Cryogenic Sample Vitrification Using Directed Liquid Coolant Jets
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Solution Overview
Problem
Existing methods for preparing samples under cryogenic conditions for imaging or diffraction experiments often result in artifacts due to the reinforcement features of the sample support, and there is a need for improved vitrification techniques that can achieve extreme cooling rates without the use of encapsulating chemicals.
Innovation Solution
The method involves directing a jet of liquid coolant directly onto the sample, achieving rapid vitrification by cooling the sample at the center and moving towards the edges, using coolants like ethane, ethane-propane, helium, or nitrogen, and simultaneously directing jets to both sides of the support to prevent artifact formation, while also removing coolant residues with a gas to prevent crystalline ice formation.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Speed
If a jet of liquid coolant is directed onto the sample for rapid vitrification, then cooling rate is improved, but artifact formation due to support reinforcement features occurs
Solution Approach 1:
The patent applies local quality by directing the coolant jet specifically to the center of the support where the sample is located, rather than uniformly cooling the entire support. This creates a localized cooling zone that achieves rapid vitrification of the sample while minimizing the transmission of mechanical vibrations and thermal shocks from the support's reinforcement features, thereby reducing artifact formation.
Solution Approach 2:
The patent introduces a gas flow as an intermediary between the coolant jet and the sample/support structure. The gas flow serves to remove coolant residues and modulate the cooling process, preventing direct contact artifacts while maintaining the rapid cooling effect needed for vitrification.
2Stability of the object's composition
If encapsulating or fixating chemicals are used for sample preparation, then sample stability is improved, but natural state of hydrated samples is compromised
Solution Approach 1:
The patent achieves sample stability through parameter changes - specifically by controlling temperature parameters (rapid cooling to cryogenic temperatures) and pressure parameters during the vitrification process. This physical stabilization method replaces chemical fixatives, maintaining the natural state of hydrated samples while ensuring their stability for imaging or diffraction experiments.
Solution Approach 2:
The patent utilizes phase transitions - specifically the transition of water from liquid to vitreous ice state through rapid cooling. This phase transition stabilizes the sample structure without requiring chemical fixatives, preserving the natural hydrated state of biological samples while providing the stability needed for high-resolution 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
This approach enables rapid cooling rates exceeding 50,000 K/s, minimizes artifact formation, and maintains the natural state of hydrated samples, such as cells or proteins, by vitrifying them without the need for fixative chemicals, ensuring high-quality imaging or diffraction results.
Implementation Method 1
directing a jet of liquid coolant to the center of the film and onto the sample... achieving extreme cooling rates in the sample itself, e.g. cooling rates in excess of 50.000 K/s
Implementation Method 2
the sample is vitrified by directing a jet of liquid coolant to the center of the film and onto the sample
Implementation Method 3
Suitable coolants include liquid ethane, ethane-propane, helium, and nitrogen... applying a blast of cryogenic fluid
Implementation Method 4
residues of the coolant are removed from the sample, e.g. by blowing the residues off the sample by means of a gas
Data Source
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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 sample carrier, such as 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.