Cryogenic Sample Vitrification by Droplet Spraying for Electron Microscopy
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Solution Overview
Problem
Existing methods for forming cryogenic samples in electron microscopy often result in sample damage due to ice needle formation when cooling biological materials, and they require complex equipment for rapid vitrification.
Innovation Solution
Spraying an aqueous solution onto a surface maintained at cryogenic temperature within the charged particle microscope, ensuring rapid formation of vitrified samples with small droplet sizes (<10 μm) to prevent slow cooling and ice needle formation, and using a cryogenic holding device to maintain the sample holder at cryogenic temperatures.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Temperature
If biological material is cooled to cryogenic temperature using conventional methods, then the sample can be preserved for electron microscopy, but ice needles form and damage the sample material
Solution Approach 1:
The invention changes the cooling rate parameter from slow conventional cooling to ultra-rapid cooling (>10^5 K/s), which transforms the freezing process from crystalline ice formation to amorphous vitrification, eliminating ice needle damage while achieving cryogenic preservation
Solution Approach 2:
The invention utilizes the phase transition of water from liquid to amorphous solid state through ultra-rapid cooling, bypassing the crystalline ice phase that causes needle formation. This vitrification process preserves biological structures without the harmful crystallization effects
2Object-affected harmful factors
If rapid cooling is used to form vitrified ice, then ice needle formation is prevented, but the equipment required is complex and sample preparation is time-consuming
Solution Approach 1:
The invention extracts the critical cooling function from complex external vitrification equipment and integrates it directly into the electron microscope's sample stage, which already possesses cryogenic cooling capability. This eliminates the need for separate rapid-freezing apparatus while maintaining vitrification quality
Solution Approach 2:
The electron microscope's own cryogenic stage serves the dual purpose of both holding the sample and providing the ultra-rapid cooling necessary for vitrification. The system uses its inherent cooling capability to achieve the required cooling rates without external assistance
3Ease of operation
If droplet size is large, then spraying is easier, but cooling rate is too slow and vitrification cannot be achieved
Solution Approach 1:
The invention changes the droplet size parameter to a specific range (1-10 μm) that optimizes the surface-area-to-volume ratio, enabling sufficiently rapid heat extraction to achieve vitrification cooling rates while remaining practically sprayable with conventional electrospray technology
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 method allows for quick, in-situ preparation of vitrified samples without ice needle formation, enhancing the preservation of biological samples and enabling efficient sample preparation within the electron microscope environment.
Implementation Method 1
Vitrified ice is formed by cooling an aqueous solution at a rate in the order of 105 K/s to a temperature of less than the glass transition temperature of approximately 165 K
Implementation Method 2
Solidifying the solution by cooling the solution
Implementation Method 3
a beam of energetic electrons with a selectable energy of, for example, between 60 keV and 300 keV, irradiates a sample
Implementation Method 4
the surface on which the solution is sprayed has a cryogenic temperature
Data Source
AI summary
The invention relates to a method of forming a vitrified sample on a sample holder for inspection in an electron microscope. It is known to spray a solution on a grid and then immerse the grid in a cryogenic liquid, such as ethane or liquid nitrogen. The invention proposes to spray small droplets of the liquid on a cryogenic surface, such as a grid or a sample holder in vacuum. The liquid forms vitrified sample material when hitting the surface due to the low temperature of the grid or sample holder.A lamella may be excavated from the thus formed sample material, to be studied in a TEM, or the vitrified sample material may be directly observed in a SEM. In an embodiment the material may be sprayed on a cryogenic liquid, to be scooped from the liquid and placed on a grid.