Error-Robust MERFISH Probe Compositions for Fiducial-Free Alignment
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Solution Overview
Problem
Current MERFISH methods rely on fiducial markers that interfere with fluorescent imaging, are difficult to manufacture, and can be destroyed during sample preparation, leading to inaccurate image alignment and signal interference.
Innovation Solution
The use of primary nucleic acid probes with converter sequences and sandwich probes, along with readout probes, allows for improved fiducial signal generation without fiducial markers, enhancing image alignment and signal uniformity through sequential hybridization and imaging rounds.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If fiducial markers are used for image alignment, then sequential imaging data can be aligned, but the markers create bright clusters that interfere with fluorescent imaging and can be destroyed during sample preparation
Solution Approach 1:
The patent uses endogenous nucleic acid structures (such as ribosomal RNA or other abundant, stable cellular nucleic acids) as intermediary fiducial references instead of external fiducial markers. These endogenous structures serve as mediators for image alignment because they are naturally present in all cells, cannot be destroyed during sample preparation, and do not create interfering bright clusters since they are detected through the same hybridization process as the target RNAs
Solution Approach 2:
The sample's own endogenous nucleic acid structures serve the dual function of being both the biological material of interest and the fiducial reference for alignment. This self-service approach eliminates the need for separate fiducial markers, as the sample provides its own alignment references through universally present cellular components that remain intact throughout the MERFISH workflow
2Measurement precision
If fiducial markers are used for image alignment, then sequential imaging data can be aligned, but the markers are difficult to manufacture and can be destroyed during MERFISH sample preparation
Solution Approach 1:
The sample's own endogenous nucleic acid structures serve the dual function of being both the biological material of interest and the fiducial reference for alignment. This self-service approach eliminates the need for separate fiducial markers, as the sample provides its own alignment references through universally present cellular components that remain intact throughout the MERFISH workflow
Solution Approach 2:
Endogenous nucleic acid structures perform multiple functions: they serve as the biological target for transcriptomic analysis and simultaneously function as fiducial references for image alignment. This multi-functionality eliminates the need for separate fiducial marker manufacturing, as the same cellular components that are naturally present serve both purposes
3Quantity of substance
If conventional MERFISH probing is used, then RNA detection can be performed, but signal uniformity and brightness are insufficient and background signal is high
Solution Approach 1:
The patent merges the fiducial reference function with the target detection function by using the same endogenous nucleic acid structures for both purposes. This combining approach allows the detection system to simultaneously obtain alignment references and target signals from the same molecular structures, improving signal uniformity while reducing background through coordinated detection
Solution Approach 2:
The patent changes the detection parameters by using endogenous nucleic acid structures with known high abundance and stability (such as ribosomal RNA) as fiducial references. This parameter change in selecting the reference target ensures stronger, more uniform signals and lower background compared to external fiducial markers, as these endogenous structures are naturally optimized for detection
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 improves MERFISH workflows by providing accurate image alignment, increased signal brightness, and reduced background noise, resulting in enhanced spatial transcriptomic imaging and RNA detection efficiency.
Implementation Method 1
each nucleic acid probe comprising (i) a target binding sequence and (ii) one or more converter sequences complementary to a sandwich probe, wherein each unique converter sequence is assigned to two or more positions in a valid codeword assigned to a distinct nucleic acid target; wherein subpopulations of the primary nucleic acid probes hybridize to the distinct nucleic acid target
Data Source
AI summary
The present disclosure is generally directed to compositions and methods for determining nucleic acid targets in biological samples via in situ imaging and improved multiplexed error robust fluorescence in situ hybridization. The disclosure provides compositions and methods generally directed to nucleic acid probes configured to hybridize to cellular nucleic acids and improve in situ imaging signals.