Biological Specimen Clearing Reagent with Sorbitol
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Solution Overview
Problem
Current clearing techniques for biological materials often compromise the original form and structure of the material during the transparency enhancement process, either due to rapid urea permeation causing expansion, high concentration surfactants disrupting cellular structures, or thermal denaturation, and existing methods are not efficient in achieving quick transparency without significant deformation or protein denaturation.
Innovation Solution
A clearing reagent combining urea or its derivatives with sorbitol, using a surfactant at a concentration of 5% or less, to prevent expansion and maintain the original form of biological materials while enhancing transparency, and employing a method that allows the reagent to permeate without chemical denaturation or structural disruption.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If a high concentration of urea is used to rapidly increase transparency, then the transparency improvement speed increases, but the biological material expands and deforms
Solution Approach 1:
The patent applies preliminary anti-action by adding sorbitol to the clearing reagent before urea permeation begins. Sorbitol creates an osmotic pressure that counteracts the water influx caused by urea, preventing expansion before it occurs. This pre-established counterbalance allows rapid transparency improvement without shape deformation.
Solution Approach 2:
The patent changes the chemical composition parameters of the clearing reagent by combining urea with sorbitol at specific concentrations. This parameter modification enables the reagent to simultaneously achieve rapid transparency improvement and shape preservation, resolving the contradiction between speed and structural integrity.
2Productivity
If a high concentration of surfactant is used to remove lipid membranes, then the transparency increases, but cellular structures and proteins are disrupted
Solution Approach 1:
The patent modifies the surfactant concentration parameter to 5% or less, which is sufficient for lipid removal but below the threshold that causes cellular structure disruption. This parameter optimization allows transparency improvement while preserving biological material integrity.
Solution Approach 2:
The patent creates a composite clearing reagent system combining urea, sorbitol, and low-concentration surfactant. This composite formulation achieves synergistic effects where each component contributes to transparency improvement without the harmful side effects of high concentrations, maintaining cellular structure reliability.
3Productivity
If electrophoresis is used to remove lipid components, then transparency is achieved, but thermal denaturation of proteins occurs
Solution Approach 1:
The patent replaces the electrophoresis mechanical system with a chemical clearing reagent system. This substitution eliminates the need for electrical current and heat generation, achieving transparency through chemical permeation and lipid removal without thermal denaturation of proteins.
Solution Approach 2:
The patent introduces sorbitol as an intermediary substance that facilitates the clearing process without generating heat. Sorbitol acts as a protective mediator that prevents protein denaturation while enabling the clearing reagent to effectively remove lipid components and achieve transparency.
4Productivity
If a complex clearing process with dedicated devices is used, then transparency is achieved, but the process complexity and device requirements increase
Solution Approach 1:
The patent extracts and eliminates the need for complex dedicated devices and multi-step processes by developing a single clearing reagent formulation that achieves transparency through simple permeation. This extraction of unnecessary complexity makes the method accessible and easy to implement.
Solution Approach 2:
The patent creates a universal clearing reagent that performs multiple functions (urea permeation, lipid removal, shape preservation) in a single formulation, eliminating the need for multiple specialized devices and processes. This multi-functional reagent simplifies the overall clearing workflow.
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 allows for rapid and efficient transparency of biological materials without significant deformation or loss of fine structures, preserving the original form and fluorescence signals, and is reversible, making it suitable for detailed microscopic observations.
Implementation Method 1
Scale is free of such a problem of SeeDB that non-versatile apparatuses, such as lenses optimized for high refractive index and a two-photon microscope, are necessary
Implementation Method 2
SeeDB techniques for making a biological material transparent by adjusting a refractive index with use of a solution containing fructose
Implementation Method 3
If emphasis is placed on rapid permeation of urea or the like which is an active component for clearing treatment, then it results in treatment with urea at a high concentration. This causes expansion (deformation) of a biological material
Implementation Method 4
CLARITY performs clearing by removing light scattering of lipid membrane through causing lipid to be eluted from tissue with use of ionic surfactant sodium dodecyl sulfate (SDS) at a high concentration (4%)
Data Source
Figure 1a~1h
Figure 2a~2h
Figure 3a~3c
AI summary
A clearing reagent in accordance with an embodiment of the present invention for making a biological material transparent is a solution containing: at least one compound selected from the group consisting of urea and a urea derivative; sorbitol; and a surfactant which is contained at a concentration of 5 (w/v)% or less.