Cryogenic Sample Holder Cooling to Prevent Ice Contamination
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Solution Overview
Problem
Charged-particle microscopy faces challenges in preparing biological specimens suspended in aqueous liquids, as they tend to outgas or boil in the vacuum environment, leading to sample degradation and contamination issues due to ice crystal formation during rapid freezing, which hinders effective imaging.
Innovation Solution
A method involving a blast of cryogenic fluid applied to the backside of the sample holder before plunging into a cryogen bath to prevent water leaching and ice formation, ensuring vitrification and reducing contamination, using a controlled nozzle and cryogenic gas to achieve uniform thermal contact and amorphous solidification.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Speed
If the sample holder is plunged directly into the cryogen bath without preliminary cooling, then the freezing process is rapid, but water leaches from the sample and forms ice crystals on the backside, causing contamination
Solution Approach 1:
A blast of cryogenic gas is applied to the backside of the sample holder immediately before plunging into the cryogen bath. This preliminary cooling action prevents water from leaching out of the sample during the subsequent rapid freezing process, thereby preventing ice crystal formation and contamination on the backside of the sample
Solution Approach 2:
The cooling process is divided into two distinct stages: (1) preliminary cooling of the backside using cryogenic gas before plunging, and (2) rapid freezing of the frontside by plunging into the cryogen bath. This segmentation allows each stage to serve its specific function optimally without interfering with the other
2Ease of manufacture
If the sample holder is held vertically during plunging, then the setup is simple, but thermal contact is uneven and contamination occurs
Solution Approach 1:
Instead of plunging the sample holder vertically with the frontside facing downward, the sample holder is held horizontally during plunging. This inversion of the plunging orientation ensures that both the frontside and backside of the sample make uniform thermal contact with the cryogen bath, preventing uneven freezing and contamination while maintaining operational simplicity
3Temperature
If the aqueous liquid film is allowed to contact the cryogen directly, then freezing is achieved, but water leaches out and forms contaminating ice on the surface
Solution Approach 1:
The backside of the sample holder is pre-cooled with cryogenic gas before the aqueous liquid film contacts the cryogen bath. This preliminary anti-action creates a temperature gradient that prevents water from leaching out of the sample during freezing, thereby preventing the formation of contaminating ice on the sample surface
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 significantly reduces contamination and enhances the quality of vitrified samples, allowing for clearer imaging by preventing surfacial ice formation and maintaining the sample's amorphous state, thus improving the analysis in charged-particle microscopes.
Implementation Method 1
A blast of cryogenic fluid is applied to the backside of the sample holder before plunging into a cryogen bath
Implementation Method 2
The aqueous liquid film is essentially 'spanned' across said perforation with the aid of surface tension effects
Implementation Method 3
Plunging the sample holder onto a bath of cryogen, whereby the sample holder is held with said first major surface pointing toward the cryogen
Implementation Method 4
ensuring vitrification and reducing contamination
Data Source
Figure 1A
Figure 1B
Figure 2
AI summary
A method of preparing a sample for study in a charged-particle microscope, comprising the following steps: - Providing a substantially plate-like sample holder having opposed first and second major surfaces substantially parallel to one another, comprising at least one aperture that connects said major surfaces and across which a membrane has been spanned upon said first major surface, which membrane comprises at least one perforation; - Spanning a film of aqueous liquid across said perforation, which liquid comprises at least one study specimen suspended therein; - Plunging the sample holder onto a bath of cryogen, whereby the sample holder is held with said first major surface pointing toward the cryogen and arranged substantially parallel to an exposed surface of the cryogen, further comprising the following step: - Applying a blast of cryogenic fluid to said film from a nozzle pointing toward said second major surface, immediately prior to the film making contact with said cryogen.