Cryo-Grid Preparation Stage for Lossless Cryo-EM Sample Vitrification
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Solution Overview
Problem
Current methods for preparing samples for electron microscopy require large sample volumes and involve significant sample loss through blotting steps, which are not representative and inefficient, especially for specimens available in sub-microliter volumes or incompatible with ink-jet technology.
Innovation Solution
A cryo-grid preparation system utilizing a high-precision liquid handling system with a dispensing head capable of aspirating and dispensing nanoliter volumes, a temperature-controlled stage, and a transfer mechanism to vitrify samples directly on a support structure without blotting, using a cryogenic liquid to transform the sample into an amorphous solid.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Extent of automation
If commercial vitrification robots are used for cryo-EM sample preparation, then automation and control are improved, but large sample volumes (around 3 μl) are required and massive blotting steps remove more than 99% of the sample
Solution Approach 1:
The invention changes the sample volume parameter from microliter scale to nanoliter scale (10^-6 to 10^-9 liters), and eliminates the blotting step entirely by using a different preparation methodology that deposits sample directly onto the grid without requiring excess fluid removal
Solution Approach 2:
The invention utilizes phase transition of the cryogenic liquid (liquid to solid) to vitrify the sample directly on the grid. The sample is deposited as a liquid film and then rapidly frozen by contact with cryogenic liquid, transforming from liquid to amorphous solid state without requiring blotting
2Quantity of substance
If ink-jet picoliter dispensing is used to decrease sample consumption, then sample volume is reduced, but ink-jet type dispensing requires a filled sample reservoir which limits the minimal sample volume available
Solution Approach 1:
The invention extracts the sample directly from a sub-microliter reservoir (such as a single-use PCR tube) using a capillary-based liquid handling system. The capillary aspirates and dispenses nanoliter volumes directly without requiring a filled reservoir, enabling work with extremely limited sample volumes down to the nanoliter scale
Solution Approach 2:
The invention replaces the ink-jet mechanical dispensing system with a capillary-based liquid handling system. The capillary system uses surface tension and pressure differential mechanisms rather than ink-jet droplet ejection, enabling precise nanoliter dispensing from minimal sample volumes without the reservoir filling constraint
3Ease of manufacture
If blotting steps are used to remove excess sample liquid, then sample preparation is simplified, but preferential removal of sample sub-classes occurs leading to unrepresentative sample population
Solution Approach 1:
The invention takes out (eliminates) the blotting step entirely from the sample preparation process. Instead of removing excess liquid through blotting, the method deposits the precise nanoliter volume directly onto the grid and vitrifies it in place, preserving the complete sample population without preferential removal of any sub-classes
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 system enables lossless cryo-EM preparation consuming minimal sample volumes (few nanoliters) without blotting, ensuring representative samples and reducing sample consumption, while allowing for precise control and handling of sensitive specimens.
Implementation Method 1
a (high-precision) liquid handling system comprising a dispensing head, wherein said liquid handling system is configured to aspirate and dispense a volume (preferably as low as 1 nl) of a (e.g. heterogeneous) sample
Implementation Method 2
a temperature-controlled stage that is configured to keep said support structure at a pre-defined temperature when the support structure is arranged on the temperature-controlled stage (e.g. at a dew point temperature of the environment, e.g. in the vicinity of the support structure, with the possibility of establishing a small offset temperature to induce evaporation/condensation)
Implementation Method 3
the sample on the support structure contacts the cryogen (preferably the cryogen comprises ethane or a mixture of ethane and propane) so that the sample (e.g. liquid film) on the support structure is preferably transformed into a film of amorphous solid water
Data Source
Figure 1A~1D
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AI summary
The invention relates to a preparation system for preparing a sample for electron microscopy, comprising: - a liquid handling system (0) comprising a dispensing head (1), wherein said liquid handling system (0) is configured to aspirate and dispense a volume of a sample via the dispensing head (1), - a support structure (2) that is configured to accommodate the sample, a temperature-controlled stage (4) that is configured to keep said support structure (2) at a pre-defined temperature when the support structure (2) is arranged on the temperature-controlled stage (4), a first adapter (3) configured to hold said support structure (2), a transfer mechanism (60) that is configured to be connected to the first adapter (3) holding the support structure (2) and to move said support structure (2) into a container (8) containing a liquid cryogen (80) so that the sample on the support structure (2) contacts the cryogen (80). Furthermore, the invention relates to a corresponding method.