Analyte Detection Using Diluted Readout Signals
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Solution Overview
Problem
Existing in situ analyte detection methods often yield detection signals outside of an optimal range, leading to optical overcrowding and reduced image analysis quality due to large or high-intensity signals.
Innovation Solution
A method involving primary probes with binding and barcode regions, and intermediate probes with hybridization, spot-calling, and decoding regions, to detect analytes in biological samples by generating and analyzing signal codes associated with the analytes.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If existing in situ analyte detection methods are used, then analyte detection is achieved, but detection signals become too large or high-intensity causing optical overcrowding and reduced image analysis quality
Solution Approach 1:
The patent segments the detection signal into multiple components: primary probes bind to analytes with barcode regions, intermediate probes bind to barcodes with spot-calling regions, and separate detectable probes bind to spot-calling regions. This segmentation distributes the signal across multiple detection steps, preventing optical overcrowding while maintaining measurement precision.
Solution Approach 2:
The patent introduces intermediate probes as mediators between primary probes and detectable probes. The intermediate probes contain hybridization regions that bind to barcode regions and spot-calling regions that bind to detectable probes. This intermediary step dilutes and distributes the signal, preventing optical overcrowding while enabling accurate analyte detection.
2Reliability
If signal amplification is performed to enhance detection sensitivity, then detection sensitivity improves, but signal overcrowding increases reducing image quality
Solution Approach 1:
The patent divides the signal amplification and detection process into separate stages: primary probe binding, intermediate probe hybridization to barcodes, and detectable probe binding to spot-calling regions. This segmentation allows controlled signal amplification at each stage without causing overall signal overcrowding, maintaining both detection sensitivity and image quality.
Solution Approach 2:
The patent adds spatial dimensionality to signal detection by using spot-calling regions that localize signals to specific positions, and by performing detections at different stages. This dimensional approach distributes signal quantity across space and time, preventing overcrowding while maintaining detection sensitivity.
3Adaptability or versatility
If multiple analytes are detected simultaneously, then multiplexing capability improves, but optical overcrowding increases reducing analysis quality
Solution Approach 1:
The patent segments the detection of multiple analytes into distinct detection steps using unique barcode regions for each analyte type. Primary probes with analyte-specific barcodes are detected separately through intermediate probe hybridization and spot-calling, allowing multiplexing without signal overlap or optical overcrowding.
Solution Approach 2:
The patent uses barcode copying where intermediate probes hybridize to barcode regions and create copies of the barcode information in spot-calling regions. This copying mechanism allows multiple analyte signals to be read out separately without direct optical overlap, maintaining image analysis quality while enabling 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 method enables accurate detection of analytes by reducing signal overcrowding and improving image analysis quality, allowing for precise localization and identification of multiple analytes in a sample.
Implementation Method 1
each intermediate probe comprises (i) a hybridization region configured to hybridize to the barcode region of a primary probe of the plurality of primary probes or a complement of the barcode region
Data Source
AI summary
In some aspects, the present disclosure generally relates to methods and compositions for detecting a plurality of molecules of one or more analytes in a sample. In some aspects, the present disclosure relates to methods for determining the locations and identities of analytes in a biological sample using detectable probes. In some aspects, the present disclosure relates to methods for reducing the crowding of signals that are used for determining the locations of the analytes (e.g., spot-calling signals). The methods herein have particular applicability in the detection of spot-calling signals and barcode sequences in sequencing-by-hybridization (SBH) methods, including those relying on combinatorial labeling schemes and decoding of the barcodes by sequential cycles of spot-calling and/or decoding using hybridization probes.