Cryo-EM Grid Edge Wicking for Uniform Ice Thickness
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Solution Overview
Problem
Current methods for preparing electron microscopy samples result in inconsistent ice thickness and distribution due to direct contact with absorbent materials, leading to reproducibility issues and uneven sample preparation for cryo-EM.
Innovation Solution
An apparatus and method that thins the aqueous suspension on an electron microscopy grid by draining excess liquid to the perimeter, assisted by air flow, acceleration, or temperature gradients, without direct contact with absorbent materials, allowing for controlled vitrification and maintaining structure-friendly conditions for biological samples.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Loss of substance
If an absorbent material is pressed onto the entire area of an electron microscopy grid to remove excess liquid, then the liquid removal efficiency is improved, but the ice thickness becomes inconsistent and the sample preparation repeatability deteriorates
Solution Approach 1:
The absorbent material is segmented into multiple discrete pieces positioned at specific locations around the grid perimeter rather than covering the entire grid area. This segmentation allows controlled liquid removal at edges while preserving the central imaging area, resolving the contradiction between liquid removal efficiency and ice thickness consistency.
Solution Approach 2:
Different regions of the grid are treated differently: absorbent material is placed only at peripheral regions for liquid removal, while the central imaging region is left untouched to maintain consistent ice thickness. This local differentiation resolves the contradiction by applying liquid removal only where needed without affecting the critical imaging area.
2Measurement precision
If multiple samples are prepared to find areas with sufficient contrast, then the contrast quality is improved, but the sample preparation time and complexity increase
Solution Approach 1:
The absorbent material is pre-positioned on the grid before sample deposition, and the liquid removal process is optimized to achieve uniform ice thickness in a single preparation step. This preliminary setup and optimized process reduce the need for multiple sample preparations and searching, thereby improving contrast quality while reducing preparation time.
3Reliability
If affinity electron microscopy grids are used to bind molecules to the substrate, then the molecular stability is improved, but the liquid removal process becomes more complex
Solution Approach 1:
The absorbent material is segmented into small discrete pieces positioned at the grid perimeter, which simplifies the liquid removal process while maintaining compatibility with affinity grids. The segmented approach allows gentle, localized liquid removal that does not disrupt molecule-substrate binding, thus maintaining molecular stability while reducing process complexity.
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 improves the repeatability, control, and throughput of sample preparation by eliminating tortuous boundary conditions and preventing molecular diffusion or denaturation, resulting in more consistent and effective cryo-EM imaging.
Implementation Method 1
contacting an edge of the electron microscopy grid with an absorbent material, allowing a thickness of the aqueous suspension to decrease over a period of time
Implementation Method 2
freezing the aqueous suspension
Implementation Method 3
The resulting thin aqueous suspension is then immersed in a cryogenic liquid for vitrification of the aqueous suspension
Implementation Method 4
a flow of air (e.g., humid air) to push aqueous suspension away from the imaging area
Implementation Method 5
a temperature gradient or surfactant gradient across the aqueous suspension disposed on the electron microscopy grid to create a gradient in surface tension and subsequent flow
Data Source
AI summary
This disclosure provides systems, methods, and apparatus related to electron microscopy sample preparation. In one aspect, a method includes providing an electron microscopy grid. An aqueous suspension including an electron microscopy sample is deposited on the electron microscopy grid. An edge of the electron microscopy grid is contacted with an absorbent material. A thickness of the aqueous suspension is allowed to decrease over a period of time. The aqueous suspension is then frozen.