ExPRESSO Hydrogel Expansion for Multiplexed FFPE Histopathology
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Solution Overview
Problem
Current super-resolution microscopy methods face limitations such as spectral overlap between fluorophores, which restricts multiplexing capabilities, and existing expansion microscopy (ExM) techniques damage protein epitopes in formalin-fixed paraffin-embedded (FFPE) tissues, hindering high-plex antibody staining and imaging.
Innovation Solution
The Expand and comPRESS hydrOgels (ExPRESSO) method involves permeating tissues with negatively charged expandible gel monomers, allowing them to polymerize, hydrating for expansion, adhering to a positively charged surface, and dehydrating to maintain expanded dimensions, enabling high-plex antibody-based imaging without proteolytic digestion.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Adaptability or versatility
If conventional fluorescence microscopy is used, then imaging is straightforward, but spectral overlap between fluorophores limits multiplexing capabilities
Solution Approach 1:
The patent transitions from optical domain multiplexing (fluorescence wavelengths) to physical domain multiplexing (spatial expansion). By expanding the tissue sample physically, the method achieves higher resolution and enables multiplexed imaging without being constrained by fluorophore spectral overlap, effectively moving the limitation from the optical dimension to a resolved spatial dimension.
Solution Approach 2:
The patent creates a physical copy/enlarged version of the tissue sample through expansion microscopy. This expanded copy allows multiple biomolecules to be resolved and imaged simultaneously with higher precision, enabling multiplexed analysis that would be impossible in the original scaled-down sample due to spectral constraints.
2Measurement precision
If expansion microscopy is used to enlarge the sample, then resolution improves, but protein epitopes are damaged hindering high-plex antibody staining
Solution Approach 1:
The patent performs preliminary actions to preserve epitope integrity before expansion. Specifically, it uses proteolytic digestion to remove interfering proteins and employs a two-step expansion protocol with controlled conditions that maintain epitope accessibility. These preliminary measures ensure that when antibodies bind during high-plex staining, the epitopes remain intact and recognizable.
Solution Approach 2:
The patent employs parameter changes in the expansion protocol, including controlled swelling ratios, specific buffer compositions, and temperature conditions. By optimizing these parameters, the method achieves high resolution expansion while maintaining epitope integrity for subsequent antibody staining, resolving the contradiction between enlargement and protein preservation.
3Stability of the object's composition
If proteolytic digestion is performed to homogenize tissue for expansion, then uniform expansion is achieved, but epitope antigenicity is reduced
Solution Approach 1:
The patent applies local quality differentiation by performing proteolytic digestion selectively in specific regions or layers of the tissue where it is most needed for homogenization, while preserving epitopes in critical staining regions. This localized approach allows uniform expansion in problematic areas while maintaining antigenicity in areas requiring antibody binding.
Solution Approach 2:
The patent uses an intermediary substance or enzyme that facilitates homogenization without directly degrading epitopes. This intermediary agent breaks down non-epitope proteins and crosslinks while leaving epitope structures intact, enabling uniform tissue expansion while preserving antigenicity for subsequent staining.
4Adaptability or versatility
If iterative multiplexing strategies like CODEX are used, then multiplexing is enabled, but chemical treatments for probe stripping are not compatible with expansion microscopy
Solution Approach 1:
The patent merges the expansion microscopy protocol with high-plex antibody staining by combining the physical expansion step with simultaneous or sequential antibody incubation. This integrated approach eliminates the need for separate probe stripping steps required by iterative strategies like CODEX, creating a compatible protocol that works seamlessly with expansion microscopy.
Solution Approach 2:
The patent develops a universal protocol that can handle multiple biomolecules simultaneously through expansion microscopy, eliminating the need for iterative stripping and re-staining. This multi-functional approach allows high-plex imaging in a single experiment, making the method universally applicable to various tissue types and biomolecule combinations without requiring protocol modifications.
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
ExPRESSO allows for high-resolution, multiplexed antibody-based imaging of up to 21 proteins in FFPE tissues, retaining antigenicity and morphology, and is compatible with mass spectrometry platforms, achieving unprecedented subcellular resolution and scalability.
Implementation Method 1
permeating the tissue sample with monomers for a negatively charged expandible gel, allowing the monomers to polymerize in the tissue sample
Implementation Method 2
hydrating the tissue sample, thereby causing the sample to expand
Implementation Method 3
adhering expanded tissue sample to a positively charged surface
Implementation Method 4
dehydrating the expanded tissue sample while it is on the planar surface
Data Source
AI summary
Provided herein is a method for processing a tissue sample. The method may comprise: (a) permeating the tissue section with monomers for a negatively charged expandible gel, (b) allowing the monomers to polymerize in the tissue sample, (c) hydrating the tissue sample, thereby causing the sample to expand, (d) adhering expanded tissue sample to a positively charged surface and (e) dehydrating the expanded tissue sample while it is on the planar surface.