Kits and methods for super-resolution microscopy
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Solution Overview
Problem
Existing super-resolution microscopy techniques face challenges in achieving reproducible 4-fold and larger expansions, require expensive equipment, and involve complex methods that are difficult to reproduce, especially in 3D imaging.
Innovation Solution
A modified expansion microscopy (mExM) method using a chemically and biochemically inert mold and protein digestion buffer, with manual sealing to exclude oxygen, enabling 4-fold and 12-fold expansions with sodium dodecyl sulfate and buffering components, and manual sealing to avoid active deoxygenation.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Manufacturing precision
If conventional expansion microscopy methods are used, then some expansion is achieved, but reproducible 4-fold and larger expansion cannot be achieved
Solution Approach 1:
The patent employs an oxygen-scavenging system using glucose oxidase, catalase, and glucose to create an oxygen-free environment during polymerization. This inert atmosphere prevents oxygen inhibition of polymerization, enabling consistent and reproducible 4-fold and larger expansion folds that were not achievable with conventional methods.
Solution Approach 2:
The patent modifies key parameters including using high concentration acrylamide (20% w/v), specific crosslinker ratios (0.15% w/v BIS), and controlled polymerization conditions (4°C overnight). These parameter changes ensure consistent gel formation and expansion, achieving reproducible 4-fold and larger expansion across different samples.
2Measurement precision
If super-resolution microscopy equipment is used, then better resolution is achieved, but expensive equipment is required
Solution Approach 1:
The patent creates physical copies of the specimen at expanded scales (4-fold or greater). By expanding the biological sample itself rather than enhancing the microscope, the method enables super-resolution imaging with conventional microscopes, eliminating the need for expensive super-resolution microscopy equipment.
Solution Approach 2:
The patent transitions from attempting to resolve fine details in the original dimension to physically expanding the specimen in three dimensions. This dimensional approach allows conventional microscopes to achieve super-resolution by imaging expanded structures that are magnified to resolvable scales.
3Measurement precision
If mathematical image processing is used, then resolution is improved, but it is difficult to determine whether the processed image is real
Solution Approach 1:
The patent replaces mathematical/image processing methods with a physical expansion method. By physically expanding the specimen 4-fold or greater, the resolution improvement is achieved through physical magnification rather than computational algorithms, providing direct visual evidence of real structures without relying on image processing artifacts.
4Reliability
If active deoxygenation equipment is used, then polymerization proceeds correctly, but expensive and complex equipment is required
Solution Approach 1:
The patent employs a self-service oxygen scavenging system where glucose oxidase and catalase enzymes automatically consume oxygen in the presence of glucose, creating an oxygen-free environment without requiring external deoxygenation equipment. This biochemical self-service approach replaces expensive mechanical deoxygenation systems.
Solution Approach 2:
The patent introduces glucose as an intermediary substrate that the enzyme system consumes along with oxygen. The glucose-glucose oxidase-catalase system acts as a biochemical intermediary that mediates oxygen removal, replacing the need for direct mechanical or chemical deoxygenation equipment.
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
Achieves stable and reproducible 5 nm accuracy with super-resolution microscopes and 20 nm accuracy with regular wide-field microscopes, simplifying the method steps and reducing equipment requirements.
Implementation Method 1
The instructions include a written or visual representation of execution of the exM method by conducting the at least one method step that involves polymerization in the mold by forming a hand-made seal with a manual sealing material and excluding visible oxygen bubbles form the mold upon sealing
Implementation Method 2
The instructions include a description an expanded microscopy (ExM) method that includes at least one method step that involves polymerization in the absence of oxygen
Implementation Method 3
The protein digestion buffer includes sodium dodecyl sulfate and a buffering component. The instructions include a description of an ExM method that includes at least one method step that involves a digestion step utilizing a protein digestion agent
Implementation Method 4
The mold is dimensioned to receive a cell of interest with sufficient volume to undergo at least a 12-fold increase in cell volume of the cell of interest
Data Source
AI summary
Conventional expanded microscopy (ExM) is improved upon to show reliable and reproducible 4-fold and 12-fold modified expansion microscopy (mExM). Kits and methods are disclosed to achieve these improvements. The kits can include a sealable mold and instructions for using it in the improved methods. The kits can include a protein digestion buffer including sodium dodecyl sulfate and instructions for using the buffer in the improved methods. The improved methods can include a simplified approach to executing oxygen-sensitive reactions within the methods. The improved methods can include improved protein digestion chemistries, which can facilitate the improvements in reliability and reproducibility.