Expansion Microscopy MERFISH RNA Resolution
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Solution Overview
Problem
Current microscopy techniques face challenges in accurately imaging and resolving a large number of high-abundance nucleic acids within cells due to overlapping fluorescent signals caused by the diffraction limit, limiting the density of RNAs that can be simultaneously measured.
Innovation Solution
The use of expansion microscopy, where cells are embedded in an expandable material like a polymer gel, allowing physical expansion that separates molecules and enhances resolution, combined with multiplexed error-robust fluorescence in situ hybridization (MERFISH) to improve the detection efficiency of nucleic acids.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If conventional microscopy techniques are used to image nucleic acids, then the imaging process is simple, but the resolution is limited due to overlapping fluorescent signals from the diffraction limit
Solution Approach 1:
The patent applies dimensionality change by embedding cells in an expandable hydrogel matrix that physically expands the sample in three-dimensional space. This expansion separates molecules that were previously overlapping in the diffraction-limited optical space, effectively adding a physical expansion dimension to resolve the resolution limitation without requiring complex optical systems.
Solution Approach 2:
The expandable hydrogel matrix serves as an intermediary medium between the cells and the imaging system. This intermediary material enables physical separation of nucleic acids through controlled expansion while maintaining the integrity of biological structures, allowing conventional microscopes to achieve super-resolution capability without modifying the imaging system itself.
2Quantity of substance
If the density of RNAs is increased to measure more transcripts, then more biological information is obtained, but the overlapping fluorescent signals increase making accurate identification difficult
Solution Approach 1:
By physically expanding the sample in three-dimensional space through hydrogel expansion, the patent creates additional spatial separation between high-density RNA molecules. This expansion maintains the high quantity of RNA molecules while resolving their individual fluorescent signals by increasing the physical distance between them beyond the diffraction limit.
Solution Approach 2:
The patent segments the overlapping fluorescent signals by physically separating individual RNA molecules through hydrogel expansion. Each RNA molecule becomes spatially isolated from others, allowing their fluorescent signals to be resolved and identified independently even when maintaining high RNA density for comprehensive transcriptome coverage.
3Adaptability or versatility
If sequential rounds of smFISH are performed to identify RNA species, then multiplexed imaging is achieved, but the time required for imaging increases
Solution Approach 1:
The patent applies preliminary action by performing physical expansion of the sample before conducting the sequential smFISH imaging rounds. This pre-expansion step permanently separates the RNA molecules in space, so that subsequent imaging rounds can proceed more efficiently with better signal resolution, reducing the total time required for multiplexed imaging compared to imaging unexpanded samples.
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 significantly increases the detection efficiency of nucleic acids, achieving near 100% accuracy and efficiency in resolving individual RNA molecules, even in high-abundance libraries, and allows for simultaneous imaging of RNAs and proteins in expanded samples.
Implementation Method 1
expansion microscopy, where cells are embedded in an expandable material like a polymer gel, allowing physical expansion that separates molecules and enhances resolution
Implementation Method 2
multiplexed error-robust fluorescence in situ hybridization (MERFISH) to improve the detection efficiency of nucleic acids
Implementation Method 3
multiplexed error-robust fluorescence in situ hybridization (MERFISH)
Data Source
AI summary
The present invention generally relates to microscopy, and to systems and methods for imaging or determining nucleic acids or other desired targets, for instance, within cells. In certain aspects, a sample is contained within an expandable material, which is expanded and imaged in some fashion. Expansion of the material improves the effective resolution of the subsequent image. This may be combined, for example, with other super-resolution techniques, such as STORM, and/or with techniques such as MERFISH for determining nucleic acids such as mRNA within the sample, for example, by binding nucleic acid probes to the sample. Other aspects are generally directed to compositions or devices for use in such methods, kits for use in such methods, or the like.