Expansion Microscopy Tissue Preparation via Segmented Fixation
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Solution Overview
Problem
Current methods for imaging biomolecules in tissue samples face challenges such as localization error due to the finite size of targeting reagents and the fixation tradeoff, where strong fixation preserves tissue structure but reduces the ability of targets to bind imaging reagents, limiting imaging quality.
Innovation Solution
A method involving treating the tissue sample with a chemical fixative, attaching target biomolecules to a linear polymer, and embedding them in a swellable hydrogel, allowing for ultrahigh effective imaging resolution and multiple rounds of antibody or affinity reagent application and washout.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Stability of the object's composition
If strong fixation is applied to preserve tissue structure, then ultrastructure quality is improved, but the ability of targets to bind imaging reagents deteriorates due to increased chemical modification and steric hindrance
Solution Approach 1:
The patent segments the fixation process into two distinct stages: (1) mild initial fixation to preserve basic tissue structure, and (2) strong fixation applied only after expansion to preserve ultrastructure. This segmentation allows each fixation stage to serve its specific purpose without the negative effects of premature strong fixation, thereby resolving the contradiction between structure preservation and reagent binding ability.
Solution Approach 2:
The patent applies preliminary mild fixation before expansion to preserve basic tissue structure while maintaining epitope accessibility. This preliminary action allows imaging reagents to bind effectively to targets before the strong fixation is applied post-expansion, thus resolving the contradiction by performing the necessary binding-friendly fixation first.
2Measurement precision
If expansion microscopy is applied to reduce localization error, then imaging resolution is improved, but the fixation tradeoff limitation persists
Solution Approach 1:
The patent applies dynamics by making the fixation strength adaptive to the expansion stage. Mild fixation is used in the pre-expansion state to maintain binding capability, while strong fixation is dynamically applied after expansion to preserve ultrastructure. This dynamic approach allows high-resolution imaging through expansion while maintaining antibody staining quality at each stage.
Solution Approach 2:
The patent changes the fixation parameter (strength) based on the expansion state. Before expansion, weak fixation parameters are used to preserve epitope accessibility. After expansion, strong fixation parameters are applied to preserve ultrastructure. This parameter change resolves the contradiction by optimizing fixation strength for each imaging stage.
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
This approach enables high-quality antibody staining and ultrahigh effective imaging resolution by minimizing localization error and overcoming the fixation tradeoff, allowing for stable and detailed imaging of biomolecules within the tissue sample.
Implementation Method 1
treating the tissue sample with a chemical fixative
Implementation Method 2
embedding the tissue sample and the target biomolecule within the swellable hydrogel
Implementation Method 3
treating the tissue sample with a polymerization solution to be polymerized to generate the linear polymer
Data Source
AI summary
Methods of imaging biomolecules from a tissue sample are described, including methods for preparing a tissue sample for imaging using expansion microscopy, while achieving ultrahigh effective imaging resolution.