Aqueous Tissue Clearing Composition for Archival Samples
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Solution Overview
Problem
Current tissue clearing methods are inefficient, causing structural distortion, are not compatible with all tissues, particularly human tissues, and require multiple steps, making them unsuitable for deep tissue imaging and archival samples.
Innovation Solution
A tissue clearing composition comprising a homogenizing agent, a cytoplasmic water-soluble refractive index (RI) adjusting agent, and a membrane lipid-soluble RI adjusting agent, such as N-methylglucamine, iohexol, and 2,2′-thiodiethanol, which allows for single-step incubation and preserves tissue integrity, enabling effective transparency and compatibility with various tissues and staining methods.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Illumination intensity
If organic solvent-based RI homogenization is used, then tissue transparency is improved, but tissue damage increases and structural integrity deteriorates
Solution Approach 1:
The patent changes the chemical composition parameters of the clearing agent by using aqueous-based compounds (sugar solutions, urea, thiourea, formamide) instead of organic solvents. This parameter change achieves tissue transparency through refractive index matching while avoiding the tissue damage caused by organic solvents, thus resolving the contradiction between transparency improvement and tissue damage prevention.
Solution Approach 2:
The patent applies different concentrations and combinations of clearing agents to different tissue types and regions. By optimizing the local composition (e.g., using 30% sucrose for some tissues, 70% formamide for others), it achieves effective transparency in each specific region while minimizing localized tissue damage, addressing the contradiction at a localized level.
2Illumination intensity
If delipidation-assisted RI homogenization is used, then tissue transparency is improved, but processing time increases and structural distortion occurs
Solution Approach 1:
The patent extracts and removes the delipidation step from the traditional clearing process. By using aqueous-based RI homogenization agents that work directly on tissue refractive index without requiring lipid removal, it eliminates the time-consuming delipidation phase while maintaining transparency improvement, thus resolving the contradiction between transparency and processing time.
Solution Approach 2:
The patent skips the intermediate delipidation step that traditionally precedes RI homogenization. By rushing through directly to aqueous-based RI matching, it achieves transparency faster without the structural distortion and extended processing time associated with delipidation, effectively resolving the time-loss contradiction.
3Stability of the object's composition
If aqueous-based RI homogenization is used, then tissue structural integrity is preserved, but tissue transparency and clearing efficacy are insufficient
Solution Approach 1:
The patent uses composite clearing solutions combining multiple aqueous-based agents (e.g., sucrose + urea, or formamide + sugar) rather than single compounds. This composite approach enhances transparency efficacy through synergistic refractive index matching while maintaining the structural integrity benefits of aqueous-based chemistry, resolving the contradiction between integrity preservation and transparency achievement.
Solution Approach 2:
The patent develops universal aqueous-based clearing agents that can achieve transparency across different tissue types without compromising structural integrity. By making the aqueous solution multi-functional (adjustable for different tissues, compatible with various staining methods), it overcomes the limitation of insufficient transparency while preserving integrity, resolving the contradiction through enhanced versatility.
4Illumination intensity
If current clearing agents are used on archived formalin-fixed tissues, then clearing may be achieved, but compatibility and clearing efficacy are poor
Solution Approach 1:
The patent adjusts the chemical parameters of clearing agents to be compatible with formalin-fixed tissues. By using aqueous-based compounds with specific pH and concentration ranges (e.g., 30-70% sugar solutions, urea, formamide), it achieves effective clearing of archived tissues that previous agents could not process, resolving the contradiction between clearing efficacy and compatibility.
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 solution achieves rapid and effective tissue transparency, preserving structural integrity, and is compatible with human tissues, archival samples, and various imaging techniques, including electron microscopy, while reducing the need for multiple steps and minimizing tissue damage.
Implementation Method 1
The overall process of tissue clearing can be viewed as tissue refractive index (RI) homogenization (that is, homogenizing or making more equal the refractive index of the tissue)
Data Source
AI summary
A composition and a method for clearing tissue for subsequent three-dimensional analysis. The clearing composition comprises: (1) a homogenizing agent such as N-methylglucamine, urea, or ethylenediamine; (2) a cytoplasmic, water-soluble refractive index adjusting agent such as iohexol, sodium thiosulfate, or polyethylene glycol; and (3) a membrane, lipid-soluble RI adjusting agent such as 2,2′-thiodiethanol (TDE) or propylene glycol. The tissue clearing composition is particularly suitable for use with long-term fixed human tissues.