Method for preparing samples for imaging or diffraction experiments under cryogenic conditions
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Solution Overview
Problem
Current methods for preparing samples under cryogenic conditions for imaging or diffraction experiments often result in artifact formation and inefficient vitrification processes, particularly due to the lack of precise control over coolant application and temperature management.
Innovation Solution
The method involves directing a jet of liquid coolant directly onto the sample on a film support, using coolants like ethane or nitrogen, with synchronized jets to prevent artifact formation, and subsequent removal of coolant residues using a gas to maintain the sample in a vitrified state, while also employing plasma treatment to render the support hydrophilic for even sample application and using a Peltier element to control temperature above the dew point.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If conventional plunge freezing methods are used, then samples can be vitrified, but artifact formation occurs and reproducibility is poor
Solution Approach 1:
The patent replaces the conventional mechanical plunge freezing system with a jet freezing system that uses focused liquid coolant jets delivered by computer-controlled positioning. This substitution enables precise control over freezing parameters, eliminating the variability and artifact formation associated with manual plunge freezing methods.
Solution Approach 2:
The patent implements precise control and optimization of freezing parameters including jet temperature, flow rate, duration, and positioning. By systematically controlling these parameters, the method achieves reproducible vitrification while minimizing ice crystal formation and other artifacts that occur with uncontrolled conventional methods.
2Speed
If liquid coolant is applied to vitrify samples, then rapid freezing is achieved, but coolant residues contaminate the sample
Solution Approach 1:
The patent extracts and removes coolant residues from the vitrified sample using a gentle gas stream or wicking material after the jet freezing process. This separation step eliminates contamination while preserving the rapid freezing benefits, as the coolant removal occurs after vitrification is complete.
Solution Approach 2:
The patent introduces an intermediary substance (gas stream or wicking material) to mediate between the liquid coolant and the sample. This intermediary removes the coolant residues without directly contacting or damaging the vitrified sample, thus eliminating contamination while maintaining freezing speed.
3Ease of manufacture
If sample is applied to hydrophobic support, then application is simple, but sample distribution is uneven
Solution Approach 1:
The patent changes the surface property parameter of the support from hydrophobic to hydrophilic through plasma treatment or chemical modification. This parameter change enables uniform sample distribution by improving wetting and adhesion, while the overall application process remains simple and maintains its ease of manufacture.
4Device complexity
If support temperature is not controlled, then device complexity is reduced, but sample preparation quality deteriorates
Solution Approach 1:
The patent implements localized temperature control specifically at the sample support region using Peltier elements or Peltier-controlled holders, rather than controlling the entire device temperature. This localized approach ensures high-quality vitrification where needed while minimizing overall device 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 ensures rapid and effective vitrification of samples with minimal artifact formation, maintaining the sample in a stable cryogenic state for precise imaging or diffraction experiments by controlling coolant application and temperature, enhancing reproducibility and preventing crystalline ice formation.
Implementation Method 1
vitrifying the sample
Implementation Method 2
directing a jet of liquid coolant to the center of the film and onto the sample
Implementation Method 3
using a Peltier element to control temperature above the dew point
Implementation Method 4
employing plasma treatment to render the support hydrophilic for even sample application
Implementation Method 5
subsequent removal of coolant residues using a gas to maintain the sample in a vitrified state
Data Source
Figure 1
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AI summary
The invention relates to a method of and apparatus for preparing a sample for imaging or diffraction experiments under cryogenic conditions, comprising the steps of applying a sample to a film on a support, in particular a grid (3) comprising such a film on a support, or removing residual medium, typically liquid, from an incubated sample on a film on a support (3), and vitrifying the sample. The sample is vitrified by directing a jet (50, 51) of liquid coolant to the center of the film (3) and onto the sample.