Microfluidic Cryo TEM Sample Preparation Without Blotting
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Solution Overview
Problem
The preparation of TEM samples is complex and time-consuming, particularly due to the need for a blotting step that introduces high shear and limits the ability to examine certain specimens, especially biological samples, which can deform structures and hinder the visualization of fine details.
Innovation Solution
An automated system that forms a sample liquid droplet at the end of a capillary, allowing controlled deposition and retraction without a blotting step, using low shear rates and computer-controlled microfluidics to position and control the droplet size, followed by vitrification in liquid ethane for direct visualization of aggregates within milliseconds.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Manufacturing precision
If a blotting step is used to remove excess sample fluid, then the sample droplet size is controlled, but high shear is introduced that deforms biological structures
Solution Approach 1:
The patent extracts and removes the harmful blotting step from the sample preparation process. By using a microfluidic device that allows controlled deposition of sample droplets directly onto the grid without requiring blotting, the harmful shear forces are eliminated while still achieving proper droplet size control through the microfluidic channel geometry and flow rate management.
Solution Approach 2:
The patent employs hydraulic principles through microfluidics to control sample delivery. The microfluidic device uses controlled fluid flow through channels with specific dimensions to regulate droplet formation and deposition, replacing the mechanical blotting action with a hydraulic control mechanism that imposes minimal shear on the sample.
2Ease of manufacture
If traditional sample preparation methods are used, then samples can be prepared, but the process is complex and time-consuming
Solution Approach 1:
The patent merges multiple preparation steps into a single integrated microfluidic device. The device combines sample loading, droplet formation, controlled deposition onto the grid, and excess fluid removal in one continuous operation, eliminating the need for separate blotting steps and reducing overall preparation time and complexity.
Solution Approach 2:
The patent changes the key parameter of fluid removal from mechanical blotting to controlled capillary action and surface tension effects within the microfluidic channel. By adjusting channel dimensions, surface properties, and flow rates, the system achieves proper droplet sizing without time-consuming blotting operations.
3Quantity of substance
If filter paper blotting is used to remove excess fluid, then droplet size is reduced, but the process introduces high shear and takes considerable time
Solution Approach 1:
The patent replaces the mechanical blotting system (filter paper contact and pressing) with a microfluidic hydraulic system. The microfluidic device uses controlled fluid flow, capillary action, and pressure differential to remove excess fluid, eliminating the high shear mechanical action of blotting while achieving faster fluid removal through optimized channel design and flow control.
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 eliminates the need for blotting, reduces sample deformation, and accelerates the preparation process, enabling the visualization of previously unobserved structures and providing a unique window for examining temporal evolution of aggregate morphologies.
Implementation Method 1
formation of a sample liquid droplet at the end of a capillary
Implementation Method 2
formation of a sample liquid droplet at the end of a capillary
Implementation Method 3
The biological material is spread on an electron microscopy grid and is preserved in a frozen-hydrated state by rapid freezing, usually in liquid ethane near liquid nitrogen temperature
Implementation Method 4
preserved in a frozen-hydrated state by rapid freezing
Data Source
AI summary
A method and system is provided for automatically preparing transmission electron microscopy (TEM) samples for examination by depositing extremely small samples onto a grid without need for a blotting step. A sample liquid droplet is formed at the end of a capillary, wherein a portion of the liquid is transferred to the TEM sample grid by contact. The excess volume in the liquid droplet is then retracted by an adjacent capillary. After a predetermined time interval, the retraction capillary is moved toward the drop of the sample to remove the excess volume. As compared to a conventional machine, where the blotting procedure can deform the structure of the molecule of interest, the present invention utilizes a very low shear rate for removal of the excess sample fluid.