Pressure-Driven Cryofixation Device for Vitrification
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Solution Overview
Problem
Conventional methods for ultra-rapid cryofixation of biological samples are limited by ice crystal formation, poor cooling rates, and the need for cryoprotective substances, which alter the sample and restrict continuous observation under microscopy.
Innovation Solution
A method and device utilizing a pressure tank with a cooling liquid and gas under high pressure to rapidly decrease the sample temperature, preventing ice crystal formation and allowing continuous observation by inverse microscopy without cryoprotective substances.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Temperature
If conventional cryofixation methods are used, then sample cooling is achieved, but ice crystal formation occurs and sample integrity is compromised
Solution Approach 1:
The patent uses a pressure-driven liquid delivery system where a cooling liquid is forced through a nozzle onto the sample carrier under controlled pressure. This hydraulic approach enables precise control of cooling liquid flow rate and contact pressure, achieving rapid and uniform cooling that prevents ice crystal formation while maintaining sample integrity.
Solution Approach 2:
The patent exploits the phase transition of the cooling liquid from liquid to solid upon contact with the sample carrier, absorbing latent heat of fusion to achieve extremely rapid cooling rates. This phase change mechanism enables the system to reach cooling rates sufficient for vitrification without requiring cryoprotective substances.
2Reliability
If cryoprotective substances are used to prevent ice crystal formation, then sample integrity is improved, but the native molecular state is altered
Solution Approach 1:
The patent changes the physical parameter of cooling rate to such an extreme value that it becomes the dominant factor in preventing ice crystal formation, rendering cryoprotective substances unnecessary. By achieving cooling rates high enough to induce vitrification directly, the system preserves the native molecular state without chemical modification.
3Measurement precision
If rapid cooling is applied to prevent molecular motion, then localization precision is improved, but ice crystal formation occurs
Solution Approach 1:
The patent creates a copy of the liquid cooling approach used in other cryofixation methods but adapts it for inverted microscopy by delivering cooling liquid from above rather than from the side. This modified copying approach maintains the effective rapid cooling mechanism while adapting it to the specific geometric constraints of inverted microscope configurations.
4Temperature
If the sample is cooled rapidly, then vitrification is achieved, but continuous observation during cooling becomes difficult
Solution Approach 1:
The patent segments the cooling process into distinct phases: a rapid initial cooling phase that achieves vitrification, followed by a controlled equilibration phase. The liquid delivery system can be modulated to provide different flow rates at different stages, enabling both rapid vitrification and subsequent stable cryogenic conditions suitable for continuous microscopic observation.
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
Enables rapid and controlled temperature adjustment, achieving vitrification without ice crystals, allowing for detailed microscopic analysis of biological samples before, during, and after cryofixation, preserving native molecular states.
Implementation Method 1
supplying the at least one liquid (2) from the pressure tank (400) to the upper side (11) of the carrier substrate (10) under pressure... wherein the temperature of the sample (1) is decreased, preferably rapidly decreased
Implementation Method 2
providing in a pressure tank (400) at least one liquid (2) having a temperature below its critical temperature and a gas (500) having a pressure higher than the atmospheric pressure; and supplying the at least one liquid (2) from the pressure tank (400) under pressure
Data Source
Figure 1
Figure 2
Figure 3A~3C
AI summary
The present invention relates to method for decreasing temperature of a sample, in particular a method for ultra-rapid cryofixation of a sample for time and spatially resolved microscopic measurements as well as a device for ultra-rapid cryofixation of a sample at any particular time point of interest on a microscope. Said method and device are particularly useful for studying native molecular organization as well as (bio)chemical reactions within living cells with spatial and spectroscopic resolution beyond the fundamental limits caused by molecular motion at positive Celsius temperatures.