Quencher Compound for Autofluorescence Reduction in Tissue Imaging
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Solution Overview
Problem
Existing methods for in situ analysis of biological samples suffer from low sensitivity, specificity, and detection efficiency due to background autofluorescence, which complicates the detection of analytes of interest.
Innovation Solution
A method involving a quencher comprising a quenching moiety and a targeting moiety is used to reduce autofluorescence in tissue samples. The targeting moiety binds to endogenous biological moieties, targeting the quencher to these sites, thereby reducing autofluorescence and enhancing the signal-to-noise ratio for analyte detection.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If standard fluorescence imaging methods are used for in situ analysis of biological samples, then the detection of analytes can be performed, but the sensitivity and signal-to-noise ratio are reduced due to background autofluorescence
Solution Approach 1:
The patent extracts and removes the harmful autofluorescence signal from the biological sample by treating it as a separate target. A quencher compound is used to specifically bind to and eliminate the autofluorescent background, allowing the analyte signal to be detected with high sensitivity and clarity.
Solution Approach 2:
The patent introduces a quencher compound as an intermediary substance that mediates between the autofluorescence and the detection system. This quencher binds to the autofluorescent moieties in the sample, preventing them from interfering with the analyte detection while allowing the analyte signal to pass through unaffected.
2Productivity
If multiple imaging cycles are performed to enhance detection, then the detection efficiency improves, but the autofluorescence background persists and complicates analysis
Solution Approach 1:
The patent applies preliminary action by treating the sample with the quencher compound before performing the fluorescence imaging cycles. This pre-treatment eliminates the autofluorescence background in advance, ensuring that subsequent imaging cycles proceed with a clean signal and improved detection efficiency without the complicating presence of background autofluorescence.
3Measurement precision
If conventional probe-based assays are used, then the analysis can be performed, but the sensitivity and specificity are reduced in the presence of autofluorescence
Solution Approach 1:
The patent converts the harmful autofluorescence into a beneficial target for the quencher compound. By designing the quencher to specifically recognize and bind to autofluorescent moieties, the previously problematic background signal becomes the focus of the quenching action, effectively transforming the harm into a controlled and manageable interaction that enhances overall detection sensitivity.
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
The method achieves significant reduction of autofluorescence by at least 50% and improves the signal-to-noise ratio and detectable object count density, allowing for more accurate analysis of biological samples over multiple imaging cycles.
Implementation Method 1
a quenching moiety (e.g., a quencher dye)
Implementation Method 2
the targeting moiety binds and/or reacts with a biological moiety endogenous in the tissue sample
Data Source
AI summary
The present disclosure generally relates to methods and compositions for in situ analysis or detection of analytes in a biological sample. More specifically, the present disclosure relates to methods for reducing autofluorescence in tissue samples, methods for analyzing tissue samples, and compounds for use in the same. The methods and compounds of the present disclosure may be especially suitable for analytical methods employing fluorescence in situ hybridization techniques over multiple cycles of imaging.