PBS Pretreatment Prevents Floating Damage During Organoid Clearing
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Solution Overview
Problem
Existing methods for clearing biological samples, particularly spheroids or organoids with sizes of 1 mm or less, face issues such as floating due to density differences, leading to structural damage and loss, and lack effective techniques for rapid three-dimensional imaging and quantification.
Innovation Solution
A pretreatment method using phosphate-buffered saline (PBS) followed by a clearing agent represented by Formula 1, which includes compounds like CHAPS, urea, and agarose, to stabilize the samples and enhance clearing efficiency while minimizing damage.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If 40% sucrose solution is used for pretreatment to preserve protein, then protein integrity is improved, but small spheroids or organoids float due to density difference causing structural damage
Solution Approach 1:
The patent changes the density parameter of the pretreatment solution by replacing 40% sucrose solution with 30-70% glycerol solution. Glycerol has a density closer to that of small spheroids and organoids, preventing them from floating while still providing effective protein preservation and clearing effects.
Solution Approach 2:
The patent introduces glycerol as an intermediary substance that serves dual functions: it acts as a density matching medium to prevent floating and as a clearing agent to enable transparentization. This intermediary resolves the conflict between protein preservation and preventing structural damage.
2Reliability
If 40% sucrose solution is used for pretreatment, then protein preservation is improved, but sample loss increases due to floating and fragility
Solution Approach 1:
The patent changes the density parameter of the pretreatment solution by replacing 40% sucrose solution with 30-70% glycerol solution. Glycerol has a density closer to that of small spheroids and organoids, preventing them from floating while still providing effective protein preservation and clearing effects.
3Measurement precision
If confocal microscope is used for three-dimensional imaging, then thickness information is obtained, but imaging depth is limited to several tens of micrometers
Solution Approach 1:
The patent uses optical clearing agents (glycerol-based solutions) that change the optical properties of the tissue by reducing light scattering. This allows light to penetrate deeper into the tissue while maintaining image quality, enabling imaging of thick tissues (several hundred micrometers or more) that would otherwise be inaccessible to confocal microscopes.
Solution Approach 2:
The patent replaces the mechanical limitation of confocal microscope depth with an optical solution. By using optical clearing agents to transparentize the tissue, the system overcomes the physical barrier of light penetration depth, enabling three-dimensional imaging of thick organoids without requiring mechanical sectioning.
4Loss of information
If series of cutting and pasting processes is used to acquire internal information, then comprehensive imaging is achieved, but sample damage increases exponentially
Solution Approach 1:
The patent replaces the mechanical process of cutting and pasting tissue sections with an optical clearing method. By using glycerol-based clearing agents to transparentize the entire organoid, the system enables direct three-dimensional imaging of the complete internal structure without mechanical disruption, thereby avoiding exponential sample damage.
Solution Approach 2:
The patent eliminates the need for segmenting the tissue into thin sections by using optical clearing to make the entire thick organoid transparent. This allows the complete three-dimensional structure to be imaged as a single intact sample, avoiding the cumulative damage from repeated sectioning and reconstitution processes.
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 method effectively prevents structural damage, allows for clear imaging of deep tissue parts, and enables rapid three-dimensional imaging and quantification of small biological samples, facilitating high-throughput drug screening and evaluation.
Implementation Method 1
clearing the resulting pretreated biological sample by bringing the pretreated biological sample into contact with a clearing agent
Implementation Method 2
pretreating a biological sample that is fixed and has a size of 1 mm or less with a pretreatment solution including phosphate-buffered saline (PBS)
Data Source
AI summary
A clearing pretreatment method of a biological sample having a size of at most 1 mm, that is, a spheroid or organoid, according to the present invention uses a phosphate-buffered saline (PBS) rather than a conventional sucrose solution as a pretreatment solution, thus solving the problem in which the spheroid or organoid floats above the surface of water due to the density difference such that the structure of the sample is damaged. Therefore, there is an effect in that the spheroid or organoid can be transparentized while maintaining the original shape thereof, thus making it possible to image deep parts.