Cryo-EM Grid Preparation via Automated Dispensing
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Solution Overview
Problem
Conventional cryo-EM sample preparation methods are unreliable, labor-intensive, and wasteful, as they struggle to consistently control the thickness of the vitreous ice layer, leading to adverse sample changes and high material loss during the preparation of biological samples for electron microscopy.
Innovation Solution
A cryogenically-cooled sample preparation apparatus with a movable and rotatable sample dispenser that automatically deposits and vitrifies liquid samples on a grid, eliminating the need for manual handling and blotting, and allowing for precise control of ice layer thickness through adjustable deposition rates and cryogen circulation.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Manufacturing precision
If manual blotting and plunge freezing methods are used, then sample preparation can be performed with simple equipment, but the ice layer thickness cannot be reliably controlled and sample integrity deteriorates
Solution Approach 1:
The patent replaces manual mechanical blotting operations with an automated dispensing system that uses computer-controlled liquid ejection to deposit precise volumes of sample onto the grid. This substitution eliminates the variability of manual blotting and enables precise control over ice layer thickness through programmable deposition parameters.
Solution Approach 2:
The patent controls ice layer thickness by adjusting deposition parameters including liquid volume, deposition speed, and temperature. By systematically varying these parameters, the system optimizes ice layer formation to achieve consistent thickness while maintaining sample integrity during the vitrification process.
2Productivity
If conventional manual preparation methods are used, then device complexity remains low, but labor intensity and time consumption increase
Solution Approach 1:
The patent implements an automated system where the dispensing apparatus independently performs sample deposition, volume control, and grid preparation without manual intervention. The system self-regulates deposition parameters and automatically vitrifies samples, eliminating labor-intensive manual operations while managing complexity through integrated automation.
Solution Approach 2:
The patent performs preliminary actions by pre-cooling the grid and controlling sample deposition conditions before vitrification occurs. The system prepares the sample environment in advance with controlled temperature and deposition parameters, enabling rapid automated processing while reducing the need for complex post-deposition adjustments.
3Manufacturing precision
If rapid vitrification is implemented, then sample integrity is maintained, but control over ice layer thickness becomes difficult
Solution Approach 1:
The patent incorporates feedback control by monitoring deposition parameters and adjusting subsequent deposition actions based on observed ice layer formation. The system uses real-time data from temperature sensors and deposition metrics to dynamically adjust vitrification conditions, maintaining both rapid processing and precise thickness control through closed-loop regulation.
Solution Approach 2:
The patent employs dynamic control of deposition and vitrification parameters during the process. The system adjusts deposition speed, liquid volume, and cooling rate in real-time based on the stage of sample preparation, enabling rapid vitrification while maintaining precise control over final ice layer thickness through adaptive parameter modulation.
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 method enables rapid, consistent, and efficient formation of cryo-EM grids with controlled ice layer thickness, reducing sample loss and adverse sample changes, while allowing for immediate imaging without the need for additional processing steps.
Implementation Method 1
a cryogenically-cooled stage that is configured to removably receive a sample deposit surface such that the deposit surface is cryogenically cooled through direct contact with the stage
Implementation Method 2
a sample dispenser that is at least one of laterally, longitudinally, vertically, or rotationally movable with respect to the stage. The sample dispenser is configured to deposit a liquid sample onto the sample deposit surface at a selected rate of deposition
Implementation Method 3
During the sample preparation stage, sample proteins 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
Data Source
AI summary
An electron microscope (EM) preparation and imaging system including an EM device and a sample preparation device for forming a vitreous ice layer containing a liquid sample through vitrification, which are located within a sealable environment. The sample preparation apparatus includes a cryogenically-cooled stage that receives a sample deposit surface, such as a cryo-EM grid, which is cryogenically cooled through direct contact with the stage. A sample dispenser is movable with respect to the stage and is configured to deposit a liquid sample onto the sample deposit surface at a selected rate of deposition. Once the liquid sample is deposited onto the sample deposit surface by the sample dispenser, it is vitrified automatically in place.