DNA Exchange Imaging with Rolling Circle Signal Amplification
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Solution Overview
Problem
Existing multiplexed imaging methods face limitations in signal strength and require specific means of switching between targets due to spectral overlap between fluorophores, and existing signal amplification methods are not compatible with DNA exchange immunofluorescence.
Innovation Solution
A method involving DNA exchange imaging with rolling circle amplification and labeled imager strands to enhance multiplexing capability, allowing simultaneous or sequential imaging of multiple targets using decodable and undecodable amplification products, and methods to extinguish or remove the signal.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Quantity of substance
If multiple fluorophores are used for multiplexed imaging, then the number of distinct species that can be visualized increases, but spectral overlap between fluorophores limits the multiplexing power and signal strength
Solution Approach 1:
The imaging process is segmented into multiple sequential rounds, with each round detecting a subset of targets using distinct fluorophores. After imaging, fluorophores are selectively removed or inactivated, allowing the same fluorophores to be reused in subsequent rounds for detecting different targets. This temporal segmentation resolves spectral overlap by ensuring only one set of fluorophores is active at any given time.
Solution Approach 2:
The system changes the state of fluorophores from bound to unbound, or from fluorescent to non-fluorescent, between imaging rounds. This parameter change allows the same physical fluorophores to be reused for detecting different targets in different rounds, effectively increasing multiplexing capability without requiring proportionally more fluorophores, thereby maintaining signal strength.
2Reliability
If signal amplification methods are applied to enhance detection sensitivity, then signal strength improves, but existing amplification methods are not compatible with DNA exchange immunofluorescence
Solution Approach 1:
A DNA intermediary system is introduced where target-specific binding partners are linked to nucleic acid docking strands. Amplification occurs through hybridization of complementary imager strands to these docking strands, rather than through traditional enzymatic amplification methods. This intermediary approach maintains compatibility with DNA exchange immunofluorescence while enabling signal amplification through increased hybridization events.
3Productivity
If the same fluorophores are reused across multiple imaging rounds, then the number of imaging rounds increases and multiplexing capability improves, but specific means of switching between targets are required
Solution Approach 1:
The system uses self-specific binding between imager strands and docking strands to automatically select which targets are detected in each round. Each imager strand is designed with sequence specificity for particular docking strands, so the binding occurs automatically without requiring external switching mechanisms. This self-service approach simplifies the overall system complexity while enabling flexible multiplexing.
4Reliability
If rolling circle amplification is used to amplify docking strands, then signal intensity improves, but the method requires compatibility with nucleic acid-based target-specific binding partners
Solution Approach 1:
The nucleic acid docking strands serve multiple functions: they act as targets for imager strand hybridization, serve as templates for rolling circle amplification, and provide a universal platform for linking to various target-specific binding partners. This multi-functionality allows the same nucleic acid-based system to work with different types of binding partners (antibodies, aptamers, etc.) while maintaining compatibility with rolling circle amplification for signal intensification.
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
Enables high multiplexing capability with improved signal intensity and flexibility in imaging multiple targets, overcoming spectral overlap and compatibility issues with existing amplification methods.
Implementation Method 1
amplifying the different docking strands with rolling circle amplification to produce amplified strands comprising concatemeric repeats of the different docking strands
Implementation Method 2
contacting the tissue sample with labeled imager strands having complementarity to the docking strands
Implementation Method 3
labeled imager strands having complementarity to the docking strands, wherein each of the labeled imager strands comprise an imager strand with a different fluorescent label attached thereto
Data Source
Figure 1A~1C
Figure 1D
Figure 1E
AI summary
The present application provides certain advantageous ways of conducting multiplexed imaging.