Cryo-EM Sample Support Grid for Faster Vitrification Handling
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Solution Overview
Problem
Current cryo-electron microscopy sample preparation and handling techniques are prone to errors, damage, and inefficiencies, leading to poor reproducibility and high costs, particularly due to manual handling issues and suboptimal cooling rates, resulting in crystalline ice formation and sample contamination.
Innovation Solution
The design of sample supports with modified grids and foils that include solid gripping areas, reduced grid bar widths and thicknesses, unique markings for orientation and tracking, and optimized cooling systems to enhance vitrification and reduce sample motion, along with automated handling tools to minimize damage and improve cooling rates.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Ease of operation
If manual handling of grids and foils is used, then sample preparation can be performed, but grids and foils are routinely bent, torn, and damaged
Solution Approach 1:
The grid is divided into a solid outer edge region and a mesh pattern region. The solid outer edge region serves as a dedicated handling zone that can be gripped by tweezers without risking damage to the fragile foil, while the mesh region maintains imaging functionality. This segmentation isolates the handling function from the imaging function, protecting the foil during manual operations.
Solution Approach 2:
Different regions of the grid are assigned different properties: the outer edge region has solid structure for mechanical strength and handling, while the central region has mesh pattern with holes for sample imaging. The foil is selectively applied only where needed for imaging, leaving the handling region as solid grid for robust manipulation.
2Strength
If standard grid bar widths and thicknesses are used, then grid strength is maintained, but cooling rates are suboptimal and crystalline ice forms
Solution Approach 1:
The grid bar width and thickness are reduced only in specific regions where the foil is applied and sample imaging occurs, while maintaining standard dimensions in the solid outer edge region for handling. This localized modification optimizes cooling rates at the sample location without compromising the overall structural integrity and handling capability of the grid.
Solution Approach 2:
The invention modifies grid bar dimensions in the lateral plane (width and thickness) to enhance thermal conduction. By reducing grid bar width and thickness in imaging regions, thermal resistance is decreased, allowing faster heat extraction and improved cooling rates for vitrification, while the vertical dimension (grid bar height) maintains sufficient mechanical strength.
3Device complexity
If conventional sample supports without orientation markings are used, then handling is simpler, but precise orientation relative to the electron beam cannot be determined
Solution Approach 1:
Orientation markings are placed specifically in the solid outer edge region of the grid, which is visible during plunge cooling and microscopy but does not interfere with the mesh pattern imaging region. These markings provide precise orientation reference without adding complexity to the sample support function or interfering with electron beam imaging.
4Strength
If thick grid bars are used, then grid strength is sufficient for handling, but sample motion occurs during plunging and imaging
Solution Approach 1:
The grid structure uses thick bars in the solid outer edge region for robust handling and thin bars in the mesh imaging region for minimal sample motion. The thick outer bars provide mechanical strength during handling and plunging, while the thin inner bars reduce thermal mass and mechanical constraints on the sample, preventing beam-induced motion during imaging.
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
The solution significantly reduces grid and foil damage, improves sample handling reproducibility, and enhances cooling efficiency, leading to better vitrification and reduced beam-induced sample motion, thus improving the quality and consistency of cryo-electron microscopy data.
Implementation Method 1
To vitrify the buffer for the best imaging, the sample-containing foil+grid is plunged at 1-2 m/s into liquid ethane at T~90 K
Implementation Method 2
the sample-containing foil+grid is plunged at 1-2 m/s into liquid ethane at T~90 K (produced by cooling gas in a liquid-nitrogen-cooled cup)
Implementation Method 3
liquid ethane at T~90 K (produced by cooling gas in a liquid-nitrogen-cooled cup)
Data Source
Figure 1(A)~1(G)
Figure 2(A)~2(B)
Figure 3(A)~3(B)
AI summary
Sample support design and sample cooling devices for single-particle cryo-electron microscopy that simplify sample preparation and handling, dramatically reduce errors and improve outcome reproducibility, and dramatically reduce overall costs. The system consisting of grid based sample support system, grid handling tools, grid blotting tools, a plunge cooling system, and jet cooling systems.