Delipidation Reagent for Biological Tissue Clearing
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Solution Overview
Problem
Current biological tissue clearing methods, such as hydrophobic, hydrogel-based, and hydrophilic methods, face limitations in efficiency, sample damage, and retention of endogenous fluorescent proteins, hindering high-resolution three-dimensional imaging of large-volume biological samples.
Innovation Solution
A delipidation composition comprising urea, N-butyldiethanolamine, and Triton X-100, with specific mass ratios, is developed to enhance delipidation efficiency and maintain tissue integrity, forming a delipidation reagent that accelerates the clearing process while preserving structural features and fluorescent proteins.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If hydrophobic clearing methods are used, then clearing ability is improved, but endogenous fluorescent proteins are damaged and refractive index matching becomes difficult
Solution Approach 1:
The patent changes the chemical composition parameters of the clearing reagent by using a hydrophilic organic solvent system (containing urea, sucrose, antipyrine, and trihydroxyethylamine) instead of traditional hydrophobic solvents. This parameter change enables the reagent to achieve both strong clearing ability and preservation of endogenous fluorescent proteins, while also providing refractive index matching (1.46-1.50) suitable for high numerical aperture objective lenses.
2Stability of the object's composition
If hydrogel-based clearing methods are used, then refractive index matching is improved, but tissue clearing procedure becomes complicated and sample damage increases
Solution Approach 1:
The patent extracts and eliminates the hydrogel formation step from the traditional CLARITY method. By removing the acrylamide monomer polymerization step and electrophoresis/detergent treatment, the invention simplifies the procedure while maintaining good refractive index matching (1.46-1.50) and reducing sample damage through a more gentle chemical treatment process.
3Object-affected harmful factors
If hydrophilic clearing methods are used, then biocompatibility and fluorescent protein retention are improved, but clearing speed becomes slow
Solution Approach 1:
The patent creates a composite clearing reagent system combining multiple hydrophilic organic solvents (urea, sucrose, antipyrine, trihydroxyethylamine) in specific ratios. This composite formulation leverages the synergistic effects of different components to achieve both high biocompatibility for fluorescent protein retention and enhanced clearing speed, overcoming the limitation of slow clearing in traditional hydrophilic methods like CUBIC.
4Measurement precision
If traditional sectioning and reconstruction methods are used, then three-dimensional imaging is achieved, but imaging efficiency decreases and sample integrity is compromised
Solution Approach 1:
The patent performs preliminary tissue clearing and transparency enhancement before imaging, allowing the entire three-dimensional structure to be visualized in situ without physical sectioning. This preliminary action of rendering the tissue transparent enables direct high-resolution three-dimensional imaging, eliminating the need for subsequent sectioning, mounting, and computational reconstruction steps.
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 delipidation reagent achieves faster clearing speeds with improved tissue transparency and structural preservation, outperforming existing methods by maintaining the integrity and fluorescent protein retention of biological samples at the cellular and subcellular levels.
Implementation Method 1
a delipidation composition comprising urea, N-butyldiethanolamine and Triton X-100
Implementation Method 2
Triton X-100
Implementation Method 3
refractive index matching on biological tissues
Data Source
AI summary
A degreasing composition for clearing biological tissue, a use in the preparation of a degreasing reagent, a degreasing reagent, a clearing kit comprising the degreasing composition or the degreasing reagent, a biological tissue clearing treatment method using the degreasing reagent, and an imaging method for clearing biological tissue comprising the steps of the biological tissue clearing treatment method. The degreasing composition comprises urea, N-butyldiethanolamine and Triton X-100, wherein the mass ratio of urea, N-butyldiethanolamine and Triton X-100 is (0.6-5):(0.3-3):1. The degreasing composition has a fast degreasing speed, good degreasing capabilities for large-volume biological samples, and may retain the structural features of biological tissues well from the cellular level to the subcellular level.