Tissue-Hydrogel Hybrid Expansion for Subcellular Imaging
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Solution Overview
Problem
Current methods fail to effectively reconstruct the complex interactions of diverse cells in biological systems at a systems-level, lacking tools for holistic reconstruction of cellular connectivity and molecular details across multiple scales.
Innovation Solution
The Magnified Analysis of Proteome (MAP) method creates a size-adjustable tissue-hydrogel hybrid that preserves three-dimensional proteomic libraries and organ-wide cellular connectivity, allowing for multi-resolution imaging by preventing intra- and inter-protein crosslinking and denaturing proteins to enable natural expansion, using high concentrations of acrylamide monomers.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Volume of moving object
If conventional tissue processing methods are used, then tissue structure is preserved, but tissue expansion and multi-resolution imaging are not achieved
Solution Approach 1:
The patent changes the physical and chemical parameters of the tissue by infusing hydrogel monomers and inducing denaturation, transforming the tissue from its native state to an expanded hydrogel-hybrid state. This parameter change enables volume expansion while preserving structural information through the hydrogel network that forms during the process.
Solution Approach 2:
The patent creates a composite material by combining tissue with hydrogel monomers (acrylamide, bis-acrylamide) to form a tissue-hydrogel hybrid. This composite structure allows the tissue to expand while maintaining structural integrity, as the hydrogel network provides a scaffold that preserves subcellular details during expansion.
2Measurement precision
If tissue is expanded to achieve multi-resolution imaging, then imaging resolution is improved, but repeated antibody staining becomes difficult due to epitope loss
Solution Approach 1:
The patent performs preliminary action by infusing hydrogel monomers and forming the hydrogel network before denaturation and expansion. This preliminary hydrogel formation creates a protective scaffold that maintains epitope accessibility throughout subsequent repeated staining cycles, preventing epitope loss that would otherwise occur during expansion.
Solution Approach 2:
The patent controls the denaturation parameters (temperature, time, detergent concentration) to achieve sufficient protein unfolding for expansion while maintaining epitope integrity. The controlled parameter changes allow repeated antibody staining by preserving the immunogenic properties of proteins throughout the expansion process.
3Volume of moving object
If high concentration of hydrogel monomers is used to prevent crosslinking, then tissue expansion is enhanced, but complexity of the processing procedure increases
Solution Approach 1:
The patent employs self-service by using the tissue's own amine groups to react with formaldehyde and subsequently with hydrogel monomers. This self-reactivity eliminates the need for external crosslinking agents or complex chemical treatments, simplifying the procedure while achieving high expansion ratios through the natural biochemical properties of the tissue.
4Volume of moving object
If proteins are denatured to enable expansion, then tissue volume increases, but protein aggregation may occur reducing imaging quality
Solution Approach 1:
The patent uses hydrogel monomers as an intermediary that binds to denatured proteins during expansion. This intermediary prevents direct protein-protein aggregation by mediating the interaction between proteins and the expanding hydrogel network, ensuring uniform protein distribution throughout the expanded tissue volume.
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
MAP allows for reversible expansion of tissues up to 4-5 fold, preserving fine subcellular details and organ-scale connectivity, enabling multiplexed imaging and repeated antibody staining without epitope loss, facilitating the study of complex biological systems.
Implementation Method 1
inducing hybridization of the hydrogel subunits to each other or to tissue
Implementation Method 2
The high concentration of hydrogel monomers reduces intra- and inter-protein crosslinking during the subsequent hydrogel-tissue hybridization step by quenching reactive methylols that form from amine residues reacting with formaldehyde
Implementation Method 3
Denaturation may be accomplished by incubating the tissue at high temperature (e.g., 80-95° C.) in the presence of detergent (e.g., 200 mM SDS) for a sufficient period of time
Implementation Method 4
The denatured and/or dissociated proteins are hybridized to the surrounding hydrogel but not to themselves and thus are able to expand along with the hydrogel. Importantly, the expansion is linear, intending that it occurs to about the same degree in all dimensions or directions
Data Source
AI summary
This disclosure provides methods for producing size-adjustable tissue-hydrogel hybrids or cell-hydrogel hybrids for imaging cellular and subcellular details and system-scale (e.g., tissue or organism level) intercellular connectivity.