Cryo-EM Grid Preparation with EMF Protein Orientation
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Solution Overview
Problem
The conventional cryo-EM sample preparation methods are unreliable, labor-intensive, and costly, with challenges in controlling the thickness of the vitreous ice layer, leading to protein aggregation, dissociation, and preferential orientations, which affect the accuracy of cryo-EM imaging and analysis.
Innovation Solution
A method and apparatus that involves depositing a liquid sample onto a cryo-EM grid using a dispenser, exposing it to an electromagnetic field (EMF) to re-orient proteins, and using a gas jet to shape and thin the sample before vitrification, allowing for precise control of the ice layer thickness and protein orientation.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Manufacturing precision
If conventional manual blotting and plunge freezing methods are used, then sample preparation can be performed with simple equipment, but the thickness of the vitreous ice layer cannot be reliably controlled, leading to protein aggregation, dissociation, or preferential orientations
Solution Approach 1:
The patent replaces manual mechanical blotting operations with an automated dispensing system that uses precise volumetric control to deposit sample onto the grid. The gas jet system substitutes mechanical blotting pressure with controlled gas flow to achieve consistent sample thinning, eliminating the variability inherent in manual operations.
Solution Approach 2:
The invention controls ice layer thickness by precisely controlling the volume of liquid sample deposited and the parameters of gas jet application (flow rate, duration, pressure). By changing these parameters systematically, the system achieves reliable control over the final ice layer thickness without requiring complex mechanical adjustment mechanisms.
2Reliability
If samples are deposited too thickly, then proteins are fully encapsulated by vitreous ice, but the ice layer becomes too thick for sufficient electron transmission, causing poor image quality
Solution Approach 1:
The system deposits a controlled amount of sample that is slightly excessive to ensure full protein encapsulation, then uses gas jet thinning to remove the excess. This approach ensures that all proteins are encapsulated while achieving the optimal thinness for electron transmission by removing only the necessary amount of excess material.
3Manufacturing precision
If samples are deposited too thinly, then electron transmission is improved, but proteins may extend through and become exposed at the air-water interface, causing shape alteration or composition changes
Solution Approach 1:
The system uses feedback control where the deposited sample volume and gas jet parameters are adjusted based on observed outcomes. The dispensing amount and gas flow are tuned to achieve the optimal balance where proteins remain fully encapsulated while maintaining sufficient thinness for electron transmission, preventing interface exposure.
4Productivity
If manual blotting is used to thin the sample, then equipment complexity is minimized, but the process is labor-intensive and lacks reproducibility
Solution Approach 1:
The automated dispensing system performs the thinning operation without requiring manual blotting intervention. The system self-regulates the sample deposition and gas jet application, eliminating the need for operator skill and manual adjustment, thereby improving reproducibility and efficiency while accepting the necessary device complexity.
5Ease of manufacture
If proteins are allowed to orient preferentially during deposition, then the deposition process is simpler, but the resulting preferential orientations affect the accuracy of cryo-EM imaging and structural reconstruction
Solution Approach 1:
The system employs periodic vibration or agitation during the deposition and gas jet thinning process to prevent proteins from settling into preferential orientations. This periodic disturbance maintains random protein distribution throughout the sample, ensuring accurate cryo-EM imaging while keeping the deposition process relatively simple.
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 improves the reliability and reproducibility of cryo-EM sample preparation by ensuring random protein orientations, reducing sample loss, and enabling efficient mixing of chemical species, thereby enhancing the quality of cryo-EM images and structural reconstruction.
Implementation Method 1
using a gas jet to shape and thin the sample before vitrification
Implementation Method 2
using at least one gas jet produced by a sample shaping element
Implementation Method 3
vitrifying the sample deposited on the sample grid
Implementation Method 4
the samples must be prepared. During the sample preparation stage, sample proteins, which are presumed to be randomly oriented and homogenously dispersed in an aqueous environment, are captured in a thin layer of vitreous ice by being cooled very quickly (generally, in less than a millisecond) to cryogenic temperatures
Implementation Method 5
exposing the sample to an electromagnetic field (EMF) to re-orient proteins
Data Source
AI summary
A system for producing cryogenic electron microscopy (cryo-EM) grids. A grid holding element holds a cryo-EM grid in place while a sample deposit element deposits liquid sample from a sample supply onto the grid. A sample shaping element shapes the liquid sample and then a cryogenic sample vitrifying element vitrifies the liquid sample. The shaping element may direct a gas jet towards the grid to reduce the thickness of the liquid sample. The gas jet may mix first and second liquid samples together in midair or on the grid. A storage element stores vitrified cryo-EM grids and includes an electromagnetic field (EMF) source that creates an EMF within the storage element such that the vitrified sample is exposed to the EMF. As a result of being exposed to the EMF, a protein provided with the sample is re-oriented from a first orientation to a second orientation.