Expansion Microscopy Multiplexed RNA Detection FFPE Tissue
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Solution Overview
Problem
Current imaging technologies for analyzing RNA in clinical samples, such as FFPE tissues, are limited by the diffraction limit of conventional microscopes, which reduces the accuracy of RNA spot count quantification and the ability to systematically map transcriptional variations across a large number of genes, and existing expansion microscopy (ExM) methods are laborious and inefficient due to the use of DNA/antibody conjugates.
Innovation Solution
The method involves deparaffinization and antigen retrieval of FFPE samples, followed by contacting with gel binding moieties and a solution of polyelectrolyte gel monomers, free radical polymerization to form a gel network, proteolytic digestion, and expansion, using hybridization chain reaction (HCR) probes for RNA targets, allowing for high-speed transcriptional profiling of multiple genes.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If conventional microscopy is used for RNA imaging, then the equipment is simple and easy to operate, but the measurement precision is limited by the diffraction limit
Solution Approach 1:
The patent applies parameter changes by physically expanding the tissue sample dimensions, transforming the spatial scale of RNA transcripts from sub-diffraction to resolvable sizes. This changes the fundamental parameter of the imaging system's resolution capability without requiring complex optical components, enabling conventional microscopes to achieve super-resolution imaging.
Solution Approach 2:
The patent replaces the optical resolution mechanism with a physical expansion mechanism. Instead of improving optical resolution through complex lens systems, the invention uses mechanical expansion of the tissue matrix to separate and resolve RNA transcripts, substituting the mechanical expansion process for the optical resolution problem.
2Measurement precision
If expansion microscopy is used to overcome diffraction limit, then measurement precision improves, but the process becomes laborious and time-consuming
Solution Approach 1:
The patent applies preliminary action by performing tissue expansion and probe attachment before RNA hybridization. The tissue is expanded and probes are attached to the expansion matrix in advance, allowing subsequent RNA hybridization to proceed rapidly without requiring time-consuming steps during the imaging process itself.
Solution Approach 2:
The patent segments the expansion microscopy process into distinct modular steps: tissue expansion, probe attachment, RNA hybridization, and imaging. This segmentation allows each step to be optimized independently and performed in parallel where possible, reducing overall processing time while maintaining spatial resolution.
3Reliability
If DNA/antibody conjugates are used in ExM, then protein binding is achieved, but the process becomes inefficient and staining is dim
Solution Approach 1:
The patent introduces an intermediary expansion matrix (polymer network) that mediates between the antibodies and the RNA targets. The matrix physically separates and positions the antibodies from the RNA, allowing efficient binding without the interference of direct DNA/antibody conjugation, thereby improving both reliability and productivity.
Solution Approach 2:
The patent extracts the DNA component from the antibody conjugate system, removing the source of interference. By using antibodies bound to the expansion matrix rather than DNA/antibody conjugates, the invention eliminates the harmful effect of DNA on binding efficiency while maintaining protein detection capability.
4Adaptability or versatility
If multiple fluorescent channels are used to detect multiple transcripts, then the number of detectable genes increases, but the number of available channels is limited to 3-4
Solution Approach 1:
The patent applies dimensionality change by moving from a two-dimensional spectral unmixing approach to a three-dimensional spatial separation approach. By physically separating RNA transcripts through tissue expansion, the system can detect multiple transcripts using standard fluorescent channels without requiring complex spectral unmixing or additional channels, as spatial information provides the extra dimension for multiplexing.
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 RNA transcript data reading from FFPE clinical tissue samples, facilitating accurate diagnostic and prognostic analysis by overcoming the limitations of conventional imaging and ExM methods.
Implementation Method 1
contacting the sample with a solution comprising monomers of a polyelectrolyte gel; by free radical polymerization, polymerizing the monomers to form the polyelectrolyte gel and covalently conjugating the first and second gel binding moieties to the polyelectrolyte gel
Implementation Method 2
providing a plurality of initiator deoxyribonucleic acid (DNA) probes targeting a plurality of nucleic acid targets of interest, wherein each of the plurality of initiator DNA probes comprise (A) a sequence complementary to a sequence from one of the nucleic acid targets of interest
Implementation Method 3
contacting the sample with a first gel binding moiety and a second gel binding moiety under conditions wherein the first gel binding moiety operably links to proteins in the sample and the second gel binding moiety operably links to nucleic acids in the sample
Data Source
AI summary
The invention relates to imaging, such as in situ imaging by expansion microscopy, labelling, and analyzing biological samples, such as formalin fixed paraffin embedded (FFPE) cells and tissues, as well as reagents and kits for doing so.