Asymmetric Cryogenic Flushing for Vitrified Sample Preparation
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Solution Overview
Problem
Existing methods for vitrifying biological specimens in charged-particle microscopy often result in partially damaged samples due to uneven cooling, particularly when using grid-mounted perforated membranes, where the backside is prone to excessive flushing and damage.
Innovation Solution
A method involving deliberate asymmetry in the cooling of the sample's backside and frontside by adjusting the flush of cryogenic fluid, with the backside receiving a less vigorous flush than the frontside, using different cryogen types, phases, flow rates, or durations to optimize vitrification and reduce damage.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Temperature
If cryogenic fluid is flushed through both sides of the sample simultaneously with equal intensity, then rapid vitrification is achieved, but the backside of the sample suffers excessive flushing and damage
Solution Approach 1:
The patent applies asymmetry by deliberately creating different flushing conditions for the frontside and backside of the sample. The backside receives a less vigorous flush compared to the frontside, which resolves the contradiction by protecting the backside from excessive flushing damage while still achieving rapid vitrification through the combined cooling effect of both sides.
Solution Approach 2:
The patent applies local quality by tailoring the flushing intensity to the specific needs of different regions of the sample. The backside, which is more vulnerable to damage, receives a gentler flush, while the frontside receives a more intense flush. This localized adjustment of flushing quality optimizes vitrification while minimizing damage to each region.
2Device complexity
If the sample is cooled rapidly from one side only, then the cooling process is simpler, but uneven cooling results in partially damaged samples
Solution Approach 1:
The patent applies segmentation by dividing the cooling process into two independent cooling paths - one for the frontside and one for the backside of the sample. This segmentation allows each side to be cooled optimally while maintaining overall process simplicity, as each cooling path can be independently controlled without requiring complex coordination between multiple cooling zones.
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 the occurrence of damaged samples by tailoring the vitrification process to the asymmetrical conditions caused by the grid, achieving more consistent and effective sample preservation.
Implementation Method 1
the sample is subjected to rapid cooling using a cryogen
Implementation Method 2
achieve vitrification of the aqueous film without significant ice crystallization
Implementation Method 3
The term 'cryogen' should be interpreted as referring to a liquid at cryogenic temperatures
Implementation Method 4
suddenly immersing the sample in cryogenic fluid
Data Source
AI summary
A method of preparing a cryogenic sample (e.g. for study in a charged-particle microscope), whereby the sample is subjected to rapid cooling using a cryogen, comprising the following steps:Providing two conduits for transporting cryogenic fluid, each of which conduits opens out into a mouthpiece, which mouthpieces are arranged to face each other across an intervening gap;Placing the sample in said gap;Pumping cryogenic fluid through said conduits so as to concurrently flush from said mouthpieces, thereby suddenly immersing the sample in cryogenic fluid from two opposite sides,wherein the flush of cryogenic fluid applied from a first of said mouthpieces is different—e.g. has a different duration—to that applied from the second of said mouthpieces.