quantitation with subcellular resolution in the anatomical context of thick autofluorescent samples. 

Single-Molecule Quantitative RNA Imaging

Single-molecule HCR™ RNA-FISH enables digital RNA absolute quantitation with single-molecule resolution in the anatomical context of thick autofluorescent samples.

Single-Molecule Imaging Mode
Digital single-molecule dots
30+ split-initiator probe pairs
45-90 min
Higher (63× to 100×)
Low or medium

Subcellular Imaging Mode

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

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

Subcellular RNA Imaging

Analog subcellular voxel intensities

20+ split-initiator probe pairs

Overnight

Lower (20× to 40×)

Medium or high

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 subcellular mode (longer amplification time, lower magnification) or single-molecule imaging mode (shorter amplification time, higher magnification) depending on the expression level observed in situ. 

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

Comparison of subcellular HCR™ RNA-FISH to qPCR

While quantitative PCR (qPCR) enables analog RNA relative quantitation in vitro without anatomical context, subcellular HCR™ RNA-FISH enables analog RNA relative quantitation in situ with anatomical context.

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. 

Read-Out/Read-In Quantitative Discovery

By quantifying RNA while preserving the sample anatomy, subcellular and single-molecule HCR™ RNA-FISH enable bidirectional quantitative discovery using read-out/read-in analyses to move back and forth between expression space and anatomical space. 

Subcellular and Single-Molecule Quantitative 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

All major 

automation platforms 

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