Tissue Clearing via Acrylamide Hydrogel Perfusion
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Solution Overview
Problem
Current methods for whole-organ imaging are limited by the lack of effective techniques to render organs macromolecule-permeable and optically-transparent, hindering the understanding of structure-function relationships at cellular and organ-wide scales.
Innovation Solution
A method involving the introduction of paraformaldehyde, acrylamide hydrogel monomers, and detergent solutions into the circulatory system, followed by photoinitiator and imaging media, to create optically-clear tissues suitable for in situ immunostaining and imaging, using techniques like PACT and PARS for rapid and uniform clearing.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Illumination intensity
If traditional tissue clearing methods are used, then optical transparency is improved, but macromolecule permeability and preservation of molecular content deteriorate
Solution Approach 1:
The patent introduces an acrylamide hydrogel as an intermediary substance that permeates the tissue and forms a three-dimensional network structure. This hydrogel acts as a mediator that simultaneously improves optical transparency by replacing tissue lipids and maintains macromolecule permeability by creating a porous structure that preserves antibody accessibility and molecular content throughout the clearing process
Solution Approach 2:
The patent creates a composite tissue-hydrogel material by infusing acrylamide monomers into the tissue and polymerizing them in situ. This composite structure combines the optical clearing properties of the hydrogel with the preserved molecular architecture of the tissue, achieving both transparency and molecular content preservation that neither material could achieve alone
2Reliability
If whole-organ imaging is attempted without proper clearing, then molecular content is preserved, but optical transparency and cellular structure visualization deteriorate
Solution Approach 1:
The acrylamide hydrogel serves as an intermediary clearing agent that penetrates the entire organ through the circulatory system, replacing lipids and air pockets with a transparent gel matrix. This intermediary substance maintains porosity and molecular accessibility while providing the optical clarity needed for whole-organ imaging
Solution Approach 2:
The patent utilizes the circulatory system's hydraulic network to distribute the acrylamide monomer solution uniformly throughout the entire organ. By injecting the solution through blood vessels, the hydrogel monomers are transported hydraulically to all tissue regions, ensuring uniform clearing and transparency across the whole organ while preserving molecular content
3Productivity
If rapid clearing is performed to improve productivity, then processing time is reduced, but uniformity of clearing and molecular content preservation deteriorate
Solution Approach 1:
The patent employs continuous perfusion of acrylamide monomer solution through the circulatory system, maintaining a steady flow that continuously delivers clearing agents to all tissue regions. This continuous action ensures uniform penetration and clearing throughout the organ while maintaining molecular content preservation, achieving both speed and uniformity simultaneously
Solution Approach 2:
By utilizing the circulatory system's existing hydraulic pressure and flow pathways, the patent achieves rapid and uniform distribution of clearing agents throughout the entire organ. The blood vessel network provides pre-established channels that enable fast, uniform perfusion of acrylamide monomers to all tissue regions, maintaining both processing speed and clearing uniformity
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 the visualization of cellular structures and immunohistochemistry across various organs with improved transparency and preservation of molecular content, facilitating the study of cell-to-cell relationships and long-range neural connectivity.
Implementation Method 1
introducing a fixing solution that includes paraformaldehyde (PFA) into the circulatory system of a subject, thereby forming fixed tissue within the subject
Implementation Method 2
introducing a photoinitiator solution comprising 2,2'-Azobis[2-(2-imidazolin-2-yl)propane]dihydrochloride into the circulatory system of the de-gassed subject
Implementation Method 3
introducing a hydrogel monomer solution that includes acrylamide and phosphate buffered saline (PBS) into the circulatory system of the subject, thereby forming hydrogel-treated tissue within the subject
Implementation Method 4
introducing a detergent solution that includes sodium dodecyl sulfate (SDS) into the circulatory system of the subject, thereby forming cleared tissue within the subject
Implementation Method 5
placing the subject into a substantially air tight chamber, and introducing nitrogen into the chamber, thereby forming a de-gassed subject
Implementation Method 6
introducing imaging media into the subject's circulatory system, wherein the imaging media comprises: (1) 1-N,3-N-bis(2,3-dihydroxypropyl)-5-[N-(2,3-dihydroxypropyl)acetamido]-2,4,6-triiodobenzene-1,3-dicarboxamide or 5-(N-2,3-Dihydroxypropylacetamido)-2,4,6-triiodo-N,N'-bis(2,3-dihydroxypropyl)isophthalamide
Data Source
Figure 1A~1H
Figure 1I~1L
Figure 2A~2B
AI summary
In various embodiments, the present application teaches methods and compositions for tissue clearing in which whole organs and bodies are rendered macromolecule-permeable and optically-transparent, thereby exposing their cellular structure with intact connectivity. In some embodiments, the present application teaches PACT, a protocol for passive tissue clearing and immunostaining of intact organs. In other embodiments, the present application teaches RIMS, a refractive index matching media for imaging thick tissue. In yet other embodiments, the application teaches PARS, a method for whole-body clearing and immunolabeling.