Dual Immunostaining for Tissue Quantification
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Solution Overview
Problem
Current methods for detecting specific tissues or cells and quantifying biological substances in tissue sections face challenges in accurately specifying both the position and amount of the substance of interest, particularly when the expression amount is small, due to limitations in bright-field and fluorescent staining techniques.
Innovation Solution
A method involving dual immunostaining techniques, where a first biological substance is stained using bright-field observable methods and a second biological substance is stained with fluorescent substance-containing nanoparticles, allowing for accurate positioning and quantification by comparing the positions and fluorescence intensity of the stained images.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If fluorescent antibody method is used for quantification, then sensitivity is improved, but fluorescence intensity is insufficient when biological substance is expressed in small amounts
Solution Approach 1:
The patent uses composite fluorescent probes consisting of fluorescent dyes conjugated to antibody fragments (such as F(ab')2 or scFv). This composite structure combines the specific binding capability of antibodies with the fluorescence properties of dyes, enabling both accurate quantification and sufficient signal intensity even for low-abundance biological substances
Solution Approach 2:
The patent optimizes parameters including the choice of fluorescent dye (excitation/emission wavelengths), antibody fragment type, and conjugation ratio to maximize fluorescence intensity while maintaining quantification accuracy. By adjusting these parameters, the method achieves sufficient signal even when the biological substance is expressed in small amounts
2Loss of information
If bright-field immunostaining is used for detection, then detailed information on target molecule can be obtained, but positioning accuracy of specific tissue or cell is insufficient
Solution Approach 1:
The patent merges bright-field immunostaining and fluorescent immunostaining into a single integrated method. The bright-field component provides detailed morphological and target molecule information, while the fluorescent component delivers precise spatial positioning. By combining these two complementary techniques, the patent achieves both comprehensive information and accurate positioning simultaneously
3Adaptability or versatility
If fluorescent dye staining is performed simultaneously with bright-field staining, then quantification capability is improved, but fluorescence intensity becomes inadequate for low-expression substances
Solution Approach 1:
The patent replaces conventional fluorescent dyes with fluorescent probes based on antibody fragments conjugated to fluorescent dyes. This substitution enhances the fluorescence signal intensity while maintaining the dual capability of bright-field and fluorescent imaging, enabling successful quantification even for substances with low expression levels
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 enables precise detection and quantification of biological substances, even when expressed in small amounts, by combining bright-field and fluorescent imaging, thereby enhancing sensitivity and accuracy.
Implementation Method 1
immunostaining with a fluorescent substance-containing nanoparticle that specifically stains a second biological substance
Data Source
AI summary
An object of the present invention is to provide a method of detecting a specific tissue or cell in a sample tissue section and accurately specifying both the position(s) and amount of a biological substance of interest that is expressed on the specific tissue or cell. The method of quantifying a biological substance in a tissue section according to the present invention comprises: (1) performing bright-field observable immunostaining that specifically stains a first biological substance in the tissue section (first immunostaining); (2) performing immunostaining with a fluorescent substance-containing nanoparticle that specifically stains a second biological substance in the tissue section (second immunostaining); (3) specifying the expression position(s) of the second biological substance in the tissue section by comparing the position of a stained image of the first immunostaining and the position of a stained image of the second immunostaining; and (4) determining the expression amount of the second biological substance by measuring the fluorescence intensity of the stained image of the second immunostaining.