Expansion Microscopy Multiplexed RNA Imaging
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Solution Overview
Problem
Current imaging technologies for RNA in biological samples face limitations due to diffraction-limited resolutions and the inability to efficiently multiplex and image RNA transcripts in thick tissue sections, leading to dim staining and limited resolution of RNA targets.
Innovation Solution
The method involves using a combination of serial RNA hybridization strategies with expansion microscopy (ExM) to label nucleic acids and proteins, employing a polyelectrolyte gel that expands upon water addition, allowing for high-resolution imaging of RNA targets by hybridization chain reaction (HCR) amplification and subsequent imaging with conventional confocal microscopy.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If conventional microscopy is used for RNA imaging, then the imaging process is simple, but the resolution is diffraction-limited and cannot achieve nanoscale precision
Solution Approach 1:
The patent introduces an expansion microscopy system as an intermediary between conventional microscopy and super-resolution techniques. A polymer gel expands the tissue sample physically, creating expanded copies of RNA targets that can be imaged with conventional diffraction-limited microscopes while achieving super-resolution效果. This mediator approach resolves the contradiction by enabling nanoscale precision imaging through physical expansion rather than requiring complex super-resolution microscopy systems.
Solution Approach 2:
The patent changes the physical parameter of the tissue sample by expanding it 4-10 times in volume through polymer gel infiltration. This parameter change transforms the spatial scale of RNA targets, making them large enough to be resolved by conventional microscopes while maintaining nanoscale precision relative to the original tissue architecture.
2Quantity of substance
If multiple fluorescent dyes are used for multiplexed RNA detection, then more transcripts can be resolved, but the staining becomes dim and the number of detectable transcripts is limited
Solution Approach 1:
The patent segments the detection process into multiple sequential hybridization rounds rather than attempting to detect all transcripts simultaneously. In each round, a subset of RNA targets is detected with high signal intensity using fewer fluorescent dyes. After imaging, probes are removed and the sample is re-hybridized for the next set of targets. This segmentation approach allows multiplexed detection of thousands of transcripts while maintaining bright staining in each individual detection round.
Solution Approach 2:
The patent implements continuous useful action through serial hybridization cycles where probes are removed, washed, and new probes are hybridized in sequence. This continuous process enables the detection of many more transcripts than the number of available fluorescent dyes by repeatedly using the same detection channels with different probe sets across multiple cycles.
3Measurement precision
If tissue sections are imaged with high resolution, then spatial organization is preserved, but imaging deep into thick tissues is difficult due to scattering and absorption
Solution Approach 1:
The patent performs preliminary tissue clearing and expansion before imaging. The polymer gel infiltration and tissue expansion processes occur prior to the imaging step, transforming thick, light-scattering tissue into an expanded, cleared state that allows deep penetration of excitation and emission light. This preliminary action enables subsequent high-resolution imaging of deep tissue regions that would otherwise be inaccessible.
Solution Approach 2:
The patent changes multiple physical parameters of the tissue simultaneously: volume expansion (4-10x), light scattering properties (reduced through clearing), and refractive index matching. These parameter changes collectively enable deep tissue imaging with high spatial resolution by reducing optical path length and minimizing light scattering and absorption effects.
4Reliability
If DNA/antibody conjugates are used for ExM attachment, then proteins can be stained and expanded, but the DNA binds to nuclear DNA reducing binding efficiency and requiring lengthy procedures
Solution Approach 1:
The patent extracts and removes the problematic DNA component from the attachment chemistry. Instead of using DNA/antibody conjugates where DNA can bind to nuclear DNA and reduce efficiency, the invention uses direct antibody-gel conjugation or antibody-oligononucleotide conjugates where the oligonucleotide is designed to bind specifically to the target RNA rather than genomic DNA. This extraction of the harmful DNA-binding function resolves the contradiction by eliminating off-target binding while maintaining reliable staining.
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
This approach enables high-speed, multiplexed transcriptional profiling of thousands of genes across thick tissue sections with improved spatial resolution and signal-to-noise ratio, overcoming previous limitations in RNA imaging.
Implementation Method 1
a polyelectrolyte gel that expands upon water addition
Implementation Method 2
by free radical polymerization, polymerizing said monomers to form the polyelectrolyte gel
Implementation Method 3
contacting the sample with the plurality of initiator DNA probes under conditions wherein the sequence complementary to the sequence from one of the nucleic acid targets of interest hybridizes to that sequence from the nucleic acid target of interest
Implementation Method 4
contacting the sample with the fluorophore-labeled DNA hairpins under conditions wherein the hairpins self-assemble by HCR, in the presence of their corresponding initiator DNA probe, into fluorescent amplification polymers
Implementation Method 5
proteolytically digesting said sample
Data Source
AI summary
This invention relates to imaging, such as by expansion microscopy, labelling, and analyzing biological samples, such as cells and tissues, as well as reagents and kits for doing so.