Low Melting Agarose Matrix for Stable Expansion Microscopy
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Solution Overview
Problem
Current expansion microscopy methods are not readily amenable to efficient processing, expansion, and imaging of large biological specimens such as whole organs, due to limitations in sample stability, stiffness, and imaging speed.
Innovation Solution
The use of low melting agarose (LM agarose) in expansion solutions and composites to increase the stiffness and stability of expanded hydrogels, facilitate efficient staining, and enhance imaging capabilities for large specimens, including intact organs, by providing a supporting matrix that allows for uniform expansion and improved handling during imaging.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If conventional expansion microscopy methods are used on large biological specimens, then sample expansion and resolution improvement are achieved, but sample stability and structural integrity deteriorate
Solution Approach 1:
The patent combines hydrogel matrix with agarose to create a composite material that provides both expansion capability and structural stability. The hydrogel enables isotropic expansion while the agarose component maintains structural integrity during and after expansion, resolving the contradiction between achieving high resolution through expansion and maintaining sample stability.
Solution Approach 2:
The patent modifies the physical and chemical parameters of the expansion medium by incorporating agarose, which changes the mechanical properties of the hydrogel matrix. This parameter change allows the material to maintain appropriate stiffness and stability while still permitting the necessary expansion for super-resolution imaging.
2Measurement precision
If large biological specimens are expanded for super-resolution imaging, then imaging resolution is improved, but processing efficiency and imaging speed deteriorate
Solution Approach 1:
The patent performs preliminary stabilization of the sample within the hydrogel-agarose composite matrix before the expansion and imaging processes. This preliminary action ensures that the sample maintains its structural integrity throughout the lengthy imaging process, enabling faster and more efficient processing without compromising resolution.
Solution Approach 2:
The composite hydrogel-agarose material facilitates more efficient processing of large specimens by providing uniform expansion properties and enhanced structural support, which streamlines the imaging workflow and reduces processing time while maintaining super-resolution capability.
3Measurement precision
If hydrogel expansion is performed on intact organs, then nanoscale visualization is enabled, but sample stiffness and handling difficulty worsen
Solution Approach 1:
The patent uses a composite hydrogel-agarose system where the agarose component provides appropriate mechanical stiffness and structural support, making expanded organ samples easier to handle and manipulate while maintaining the expansion properties necessary for nanoscale visualization.
Solution Approach 2:
The patent creates a material with locally optimized properties: the hydrogel portion allows expansion and flexibility where needed, while the agarose portion provides stiffness and structural support in regions requiring handling stability, achieving both nanoscale resolution and ease of operation.
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
LM agarose enhances the integrity and imaging efficiency of large biological specimens, enabling faster and more reliable super-resolution imaging with improved sample stability and compatibility for extensive scanning and slicing, particularly for large organs like brains and other neural structures.
Implementation Method 1
ExM generally involves synthesizing a swellable polymer network (or hydrogel) throughout a biological specimen... Following gelling, the polymer-embedded sample is typically subject to homogenization to facilitate even swelling at the expansion stage. The homogenized sample can then be uniformly (isotropically) expanded in a low salt solution, water, or other solvent.
Implementation Method 2
Such expansion increases the distance between biomolecules of interest while preserving their spatial relationship, thereby allowing molecules that were previously too close together to be discerned using standard, diffraction-limited, fluorescence microscopy.
Data Source
AI summary
Matrix-assisted methods and compositions, including those based on solutions containing low melting agarose, to prepare intact organs and other samples for super resolution imaging by microscopy, and more particularly, lightsheet microscopy.