Cryo-Grid Preparation via Controlled Evaporation
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Solution Overview
Problem
Current cryo-EM grid preparation methods are inefficient, requiring large sample volumes and often leading to protein aggregation or denaturation due to blotting with filter paper, and result in varying film thickness and quality, causing image quality issues and salt artifacts from uncontrolled water evaporation.
Innovation Solution
A method involving a temperature-controlled support structure with infrared light thickness measurement to adjust film thickness, allowing for sample deposition and vitrification without blotting, using a microcapillary to create thin vitrified layers on EM grids, and a system for controlled sample evaporation and vitrification with real-time monitoring to achieve reproducible film thickness.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Quantity of substance
If blotting with filter paper is used to remove excess sample, then sample volume is reduced, but 99.9% of sample is lost and protein aggregation or denaturation occurs
Solution Approach 1:
The harmful blotting step with filter paper is completely removed from the preparation process. Instead, the method uses controlled evaporation through temperature adjustment above dew point to reduce sample volume, eliminating the need for filter paper blotting that causes 99.9% sample loss and protein damage
Solution Approach 2:
The temperature of the support structure is adjusted to a value above the dew point temperature of the environment, controlling the evaporation rate of water from the sample film. This parameter change enables gentle volume reduction without the mechanical stress and adsorption losses associated with filter paper blotting
2Manufacturing precision
If uncontrolled water evaporation occurs, then sample film thickness varies, but salt artifacts and image quality issues result
Solution Approach 1:
A feedback control system monitors the sample film thickness in real-time during evaporation using optical measurement (absorbance or interference contrast). The system automatically adjusts the temperature or evaporation rate to maintain the desired film thickness, preventing salt artifact formation while ensuring consistent quality
Solution Approach 2:
The mechanical blotting process is replaced with a controlled thermal evaporation system combined with optical feedback. This substitution enables precise, uniform thinning of the sample film without the mechanical disruption and salt concentration issues caused by filter paper contact
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 significantly reduces sample consumption, prevents protein denaturation, and improves image quality by ensuring consistent film thickness, eliminating salt artifacts and enhancing the reliability of cryo-EM imaging.
Implementation Method 1
the temperature of the support structure is adjusted to a value above the dew point temperature of the environment, such that the film thickness decreases
Implementation Method 2
at least one intensity value of the light transmitted by the support structure is measured
Implementation Method 3
the support structure is automatically inserted into a liquid cryogen dependent on the at least one measured intensity value, such that the sample is cooled down to an amorphous solid
Data Source
AI summary
Method for preparing a sample, wherein the sample is provided as a thin film on a support structure; the temperature of the support structure is adjusted to a value above the dew point temperature of the environment to decrease the film thickness, light is directed at the support structure, an intensity value of the transmitted light is measured, and the support structure is automatically inserted into a liquid cryogen dependent on the measured intensity value. The application further relates to a system comprising a support structure, a temperature-controlled stage for keeping the support structure at a pre-defined temperature, a transfer mechanism for moving the support structure into a container containing a liquid cryogen, a light source, a photodetector for measuring an intensity value of the transmitted light, and a control device for triggering the transfer mechanism dependent on the measured intensity value.