Expansion Microscopy Hydrogel for Thick Tissue Imaging
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Solution Overview
Problem
Current expansion microscopy techniques face limitations in imaging thick tissue samples, particularly fungal or bacterial samples, and lack flexibility in treatments, as traditional protease-based homogenization methods fail to break down cell walls of these organisms, restricting the ability to achieve high-resolution imaging.
Innovation Solution
A method involving permeation and polymerization of a cell or tissue sample with a polymer monomer composition containing α,β-unsaturated carbonyl monomers and enals, such as acrylates and acrolein, to form a swellable hydrogel that covalently links with biomaterials, allowing for isotropic expansion and improved mechanical homogenization, enabling high-resolution imaging of complex structures.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Adaptability or versatility
If traditional protease-based homogenization methods are used, then tissue material and proteins are digested, but cell walls of bacterial and fungal cells cannot be broken down
Solution Approach 1:
The patent changes the chemical parameters of the homogenization buffer by adding chaotropic salts (guanidine hydrochloride, urea) and detergents (SDS, Triton X-100) to the existing protease buffer system. This parameter modification enables the buffer to simultaneously digest proteins and break down cell walls of bacterial and fungal cells, achieving universal homogenization across different sample types including those resistant to traditional protease-only treatment.
2Manufacturing precision
If polymer monomer composition is permeated into thick samples, then complete infusion is achieved, but polymerization time increases
Solution Approach 1:
The patent applies preliminary action by performing extensive permeation of the polymer monomer composition into thick samples before initiating polymerization. This pre-infusion ensures complete and uniform distribution of monomers throughout the entire sample volume, eliminating gradients and ensuring homogeneous gel formation. The preliminary permeation step, though time-consuming, prevents the need for repeated treatments and ensures consistent expansion throughout thick tissues.
Solution Approach 2:
The patent maintains continuity of useful action by using a catalyzed polymerization system with ammonium persulfate and TEMED that proceeds continuously and completely once initiated. The polymerization reaction runs to completion without interruption, converting all permeated monomers into gel matrix, which maximizes the efficiency of the prior permeation effort and eliminates the need for multiple partial polymerization cycles.
3Measurement precision
If conventional optical microscopy is used, then imaging is simple, but resolution is limited by optical diffraction
Solution Approach 1:
The patent replaces the optical resolution limitation with a mechanical/chemical expansion approach. Instead of using complex super-resolution microscopy systems (STED, PALM, STORM), the invention physically expands the biological sample by incorporating it into a hydrogel matrix that swells 4-fold or more. This mechanical expansion of the sample, rather than the imaging system, brings sub-diffraction structures into the resolvable range of conventional microscopes, achieving super-resolution with standard optical equipment.
Solution Approach 2:
The patent changes the physical parameter of the sample by expanding its size through hydrogel incorporation. The biological specimen is embedded in a polymerizable monomer composition that, upon polymerization and hydration, expands the sample volume and separates closely spaced structures. This parameter change in sample dimensions transforms unresolvable nanoscale features into resolvable micrometer-scale features detectable by conventional optics.
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 rapid and complete infusion of polymer monomers into thick samples, facilitating three-dimensional visualization and imaging of complex tissue structures, including bacterial and fungal samples, with enhanced flexibility in treatment options and improved resolution.
Implementation Method 1
permeating the sample with a polymer monomer composition
Implementation Method 2
polymerizing the polymer monomer composition with the enal to form a swellable material
Implementation Method 3
resulting in covalent linking of the enal to both the swellable material and a biomaterial in the sample
Implementation Method 4
The swellable gels are then hydrated to facilitate uniform, e.g. isotropic, expansion of the gel-specimen matrix
Data Source
AI summary
Provided herein are improved expansion microscopy methods. Also provided herein are kits useful in expansion microscopy.