CryoEM Grid Vitrification Using a Cold Gas Stream Shell
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Solution Overview
Problem
Existing cryo-vitrification methods for electron microscope samples face issues such as grid distortion, difficulty in imaging before and after vitrification, limited imageable area, challenges in time-resolved experiments, and the need for specialized equipment and training, as well as limitations in using photo-activation and spectroscopic methods.
Innovation Solution
A method involving a cold gas stream with a composite flow of nitrogen or helium, surrounded by a dry warm gas stream, is used to freeze samples on an electron microscope grid, allowing imaging and exposure to triggers for time-resolved measurements without submersion in liquid cryogens.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Speed
If rapid plunge freezing into liquid cryogen is used, then vitrification speed is improved, but grid distortion and damage occur
Solution Approach 1:
The patent replaces liquid cryogen immersion with a gas-phase cryogenic system using supercritical CO2 as the cryogen and a pneumatic delivery mechanism. The cryogen is delivered through a nozzle that creates a controlled gas flow field, eliminating mechanical contact forces that cause grid distortion while maintaining rapid cooling rates for successful vitrification.
Solution Approach 2:
The patent changes the physical state parameter of the cryogen from liquid to supercritical fluid/gas phase. This parameter change allows the cryogen to deliver cooling without the mechanical impact and surface tension forces present in liquid immersion, thereby preventing grid distortion while achieving rapid vitrification through controlled thermal transfer in the gas phase.
2Reliability
If liquid cryogen spray is applied, then vitrification is achieved, but imaging difficulty increases due to liquid interference
Solution Approach 1:
The patent uses a pneumatic delivery system that propels supercritical CO2 through a nozzle to create a directed gas flow. This gas-phase approach eliminates liquid cryogen droplets and spray that interfere with optical imaging, allowing clear visualization of the grid and sample throughout the vitrification process while maintaining effective cooling.
Solution Approach 2:
By changing the cryogen delivery from liquid spray to supercritical gas flow, the patent eliminates the optical interference caused by liquid droplets. The gas phase is transparent and does not scatter or absorb light significantly, enabling continuous optical monitoring and imaging during vitrification while achieving reliable freezing results.
3Speed
If grid is rapidly translated into cryogen, then freezing speed is improved, but time-resolved experiment control deteriorates
Solution Approach 1:
The patent employs a pneumatic nozzle system that delivers supercritical CO2 in a controlled gas stream. The grid remains stationary while the cryogen is delivered through a focused gas flow, allowing precise temporal control of the freezing process. This enables synchronization with photo-triggered reactions and other time-resolved experiments, as the start of freezing can be precisely timed and controlled.
Solution Approach 2:
The patent allows preliminary actions (such as photo-triggered reactions, chemical additions, or spectroscopic measurements) to be performed on the stationary grid before cryogen delivery begins. The controlled gas-phase system provides a defined start time for freezing, enabling precise timing and control of time-resolved experiments that require events to occur at specific intervals before vitrification.
4Reliability
If enclosed vitrification system is used, then contamination is reduced, but photo-activation and spectroscopy are impeded
Solution Approach 1:
The patent uses a pneumatic gas delivery system with a nozzle that creates a localized cryogenic zone. This open gas-phase approach allows optical access from multiple directions for photo-activation and spectroscopic methods while the supercritical CO2 provides a controlled atmosphere that prevents contamination. The gas flow can be directed to protect the sample from environmental contamination without creating an enclosed chamber that blocks light.
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 minimizes grid damage, enables imaging before, during, and after vitrification, facilitates time-resolved experiments, and allows for spectroscopic analysis, overcoming limitations of traditional plunge-freezing methods.
Implementation Method 1
The cold gas stream core freezes the sample in a time duration of 100 ms or less
Implementation Method 2
the warm gas stream shell prevents condensation onto the sample
Data Source
AI summary
A method of freezing an electron microscope sample is provided. A sample is deposited on an electron microscope grid to provide an electron microscope sample-grid. The electron microscope sample-grid is exposed to a cold gas stream core of a composite gas flow. The composite gas flow includes (i) the cold gas stream core, and (ii) a dry warm gas stream shell surrounding the cold gas stream core. The cold gas stream core freezes the sample in a time duration of 100 ms or less, and the warm gas stream shell prevents condensation onto the sample. The sample and cryoEM grid does not move during the vitrification process, and rather than being submerged in cryogenic liquid during and after vitrification, the sample is instead surrounded by a cold-gas stream. Being in a cold-gas stream, the sample grid can be imaged by a high-resolution video microscope prior to, during and after vitrification.