Cryo-EM Sample Deposition with Decelerated Ions and Cryogenic Substrates
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Solution Overview
Problem
Current cryo-EM sample preparation techniques face challenges such as molecular damage, heterogeneity, and low resolution due to manual intervention, radiation sensitivity, and ice thickness issues, leading to poor imaging quality and reproducibility.
Innovation Solution
A method involving electrospray ionization to generate gas phase ions, which are decelerated to less than 20 eV per charge and deposited on a cryogenically cooled carrier substrate, minimizing molecular damage and ensuring homogeneous sample preparation.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If molecules are applied to TEM carrier substrates and rapidly submerged into liquid ethane for cryo-EM imaging, then high resolution imaging is achieved, but molecular damage occurs due to mechanical stress and thermal shock
Solution Approach 1:
The carrier substrate is pre-cooled to cryogenic temperatures (e.g., liquid nitrogen temperature) before molecule deposition. This preliminary cooling action eliminates thermal shock during the freezing process, preventing molecular damage while maintaining the ability to achieve high resolution imaging of the biomolecules
Solution Approach 2:
The traditional mechanical plunge-freezing process is replaced by a vapor-phase deposition method where molecules are delivered in a controlled manner onto the pre-cooled substrate. This substitution eliminates mechanical stress and violent thermal contact, reducing molecular damage while preserving imaging quality
2Manufacturing precision
If manual intervention is used during vitrification procedure optimization, then ice thickness conditions can be adjusted, but reproducibility is reduced due to high level of manual intervention
Solution Approach 1:
The system automatically controls the deposition process parameters including ice layer thickness, molecule concentration, and deposition rate. The automated control eliminates manual intervention variability, ensuring consistent and reproducible sample preparation while maintaining precise control over ice thickness conditions
Solution Approach 2:
The method systematically controls and optimizes deposition parameters such as vapor pressure, temperature, and flow rate to achieve optimal ice thickness. By establishing controlled parameter ranges rather than manual adjustment, the process achieves both precision in ice thickness control and high reproducibility across different samples
3Measurement precision
If the solvent layer is made thin to reduce Zheight distribution and improve reconstruction, then surface tension causes inhomogeneous particle distribution and aggregation
Solution Approach 1:
The method uses vapor-phase deposition where molecules are delivered through controlled vapor flow rather than liquid solvent. This pneumatic approach eliminates surface tension effects that cause aggregation in liquid-based methods, allowing thin uniform layers to form without inhomogeneous particle distribution while maintaining good reconstruction quality
4Ease of manufacture
If conventional electrospray ionization is used to generate gas phase ions, then molecules can be deposited on substrate, but high energy of ions causes molecular damage upon impact
Solution Approach 1:
The substrate is pre-cooled to cryogenic temperatures before ion impact. This preliminary cooling reduces the kinetic energy of ions upon impact and provides immediate cooling that prevents thermal damage, enabling successful sample preparation while minimizing molecular damage from ion bombardment
Solution Approach 2:
The method changes the energy parameter of ion impact by using cryogenic temperatures to reduce ion kinetic energy and prevent damage. By controlling the temperature parameter, the system achieves gentle deposition that preserves molecular integrity while maintaining the electrospray ionization process for sample preparation
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 preserves the native state of molecules, reduces thermal deformation, and enhances imaging resolution by creating homogeneous samples with minimized ice-related issues.
Implementation Method 1
cryogenically cooling the carrier substrate to form a cryogenically-cooled carrier substrate
Implementation Method 2
the gas phase ions are frozen on impact with the carrier substrate
Implementation Method 3
generating gas phase ions of the one or more molecule(s) by electrospray ionisation
Implementation Method 4
decelerating the gas phase ions; and receiving the gas phase ions on the cryogenically-cooled carrier substrate... wherein the step of decelerating the gas phase ions reduces the energy of each of the gas phase ions to be less than 20 eV per charge
Implementation Method 5
beam-induced motion, which likely originates from mechanical stress in the ice due to different cooling rates during plunge freezing
Data Source
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AI summary
The present invention relates to a method of preparing a sample of one or more molecule(s) for imaging with a cryo-electron microscope. The method comprises providing a carrier substrate, cryogenically cooling the carrier substrate to form a cryogenically-cooled carrier substrate, generating gas phase ions of the one or more molecule(s) by electrospray ionisation, decelerating the gas phase ions, receiving the gas phase ions on the cryogenically-cooled carrier substrate to form a sample for imaging with a cryo-electron microscope and shielding the cryogenically-cooled carrier substrate after the step of receiving the ions on the cryogenically-cooled carrier substrate. The step of decelerating the ions reduces the energy of each of the ions to be less than 20 eV per charge when received at the cryogenically-cooled carrier substrate. The present invention also relates to an apparatus for preparing a sample of one or more molecule(s) for a cryo-electron microscope.