Thermal Extrusion of High-Pressure Frozen Capillary Samples
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Solution Overview
Problem
Existing methods for forming samples from capillaries using high-pressure frozen material face challenges such as mechanical sectioning artifacts and low throughput due to the low milling rate of capillary materials compared to biological samples, particularly with FIB machining.
Innovation Solution
The method involves warming a capillary to a temperature between the glass transition temperature and the initial freezing temperature, followed by cooling it to a lower temperature, causing sample material to extrude from the capillary, which is then sectioned using a focused ion beam, allowing for forceless cutting and potentially higher throughput.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Object-affected harmful factors
If FIB machining is used to section capillary material, then mechanical sectioning artifacts are reduced, but throughput time increases due to low milling rate
Solution Approach 1:
The patent replaces mechanical sectioning (ultramicrotome cutting) and traditional FIB machining with a thermal field-based approach. By applying localized heating through the ion beam, material is selectively removed through thermal decomposition and vaporization rather than mechanical sputtering, achieving both artifact-free sectioning and improved throughput
Solution Approach 2:
The patent changes the operational parameters of the FIB system by using higher ion beam energies (30-50 keV or higher) and controlling the heating rate to achieve thermal field effects. This parameter optimization allows the ion beam to function not just as a sputtering tool but as a localized heat source that enables faster material removal while maintaining sample integrity
2Productivity
If mechanical sectioning is used to slice samples, then throughput is improved, but mechanical forces cause artifacts in the sample material
Solution Approach 1:
The patent replaces the mechanical ultramicrotome system with a thermal field-based sectioning method using the ion beam. This eliminates all mechanical contact and forces on the sample, allowing high-speed material removal without the compression, bending, or tearing artifacts that plague mechanical sectioning of vitrified samples
3Strength
If the capillary wall thickness is large compared to sample core diameter, then capillary strength is improved, but FIB milling time increases due to large amount of material to be removed
Solution Approach 1:
The patent replaces the slow mechanical sputtering process with thermal field-based material removal. The localized heating enables rapid vaporization and decomposition of capillary material, reducing the time required to mill through thick walls while maintaining precise control over the milling process
Solution Approach 2:
The patent employs periodic or pulsed ion beam application with controlled duty cycles. By alternating between milling phases and cooling phases, the system maintains high average removal rates while preventing excessive heat accumulation that could damage the sample, thereby efficiently removing large volumes of capillary material
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 enables the formation of samples without mechanical force, resulting in reduced artifacts and increased throughput by effectively extruding and sectioning the sample material, particularly suitable for transmission electron microscopy.
Implementation Method 1
warming the capillary to a temperature T2 between T1 and Tg, a subsequent step of cooling the capillary to a temperature T3 below T2, as a result of which material is extruded from the capillary
Implementation Method 2
Focused Ion Beam (FIB) machining is used for sectioning sample material, in which material is milled (sputtered away) with a focused ion beam
Implementation Method 3
the sample, contained in a holder, is first pressurized to a pressure of approximately 2100 bar, and then rapidly cooled to a temperature Tg of approximately 165 K, the glass transition temperature of water
Data Source
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AI summary
Method of forming a sample from a capillary with high-pressure frozen sample material, the method comprising: a step (102) of providing a high-pressure capillary with vitrified sample material at a temperature T1 below the glass transition temperature Tg, and a step (104) of cutting the capillary, characterized by a step (106) of warming the capillary to a temperature T2 between temperature T1 and temperature Tg, a subsequent step (108) of cooling the capillary to a temperature T3 below temperature T2, as a result of which material is extruded from the capillary, and a step (112) of freeing a sample from the extruded sample material at a temperature below temperature Tg. Vitrified biological material could be extruded from a capillary at liquid at a temperature of T = 88 K (liquid nitrogen by raising the temperature to, for example 130 K and then cooling the capillary to 88 K. Not only did this result to extrusion material, but even more surprising is that a repeated temperature cycle often results in further extrusion of sample material. From the thus extruded material a sample can be sliced by, for example, ion beam milling.