Biological Tissue Clearing Composition for Deep Imaging
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Solution Overview
Problem
Current methods for optically clearing biological samples, such as sucrose, glycerol, BABB, SCALE, SeeDB, and CLARITY, face limitations including tissue shrinkage, loss of endogenous fluorescence, poor light transmittance, and incompatibility with antibody labeling, especially when attempting to image larger samples or diverse tissues beyond the brain.
Innovation Solution
A method using a composition comprising sodium dodecyl sulfate (SDS), 3-(N,N-Dimethylmyristylammonio)propanesulfonate (SB3-14), Tween 20, Triton X-100, sodium deoxycholate, and a salt to delipidate biological samples, allowing for high-pressure perfusion and refractive index matching with 2,2′-thiodiethanol (TDE) for enhanced imaging.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Ease of operation
If traditional IHC techniques with paraffin embedding are used, then antibody labeling is enabled, but endogenous fluorescence is removed and tissue structure is damaged
Solution Approach 1:
The patent extracts and removes lipids from the tissue sample using a specific chemical composition, separating the lipid component from the protein-rich tissue structure. This allows the tissue to be cleared of light-scattering lipids while preserving the fluorescent proteins and antibody binding sites, resolving the contradiction between fluorescence retention and lipid removal
Solution Approach 2:
The patent changes the chemical parameters of the tissue environment by introducing a specific composition containing detergents and salts that selectively solubilizes lipids. This parameter change enables simultaneous achievement of lipid removal and preservation of fluorescent structures, allowing both antibody labeling and fluorescence retention
2Illumination intensity
If sucrose clearing is used to remove lipids, then light transmittance is improved, but tissue shrinkage occurs and endogenous fluorescence is lost
Solution Approach 1:
The patent changes the chemical parameters by using a specific composition with detergents (SDS, Triton X-100, Tween 20) and salts that selectively solubilizes lipids without causing tissue shrinkage. This parameter change enables lipid removal with preservation of tissue structure and fluorescence, resolving the contradiction between light transmittance improvement and structure integrity
Solution Approach 2:
The patent uses a composite clearing composition combining multiple detergents and salts that work synergistically to remove lipids while maintaining tissue structure. This composite approach achieves both improved light transmittance and preserved tissue morphology, overcoming the limitations of single-agent clearing methods
3Length of stationary object
If deep tissue imaging is attempted without clearing, then imaging depth is increased, but light scattering from lipids prevents clear visualization
Solution Approach 1:
The patent extracts lipids from the tissue sample, removing the primary cause of light scattering. This extraction enables deep tissue imaging with clear signal visualization, as the remaining tissue structure does not scatter light significantly, resolving the contradiction between imaging depth and signal clarity
4Measurement precision
If tissue is sectioned into thin slices for imaging, then imaging resolution is improved, but time consumption increases and tissue edges are damaged
Solution Approach 1:
The patent removes lipids from the intact tissue sample, eliminating the need for sectioning. This extraction enables direct imaging of whole tissues or large samples without time-consuming sectioning procedures, resolving the contradiction between imaging resolution and preparation time
Solution Approach 2:
Instead of sectioning the tissue to achieve sufficient resolution (conventional approach), the patent inverts the approach by clearing the entire tissue sample to enable imaging of thick, intact samples. This inversion eliminates the need for sectioning while maintaining or improving resolution through optical clearing
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
Enables deep tissue imaging with minimal tissue distortion, retention of endogenous fluorescence, and compatibility with antibody labeling, achieving unprecedented optical clarity and resolution for macroscopic imaging of various tissues up to several millimeters in depth.
Implementation Method 1
The composition comprises sodium dodecyl sulfate (SDS), 3-(N,N-Dimethylmyristylammonio)propanesulfonate (SB3-14), Tween 20, Triton X-100, sodium deoxycholate, and a salt
Implementation Method 2
refractive index matching with 2,2′-thiodiethanol (TDE) for enhanced imaging
Data Source
AI summary
The disclosure provides improved materials and methods for optically clearing biological tissue that is subsequently used for deep tissue imaging analysis. Also provided is a description of a microscopic image acquisition methodology in which imagery of intact tissues are acquired to rapidly acquire microscopy data on a whole-organ scale to maximize cost effectiveness for biological microscopy and minimize time spent performing such analysis.