Gas-Phase Cryo-EM Sample Prep With Uniform Amorphous Ice
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Solution Overview
Problem
Current cryo-EM sample preparation techniques face limitations such as sample deformation, preferred orientation, and low particle density due to inadequate vitrification, leading to suboptimal 3D structural analysis and increased data acquisition times.
Innovation Solution
The development of methods for controllably forming amorphous ice layers on substrates under vacuum, independent of sample deposition, allowing for uniform thickness and real-time monitoring, combined with gas-phase purification using mass spectrometry for improved sample preparation and increased image resolution.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If conventional sample preparation techniques are used, then sample vitrification is achieved, but sample deformation and preferred orientation occur due to particle migration to the air/water interface
Solution Approach 1:
The patent introduces an intermediary substance (e.g., surfactant, oil, or specialized coating) between the sample particles and the air/water interface to prevent direct contact and migration. This intermediary layer reduces surface tension effects and prevents particle deformation while maintaining vitrification quality.
Solution Approach 2:
The patent modifies physical parameters of the vitrification process, such as cooling rate, humidity control, or interface composition, to eliminate preferred orientation. By changing these parameters, particles remain randomly oriented during freezing while achieving proper vitrification.
2Reliability
If conventional sample preparation techniques are used, then sample vitrification is achieved, but particle density per grid hole is low requiring extended data acquisition times
Solution Approach 1:
The patent performs preliminary concentration and positioning of particles onto the EM grid before vitrification. Techniques such as concentration gradients, electrostatic deposition, or acoustic manipulation are used to pre-position higher densities of particles in grid holes, reducing the need for extended imaging times.
Solution Approach 2:
The patent uses multiple copies or replicates of the sample on the grid, or creates multiple ice layers with particles, to increase the effective particle density available for analysis within each grid hole.
3Quantity of substance
If ice layer thickness is increased to accommodate more particles, then particle density increases, but resolution decreases due to excessive ice thickness
Solution Approach 1:
The patent creates non-uniform ice layer thickness with local variations, ensuring that particles are embedded in optimally thin ice regions. Techniques include controlled drying, selective freezing, or topography-controlled deposition that produce local minima in ice thickness where particles reside, maintaining both density and resolution.
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, enabling more accurate and efficient cryo-EM analysis by ensuring uniform and amorphous ice formation, which maintains structural integrity and particle orientation.
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
a layer of an amorphous solid is formed on the surface of the substrate
Implementation Method 3
The substrate surface is at a temperature of −100° C. or less... a layer of an amorphous solid is formed on the surface of the substrate
Implementation Method 4
gas-phase purification using mass spectrometry for improved sample preparation
Implementation Method 5
Encasing the sample in vitreous ice (i.e., amorphous ice) helps protect the sample from radiation damage from the electron microscope
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.