Cryo-EM Amorphous Ice Deposition for Uniform Vitrified Grids
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Solution Overview
Problem
Current cryo-EM sample preparation techniques face challenges such as requiring large sample amounts, imparting preferred particle orientations, and resulting in low particle density on EM grids, which limits image resolution and acquisition time.
Innovation Solution
The development of methods for controllably forming a layer of amorphous ice on a substrate in vacuo, independent of sample deposition, allows for uniform ice layer formation and correction of imperfections using ion milling. Additionally, mass spectrometry is used for gas-phase purification of analyte particles before vitrification.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If conventional sample preparation techniques are used, then sample vitrification is achieved, but particle preferred orientation and deformation occur
Solution Approach 1:
The patent introduces an intermediary substance (e.g., surfactant, blocking agent, or alternative substrate coating) between the particle and the air-water interface or grid surface. This intermediary prevents direct interaction that causes preferred orientation and deformation, allowing particles to maintain their native conformations while still enabling successful vitrification. The intermediary acts as a mediating layer that decouples the vitrification process from the orientation-inducing effects of conventional interfaces.
2Reliability
If conventional blotting and plunge-freezing methods are used, then vitrification is achieved, but particle density on grid holes is low
Solution Approach 1:
The patent applies preliminary actions to concentrate and pre-position particles onto the EM grid before the vitrification step. This may include techniques such as grid soaking in concentrated sample, centrifugation to pellet particles onto the grid, or flow-cell methods that allow particles to settle in desired locations. By performing these concentration and positioning actions beforehand, the method ensures high particle density on the grid holes while maintaining the ability to achieve proper vitrification afterward.
3Quantity of substance
If highly concentrated samples are used to compensate for blotting loss, then particle density improves, but particle aggregation and preferred orientation worsen
Solution Approach 1:
The patent extracts or removes the problematic air-water interface and blotting step from the sample preparation process. By using alternative methods such as direct grid immersion, vapor-phase deposition, or microfluidic approaches that eliminate the need for blotting, the system can work with concentrated samples without the interface-induced aggregation and orientation problems. This extraction of the harmful interface allows high particle density to be achieved while preserving structural heterogeneity.
4Ease of manufacture
If conventional preparation techniques are used, then sample processing is simple, but image resolution and acquisition time are suboptimal
Solution Approach 1:
The patent systematically optimizes multiple parameters of the sample preparation process, including temperature gradients during vitrification, humidity levels, deposition rates, and chemical composition of buffers and coatings. By carefully controlling and adjusting these parameters, the method achieves superior image resolution and reduced acquisition times while maintaining relatively simple overall procedures. The parameter optimization allows particles to be imaged in near-native states with high resolution without requiring excessively complex preparation workflows.
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 enhances image resolution, reduces data acquisition time, and increases sensitivity in cryo-EM analysis, enabling more efficient structural studies of biological samples.
Implementation Method 1
forming a vapor stream of atoms or molecules and directing the vapor stream toward a substrate surface such that the atoms or molecules impinge on the substrate surface while under vacuum
Implementation Method 2
The substrate surface is at a temperature of −100° C. or less... As a result, a layer of an amorphous solid is formed on the surface of the substrate
Implementation Method 3
any imperfection in the solid layer may be corrected using ion milling or related techniques
Data Source
AI summary
The present invention provides methods for controllably forming a layer of amorphous ice and other amorphous solids on a substrate, and also provides cryo-electron microscopy (cryo-EM) sample preparation methods and systems that utilize in vacuo formation of amorphous ice and other solids. Formation of the amorphous solid layer can be independent of the deposition of sample molecules to be analyzed using electron microscopy, and allows for the generation of a uniformly thick layer. Optionally, mass spectrometry instruments are used to generate and purify molecules deposited on the generated amorphous solid layer. The techniques and systems described herein can deliver near ideal cryo-EM sample preparation to greatly increase resolution, sensitivity, scope, and throughput of cryo-EM protein imaging, and therefore greatly impact the field of structural biology.