Subcellular Quantitative Protein and RNA Imaging

Multiplexed, quantitative, high-resolution IF, RNA-FISH, and IF + RNA-FISH

HCR™ IHC, HCR™ RNA-FISH, and HCR™ IF + HCR™ RNA-FISH enable protein and RNA relative quantitation with subcellular resolution in the anatomical context of highly autofluorescent samples.

Subcellular Quantitative Signal

Subcellular signal provides analog quantitation in the form of fluorescence voxel intensities that scale approximately linearly with the number of protein or RNA target molecules per subcellular voxel.

Subcellular Quantitative RNA Imaging in a Whole-Mount Chicken Embryo

Redundant 2-Channel Detection

To illustrate the quantitative nature of subcellular HCR™ RNA-FISH, we detect a target mRNA using two probe sets that trigger different amplifiers carrying spectrally distinct fluorophores.

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Fluorescence voxel intensities

High Accuracy and Precision with Subcellular Resolution

The resulting scatter plot of fluorescence voxel intensities demonstrates high accuracy (linearity with zero intercept) and precision (tight scatter around the line) for subcellular voxels (2.1×2.1×2.7 µm) within a whole-mount vertebrate embryo.

Subcellular Quantitative Protein Imaging in FFPE Human Breast Tissue Sections

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Redundant 2-Channel Detection

To illustrate the quantitative nature of subcellular HCR™ IF, we detect a target protein using an unmodified 1º antibody probe and two batches of initiator-labeled 2º antibody probes that trigger different amplifiers carrying spectrally distinct fluorophores.

Fluorescence voxel intensities

High Accuracy and Precision with Subcellular Resolution

The resulting scatter plot of fluorescence voxel intensities demonstrates high accuracy (linearity with zero intercept) and precision (tight scatter around the line) for subcellular voxels (2.0×2.0×2.5 µm) within an FFPE human breast tissue section.

Subcellular Quantitative Imaging Pointers

Precision Increases with Voxel Size

Quantitative precision increases with voxel size (so long as voxels remain smaller than the expression domains under consideration). To achieve high precision with subcellular resolution we recommend averaging pixels to obtain roughly 2×2×2 µm voxels.

Precision Increases with Probe Set Size for RNA Targets

Imaging RNA targets using HCR™ RNA-FISH, precision increases with probe set size due to the benefits of automatic background suppression. For subcellular quantitative HCR™ RNA-FISH, we recommend using 20+ split-initiator probe pairs per target RNA (as permitted by target length).

Signal-to-Background Increases with Amplification Time

For subcellular protein and/or RNA imaging, we recommend overnight amplification to grow long amplification polymers that enhance the signal-to-background ratio.

Comparison of Subcellular HCR™ to qPCR

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