Electron Microscopy Grid Functionalization for Multiplexed Sample Regions
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Solution Overview
Problem
Existing electron microscopy methods face challenges in throughput, sample preparation efficiency, cross-contamination, and limited flexibility in functionalization, particularly in cryo-electron microscopy, with current techniques being time-consuming, expensive, and unsuitable for multiplexing diverse samples on a single grid.
Innovation Solution
A method for regioselective functionalization of electron microscopy grids involves creating temporary reaction chambers on the grid surface, applying modification solutions within these chambers, and removing them to achieve distinct functionalized regions, allowing for multiple analytes without cross-contamination, using affordable laboratory equipment.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Quantity of substance
If pin-printing methods are used for sample deposition, then sample deposits can be made on the EM grid, but cross-contamination occurs between deposits and the method is time-consuming
Solution Approach 1:
The EM grid surface is divided into multiple distinct quadrants or regions separated by physical barriers (such as hydrophobic coatings or raised walls). Each quadrant can independently receive different sample deposits without cross-contamination, allowing multiple samples to be processed simultaneously on a single grid while maintaining separation between them.
Solution Approach 2:
Different regions of the EM grid are given different local properties (e.g., hydrophobic vs. hydrophilic surfaces, or different chemical functionalizations) to enable selective sample deposition in specific areas. This allows different sample types to be deposited in different zones of the same grid without mixing, improving both throughput and reliability.
2Productivity
If multiple samples are deposited on a single EM grid, then throughput is increased, but cross-contamination risk increases
Solution Approach 1:
The grid surface is segmented into multiple isolated deposition zones using physical or chemical barriers. These segments allow simultaneous processing of multiple samples on one grid while preventing cross-contamination through the separating barriers, thus increasing throughput without compromising reliability.
Solution Approach 2:
Hydrophobic coatings or other intermediary substances are applied to create barriers between sample deposition zones. These intermediaries prevent direct contact between different sample solutions, eliminating cross-contamination risk while allowing multiple samples to occupy the same physical grid.
3Adaptability or versatility
If plasma treatment is used for surface functionalization, then the surface can be modified, but the type of functionalization is limited
Solution Approach 1:
The grid surface is pre-modified with a universal reactive coating (such as a silane layer or hydroxyl groups) that can subsequently react with various different functional molecules. This preliminary functionalization step enables diverse downstream functionalizations (peptides, DNA, proteins, small molecules) while maintaining high quality and reliability through a controlled, standardized initial modification process.
Data Source
Figure 1a~1b
Figure 1c~1d
Figure 1e~1g
AI summary
The invention provides a method for regioselective functionalizing a surface of an electron microscopy grid (10), therefore: a) providing a surface (20) of an electron microscopy grid (101); b) providing a plurality of wall systems (30A, 306) on the surface of an electron microscopy grid, thereby forming a plurality of temporary reaction chambers (40A, 406); c) providing a modification solution (50) in each temporary reaction chambers (40A, 406); d) modifying the parts of the surface (21A, 216) in the temporary reaction chambers (40A, 406) with the modification solution(s) (50, 50A, 506); e) removing the modification solution(s) (50, 50A, 506) from the temporary reaction chambers (40A, 406); f) removing the plurality of wall systems (30A, 306) from the surface (20). The invention further provides in an electron microscopy grid to be regioselective functionalised and an apparatus for regioselective functionalizing a surface of an electron microscopy grids.