Biological Tissue Expansion Method Preserving Fluorescence
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Solution Overview
Problem
Existing biological tissue expansion technologies face challenges such as protein denaturation causing fluorescence quenching, reduced mechanical strength, and fixed expansion ratios, which hinder high-resolution three-dimensional imaging of large-volume tissues.
Innovation Solution
A novel clearing and expansion method (CMAP) that avoids protein denaturation steps, uses delipidation and monomer permeation followed by polymerization initiated at low temperatures, allowing for adjustable expansion ratios and maintaining tissue mechanical strength.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Stability of the object's composition
If protein denaturation procedures (heating or enzymatic digestion) are used to ensure isotropic expansion, then expansion uniformity is improved, but fluorescence quenching occurs and mechanical strength is reduced
Solution Approach 1:
The patent changes the temperature parameter by conducting polymerization at low temperatures (4°C to 25°C) instead of using high-temperature heating for denaturation. This parameter change allows the tissue to expand isotropically without undergoing protein denaturation, thereby preserving fluorescence signals while achieving uniform expansion. The low-temperature polymerization initiates monomer conversion without causing thermal damage to fluorescent proteins.
Solution Approach 2:
The patent replaces the thermal/mechanical denaturation system with a chemical polymerization system. Instead of using heat or enzymatic digestion to facilitate expansion, the invention uses photo-initiated or chemical polymerization of monomers within the tissue matrix. This substitution allows expansion to occur through polymer network formation and swelling rather than protein denaturation, preserving both fluorescence and mechanical integrity.
2Stability of the object's composition
If protein denaturation procedures are used to ensure isotropic expansion, then expansion uniformity is improved, but mechanical strength is significantly reduced
Solution Approach 1:
The patent changes the temperature parameter to low conditions (4°C to 25°C) during polymerization, preventing the thermal denaturation that would compromise mechanical strength. This parameter modification enables the tissue to maintain its structural integrity while still achieving isotropic expansion through polymer network formation and osmotic swelling of the hydrogel matrix.
3Reliability
If existing expansion technologies are used, then tissue transparency is improved, but processing time lasts several weeks
Solution Approach 1:
The patent implements continuous polymerization by maintaining constant monomer supply and uninterrupted photo-initiation or chemical reaction conditions. This continuous action allows the polymerization and expansion process to proceed efficiently without the need for repeated handling, incubation cycles, or intermediate steps, thereby reducing the overall processing time from weeks to days or hours while maintaining complete tissue transparency.
4Measurement precision
If fixed expansion ratios are used in existing technologies, then imaging resolution is improved, but adaptability to different imaging systems is reduced
Solution Approach 1:
The patent introduces dynamic control of expansion ratio by adjusting monomer concentration, crosslinker concentration, polymerization conditions, or incubation time. This dynamic approach allows the expansion ratio to be optimized for different imaging systems and applications, transforming a static process into a tunable one that can adapt to various microscopy platforms while maintaining high imaging resolution.
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
CMAP preserves endogenous fluorescent proteins, enhances mechanical strength, and allows for rapid processing, enabling high-resolution three-dimensional imaging of large-volume tissues with adjustable expansion ratios.
Implementation Method 1
biological tissue sample delipidation: delipidation a fixed biological tissue sample
Implementation Method 2
The clearing and expansion method for a biological tissue according to the present invention can obtain a transparent and expanded biological tissue
Implementation Method 3
monomer incubation and permeating: incubating the delipidated biological tissue sample in a gel monomer solution to allow the monomer molecules to permeate into the degreased biological tissue sample
Implementation Method 4
polymerization: initiating polymerization of the monomer molecules that permeate into the biological tissue sample to form a polymer gel
Implementation Method 5
The basic principle of hydrogel-based biological tissue expansion technology is that the protein molecules within the biological tissue to be imaged are cross-linked to the framework structure composed of hydrogel polymer molecules, and then the framework structure of the hydrogel polymer undergoes isotropic expansion
Data Source
AI summary
Provided are a clearing and expansion method and an imaging method for biological tissue. The clearing and expansion method for biological tissue comprises: (1) degreasing a fixed biological tissue sample; (2) soaking the degreased biological tissue sample in a solution of gel monomer molecules, such that the monomer molecules permeate into the degreased biological tissue sample; (3) inducing a polymerization reaction of the monomer molecules permeating into the biological tissue sample so as to form a polymer gel; and (4) placing the biological tissue sample which has been subjected to a gelation treatment into water for expansion. The clearing and expansion method for biological tissue avoids the sample treatment steps of heating and enzymatic digestion that cause fluorescent quenching and the destruction of biological tissue, and thus has the advantages of high retention of endogenous fluorescent proteins, high mechanical strength of expanded biological tissue, a short sample treatment time, etc. Moreover, the advantage of an adjustable expansion ratio is further achieved by means of changing the components of a monomer reagent.